Structural elucidations and spectral assigments of two novel triterpene glycosides from Cephalaria paphlagonica

Article abstract of DOI:10.1080/14786410903381335

Two novel triterpene glycosides, paphlagonoside A (1) and B (2), were characterised as 28-O-[-D-glucopyranosyl(1 2) - L-arabinopyranosyl(1 4) - D-glucopyranosyl]-hederagenin (1) and 28-O-[-D-xylopyranosyl(1 4) - L-rhamnopyranosyl(1 3) - D-xylopyranosyl (1 6) - D-glucopyranosyl(1 2) - D-glucopyranosyl]-hederagenin (2) from Cephalaria paphlagonica (Dipsacaceae). In addition to these, a common natural product (hederagenin) (3) was also isolated. The structures of all compounds were identified by spectroscopic and chemical methods. The antimicrobial and antioxidant activities of the plant extracts were examined by MIC and DPPH activity methods, respectively.

Full text of DOI:10.1080/14786410903381335

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Structural elucidations and
spectral assigments of two novel
triterpene glycosides from Cephalaria
paphlagonica
S. Çapanlar a & S. Kırmızıgül a
a Chemistry Department, Faculty of Science , Ege University ,
35100 Bornova, Izmir, Turkey
Published online: 27 Aug 2010.
To cite this article: S. Çapanlar & S. Kırmızıgül (2010) Structural elucidations and spectral
assigments of two novel triterpene glycosides from Cephalaria paphlagonica , Natural Product
Research: Formerly Natural Product Letters, 24:14, 1337-1346, DOI: 10.1080/14786410903381335
To link to this article: http://dx.doi.org/10.1080/14786410903381335
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Natural Product Research
Vol. 24, No. 14, 10 September 2010, 1337–1346
Structural elucidations and spectral assigments of two novel triterpene
glycosides from Cephalaria paphlagonica
*
S. C¸ apanlar and S. K|rm|z|gul
¨
Chemistry Department, Faculty of Science, Ege University, 35100 Bornova, Izmir, Turkey
(Received 19 December 2008; final version received 20 February 2009)
Two novel triterpene glycosides, paphlagonoside A (1) and B (2), were
characterised as 28-O-[ꢀ-D-glucopyranosyl(1 ! 2)-ꢁ-L-arabinopyranosyl
(1 ! 4)-ꢀ-D-glucopyranosyl]-hederagenin (1) and 28-O-[ꢀ-D-xylopyranosyl
(1 ! 4)-ꢁ-L-rhamnopyranosyl(1 ! 3)-ꢀ-D-xylopyranosyl (1 ! 6)-ꢀ-D-glu-
copyranosyl(1 ! 2)-ꢀ-D-glucopyranosyl]-hederagenin (2) from Cephalaria
paphlagonica (Dipsacaceae). In addition to these, a common natural
product (hederagenin) (3) was also isolated. The structures of all
compounds were identified by spectroscopic and chemical methods. The
antimicrobial and antioxidant activities of the plant extracts were examined
by MIC and DPPH activity methods, respectively.
Keywords: Cephalaria paphlagonica; Dipsacaceae; paphlagonoside; triter-
pene glycoside; antimicrobial and antioxidant activity
1. Introduction
There are 93 Cephalaria species belonging to the Dipsacaceae family worldwide, of
which 23 are endemic to and widely distributed in Turkey (Bobrov, 1972). According
to the literature, alkaloids and different types of glycosidic compounds have been
reported from this species (Aliev, Movsumov, & Bagirov, 1975; Aliev, Movsumov, &
Serkerov, 1975, Aliev & Serkerov, 1976; Godevac et al., 2004, 2006a, 2006b;
Movsumov & Aliev, 1975; Movsumov, Aliev, Kondratenko, & Abubakirov, 1975;
Mustafaeva et al., 2008; Tabatadze et al., 2007). In connection with this, elucidation
of some triterpene glycosides and antimicrobial results of Cephalaria transsylvanica
were reported previously by us (K|rm|z|gul & An|l, 1994, 2002; K|rm|z|gul, An|l, &
¨
¨
Rose, 1995, 1996; Kirmiz|gul, An|l, Uc¸ ar, & Akdemir, 1996; K|rm|z|gul & Rose,
¨
¨
1997).
The present article describes the isolation and elucidation of two new triterpene
glycosides, named paphlagonosides A (1) and B (2) from the aerial parts of
C. paphlagonica Bobrov. In addition to these, hederagenin (3) was also isolated from
this plant. Their structures were established on the basis of 1D- and 2D-NMR,
ESI-MS, FT-IR and UV experiments. The antimicrobial and antioxidant activities of
the plant extracts were examined by MIC and 2,20-diphenyl-1-picrylhydrazyl
(DPPH) activity methods, respectively.
*Corresponding author. Email: suheyla.kirmizigul@ege.edu.tr
ISSN 1478–6419 print/ISSN 1029–2349 online
ꢀ 2010 Taylor & Francis
DOI: 10.1080/14786410903381335
http://www.informaworld.com

1338
S. C¸ apanlar and S. K{rm{z{gul
¨
2. Results and discussion
It is known that glycosides represent a large group of secondary metabolites which
are generally found in wild plants, vegetables and flowers. They have great
importance in the field of phytochemistry, pharmacology, biochemistry and
nutriology (Alam et al., 2008; Hostettmann & Marston, 1995; Rai & Mares,
2003). In the context of our investigation, the chemical constituents of Cephalaria
plants belonging to the Dipsacaceae have been discussed for many years. Here, two
novel glycosides have been derived from the most active BuOH phase of
C. paphlagonica Bobrov. (Dipsacaceae), according to the antimicrobial and
antioxidant activity results of the extracts.
Paphlagonoside A (1) was isolated by open silica gel CC. It was obtained as a
light brown amorphous powder and its molecular formula was assigned as
C47H76O18, as indicated by the [M þ Na]þ ion peak at m/z 952.1 by an ESI-MS
ion trap instrument in positive mode. In the UV spectrum, a main maximum at
207.0 nm was an n–ꢂ* absorption of the CH3COOH group. In the FT-IR spectrum,
the peaks at 3367.79, 2943.30, 1736.63, 1630.32 and 1069.95 cmꢀ1 indicated the
groups –OH, –C–H, –C¼O, –C¼C– and C–O–C, respectively.
1
The H-NMR spectrum of compound 1 exhibited six methyl signals at 0.55
(3H, s), 0.65 (3H, s), 0.84 (9H, s) and 1.05 (3H, s) ppm, belonging to H-24, H-25,
H-26, H-29, H-30 and H-27 protons. The C-12 proton was observed at 5.14 ppm as a
broad singlet. H-3 proton gave a signal at ꢃ 3.41 (1H, m). From these data the
aglycone was found to be hederagenin. In addition to these, the three anomeric
proton signals were determined at ꢃ 5.20 (d, J ¼ 8.4 Hz), ꢃ 4.19 (bs) and ꢃ 4.18
(d, J ¼ 7.6 Hz), belonging to glucose, arabinose and glucose, respectively.
Comparison of 13C-NMR and DEPT spectra gave eight quaternary carbon
atoms. Two specific quaternary carbon atoms in this molecule were seen at C-28 and
C-13, and their signals were assigned at 175.98 and 144.17 ppm, confirming the
esteric carbonyl carbon and olefinic carbon of aglycone, respectively. In addition to
these signals, we also observed a C-3 signal at 80.69 and C-12 signal at 122.37 ppm.
From this information, it is clear that the aglycone is a hederagenin type of
triterpene. According to the micro-hydrolysis technique, compound 1 has two
glucose and one arabinose unit. The anomeric carbon signals of these sugars were
detected at 94.74, 103.65 and 105.40 as glucose, arabinose and glucose, respectively.
The linking point of the sugar units was deduced by HMBC correlations which
were observed between the carboxylic carbon at 175.98 ppm and the anomeric
proton signal at ꢃ 5.20 (1H, d, J ¼ 8.4 Hz, Glc H-1). These findings confirmed that
the sugar moiety was linked to the aglycone through the carboxyl terminus. When we
looked at the linking points of sugars with each other, we clearly observed the
correlations between C-100 and H-40 and C-1000 and H-200. However there was no
correlation between the C-3 carbon and the anomeric proton in the HMBC
spectrum. The other important correlation was seen between C-23 and H-24. All
these findings proved that compound 1 is a monodesmosidic acyl glycoside.
On the basis of these results, the structure of paphlagonoside A (1) was assigned
as 28-O-[ꢀ-D-glucopyranosyl(1 ! 2)-ꢁ-L-arabinopyranosyl(1 ! 4)-ꢀ-D-glucopyrano-
syl]-hederagenin, a new monodesmosidic acyl glycoside (Table 1, Figure 1).
Compound 2 was isolated from the same open column as another fraction. It was
isolated as an off-white amorphous powder. The molecular formula was obtained

Natural Product Research
Table 1. 1H and 13C-NMR data of compounds 1 and 2 in DMSO (ꢃ ppm).
Compounds
1339
1
2
Position
ꢃ
13C
1H (J Hz)
ꢃ
13C
1H (J Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
33.94
25.80
80.69
42.95
46.25
17.80
36.66
46.81
49.20
41.40
23.66
122.37
144.17
42.00
27.95
23.20
46.65
47.82
38.73
30.95
32.52
32.33
63.81
13.70
17.42
16.35
26.22
175.98
33.46
23.66
1.11 m
1.68 m
3.41 m
33.74
26.07
80.14
46.73
46.26
17.77
39.59
46.89
42.95
41.38
23.41
122.36
144.15
41.99
27.89
23.20
36.63
47.42
39.45
30.94
32.47
32.27
63.05
13.79
16.30
17.39
26.21
175.99
33.46
24.04
3.44 m
–
1.05 m; 1.56 m
–
1.92 m
1.20 m
–
–
–
1.15 m
1.20 m
–
2.72 m
1.58 m
5.14 bs
2.70 d (J ¼ 9 Hz)
5.12 bs
–
–
–
–
1.30 m
0.94 m
–
1.47 m
0.90 m; 1.47 m
–
1.28 m
1.28 m
3.08 m; 3.31 m
0.55 s
0.84 s
0.65 s
1.08 s
–
–
–
1.45 m
1.38 m
1.12 m
3.06 m; 3.38 m
0.55 s
0.65 s
0.84 s
1.05 s
–
0.84 s
0.84 s
0.84 s
0.84 s
28-O-Sugar moieties
10
94.74
5.20 d (J ¼ 8.4 Hz)
3.09 m
3.27 m
3.31 m
3.28 m
3.29 m; 3.64 m
4.19 bs
2.95 m
3.10 m
3.29 m
3.59 m; 3.90 m
4.18 d (J ¼ 7.6 Hz)
3.29 m
3.14 m
2.99 m
3.03 m
94.80
74.39
76.98
74.11
77.02
61.98
103.82
74.02
74.96
70.58
76.89
66.40
106.37
72.98
81.76
68.78
5.20 d (J ¼ 8 Hz)
20
70.11
73.40
76.95
77.23
65.70
105.40
74.14
72.92
69.92
68.50
103.65
71.82
77.02
70.58
77.37
30
40
3.52 m
500
600
100
200
300
400
5
3.44 m; 3.59 m
4.30 d (J ¼ 6.8 Hz)
3.09 m
3.02 m; 3.68 m
4.20 d (J ¼ 7.6 Hz)
600
1000
2000
3000
4000
3.61 m
(Continued )

1340
S. C¸ apanlar and S. K{rm{z{gul
¨
Table 1. Continued.
Compounds
1
2
Position
ꢃ
13C
1H (J Hz)
3.41 m; 3.61 m
ꢃ
13C
1H (J Hz)
5000
61.58
68.85
100.21
68.02
69.77
79.78
68.52
18.38
101.78
71.83
79.78
71.20
65.60
3.53m
5.12 bs
10000
20000
30000
40000
50000
60000
100000
200000
300000
400000
500000
3.43 m
3.91 d (J ¼ 10.4 Hz)
1.05 d (J ¼ 6 Hz)
4.40 d (J ¼ 7.6 Hz)
3.12 m
3.28m; 3.64 m
H3C
CH3
12
CH3
28
O
CH3
OR3
1
CH3
3
R1O
H3C
23
OR2
Figure 1. Structures of compounds 1–3.
Compounds
R1
R2
R3
1
2
3
H
H
H
H
H
H
Glc(1!2)Ara (1!4) Glc-
Xyl(1!4)Rha(1!3)Xyl(1!6)Glc (1!4)Glc-
H
as C58H94O26 and the molecular ion peak was observed at m/z 1230 [M þ Na]þ by an
ESI-MS ion trap instrument in positive mode. The UV spectrum showed a specific
maximum at 208.0 nm, which was an n–ꢂ* absorption of the CH3COOH group. The
IR spectrum signals of ꢀOH, –C¼C– and C–O–C were observed at 3350.72, 1629.64
and 1055.58 cmꢀ1, respectively.
1
In the H-NMR spectrum, six methyl signals of the aglycone were exhibited at
0.55 (3H, s), 0.65 (3H, s), 0.84 (9H, s) and 1.08 (3H, s) ppm. Two other main signals,

Natural Product Research
1341
H-12 and H-3, were obtained at ꢃ 5.12 (1H, bs) and ꢃ 3.44 (1H, m). The anomeric
proton signals of the sugar moieties were determined at 5.20 (d, J ¼ 8.0 Hz), 4.30 (d,
J ¼ 6.8 Hz), 4.20 (d, J ¼ 7.6 Hz), 5.12 (bs) and 4.40 (d, J ¼ 7.6 Hz) ppm, belonging to
glucose, glucose, xylose, rhamnose and xylose, respectively.
Characteristic quaternary carbon signals of C-13 and C-28 were determined at
144.15 and 175.99 ppm. These data show that the aglycone of the glycoside is
hederagenin. In the 13C-NMR spectrum, the anomeric carbon atoms were
determined at 94.8 (C-10), 103.82(C-100), 106.37(C-1000), 100.21(C-10000
) and
101.78(C-100000) ppm for glucose, glucose, xylose, rhamnose and xylose, respectively.
These results were confirmed by HMQC spectrum. In the HMBC spectrum, the C-3
carbon atom was assigned at 80.14 ppm and there is no correlation concerning the
connection of carbohydrates from this point; in other words, this point is not
branched. Besides that, the other specific correlations between C-3 and H-24 and
C-23 and H-24 also showed that sugar moieties were not connected to C-3, but the
same HMBC spectrum revealed that the correlations between C-28 and H-10
exhibited the sugar moieties that were linked to the aglycone from the carbonyl
carbon. This means that this compound is a monodesmosidic acyl glycoside. The
connection points of the sugars with each other were also determined by the HMBC
spectrum as C-100 and 3.52 (H-40); C-1000 and 3.02, 3.68 (H-600); C-3000 and 4.20 (H-1000);
C-3000 and 5.12 (H-10000); C-100000 and 3.43 (H-40000); in addition to these, C-50000 and 5.12
(H-10000); C-50000 and 4.40 (H-100000); C-40000 and 1.05 ppm (H-60000) correlations were
observed in sugar moieties.
Thus, paphlagonoside B (2), which is named 28-O-[ꢀ-D-xylopyranosyl(1 ! 4)-ꢁ-
L-rhamnopyranosyl(1 ! 3)-ꢀ-D-xylopyranosyl(1 ! 6)-ꢀ-D-glucopyranosyl(1 ! 4)-ꢀ-
D-glucopyra-nosyl]-hederagenin, is assigned as another new monodesmosidic acyl
glycoside (Table 1, Figure 1).
The structure of the last compound (3) was determined by spectroscopic and
co-chromatographic experiments as hederagenin. Thus, the structures of the two acyl
triterpenic glycosides which have been observed rarely in natural product chemistry
will contribute markedly to this genus, in addition to providing a considerable
improvement in the chemotaxonomic markers of this family.
3. Experimental
3.1. General
The 1D and 2D NMR spectra of the compounds were recorded on a Varian AS 400
spectrometer in DMSO using TMS as an internal standard. ESI-MS measurements
were performed by a Bruker HCT Ultra ESI-MS ion trap instrument. IR spectra
were recorded on an ATI Mattson Genesis Series FT-IR in KBr. UV spectra were
run on a Shimadzu UV-160S double beam spectrophotometer using 1.0 cm quartz
cells. Optical rotations were measured in DMSO with a Rudolp Research Analitical
Autopol I automatic polarimeter. Thin layer chromatography (TLC) was carried out
using silica gel 60 F254 (Merck 5554) and reverse-phase silica gel RP-18 F254S (Merck
15685) pre-coated aluminium sheets. Open column chromatography (CC) was
carried out using silica gel 60 (0.063–0.200 mm) (Merck 7734) as a sorbent. Vacuum
liquid chromatography (VLC) and medium pressure liquid chromatography
(MPLC) were performed over reverse-phase silica gel LiChroprep RP-18

1342
S. C¸ apanlar and S. K{rm{z{gul
¨
(25–40 mm) (Merck 9303). TLC analysis of the compounds and sugar moieties was
performed on pre-coated silica gel plates. All chromatographic studies were run
using hexane, BuOH : H2O (1 : 1), CH2Cl2 : MeOH : H2O (80 : 20 : 2–61 : 32 : 7 þ 10%
MeOH) and CHCl3 : MeOH (9 : 1) solvent and solvent systems. The spots were
detected by spraying the plates with 20% H2SO4 solutions in water followed by
heating at about 110ꢁC.
For antioxidant activity studies, 2,20-diphenyl-1-picrylhydrazyl, EtOH and 3-ter-
but-4-hydroxyanisole (BHA) were used as reactives for the DPPH radical activity
method. Ascorbic acid was used as the standard.
Klebsiella pneumonie (CCM 2318), Enterococcus faecalis (ATCC 29212),
Staphylococcus epidermidis (ATCC 12228), Bacillus cereus (ATCC 7064),
Escherichia coli (ATCC 23999), Salmonella typhimurium (CCM 5445),
Pseudomonas aureginosa (ATCC 27853) and Staphylococcus aureus (ATCC 6538-
P) were used as test microorganisms on the basis of MIC values for C. paphlagonica
in the antimicrobial activity analysis. Gentamycin was used as a positive control for
bacteria.
3.2. Plant material
Cephalaria paphlagonica was collected from C
between Basaraz-Kazanc| villages about 1200–1300 m height in July 2007. A voucher
specimen was collected and identified by Prof. Dr Huseyin Sumbul and Asst. Prof.
¸ ank|r|, Turkey: Ilgaz rocky places
¨
¨
¨
R. Suleyman Gokturk. It was deposited at the Herbarium of the Research and
¨
¨
¨
Application Center of Akdeniz University, Antalya, Turkey, with the number
R.S. Gokturk 6100.
¨
¨
3.3. Extraction and isolation
Dried and powdered aerial parts of C. paphlagonica (1.76 kg) were extracted with
3 ꢂ 7000 mL of MeOH at room temperature. Each extraction was treated with
solvent overnight. The MeOH extracts were combined and the solvent was removed
by rotary evaporator under reduced pressure at ꢃ40ꢁC. The residue was extracted
with MeOH : H2O (1 : 1). The activity results showed that the MeOH : H2O extract
was more active than the other extracts for this plant. Therefore, this phase was
fractionated with a 1 : 1 / BuOH : H2O (250 : 250 mL) solvent system to obtain, in
particular, the glycosidic components successively.
The MeOH extract (100 g) was also extracted with with a 1 : 1 / BuOH : H2O
(400 : 400 mL) solvent system. The BuOH and H2O extracts were then separated
from each other. The water extract was treated with a 1 : 1 / BuOH : H2O
(400 : 400 mL) solvent system. All BuOH extracts were combined and extracted
with hexane (14 ꢂ 25 mL) to remove apolar parts of the plant. At the end of the
extractions, two main points played an important role in deciding the isolation
processes. The first was the biological activity results of the MeOH : H2O
extract, which showed higher antimicrobial and antioxidant activity than the other
fractions (Tables 2 and 3). The second was the TLC findings of the BuOH phase,
which showed a greater number of intense spots, indicating the presence of more
glycosides in that fraction than the others. These two results may indicate that the

Natural Product Research
1343
Table 2. Antimicrobial activity (MIC) results of the extracts and gentamicin (mg mLꢀ1).
Microorganisms
MeOH Hexane MeOH : H2O BuOH H2O Gentamycine
S. aureus ATCC 6538/P
S. epidermidis ATCC 12228
S. typhimurium CCM 5445
E. coli ATCC 23999
1000
750 42000
1250 42000
750
750 42000
1250 1500
750 42000
1250 42000
1250
42000
42000
42000
42000
42000
42000
42000
42000
82.5 42000
82.5 42000
82.5 42000
82.5 42000
82.5 42000
82.5 42000
82.5 42000
82.5 42000
1.0
1.0
1.0
1.0
4.0
4.0
16.0
2.0
1000
B. cereus ATCC 7064
K. pneumonie CCM 2318
E. faecalis ATCC 29212
P. aeroginosa ATCC 27853
Table 3. DPPH activity results of the extracts and vitamin C (IC50, mg mLꢀ1).
Samples
MeOH
Hexane
MeOH : H2O
BuOH
H2O
C. paphlagonica
Vitamin C
385
–
2284
–
194
–
410
–
462
11
most active compounds are present in the BuOH fraction. Therefore, isolation
studies were started with the BuOH (35.73 g) extract. This extract was chromato-
graphed by VLC using RP silica gel (230 g) column, eluting from 100% H2O to
100% MeOH, increasing the MeOH by 10%, and 10 fractions were obtained.
Fraction 8 (9.89 g) was subjected to open silica gel column and eluted with a gradient
of 80 : 20 : 2–61 : 32 : 7 / CH2Cl2 : MeOH : H2O solvent systems, giving paphlagono-
side A (1) (507.40 mg) and paphlagonoside B (2) (291.00 mg) (Table 1).
3.4. Acid hydrolysis of 1 and 2
The sugar moieties of the glycosides were identified by the micro-hydrolysis method
on TLC under hydrochloric acid vapour. Pure compounds were applied on a TLC
layer (silica gel 60 F254) and treated with concentrated HCl vapour in a closed vessel
for 45 min at 60ꢁC. After the excess HCl was removed from the plate, the sugar
references (glucose, galactose, arabinose, xylose, rhamnose, mannose and fucose)
were applied to the TLC layer. The TLC was developed by
a
CHCl3 : MeOH : H2O : gAcH /16 : 9 : 2 : 2 solvent system. Detection of the spots was
realised by heating the plates at 110ꢁC after spraying with ꢁ-naftol-H2SO4 (2%)
solution (Su et al., 2001).
3.5. Biological activities
Two different activity experiments were undertaken on the studied extracts of the
plant material. These activity methods are given below in detail.
3.5.1. Antioxidant activity
Antioxidant activity studies were prepared using the DPPH radical method for
C. paphlagonica. This method is representative of the methods employing model

1344
S. C¸ apanlar and S. K{rm{z{gul
¨
radicals in the evaluation of radical scavengers; such methods have gained high
popularity over the last decade because of their rapidity and sensitivity. The
important reagent DPPH, which has the molecular formula C18H12N5O6, was
provided from Sigma (80% D-9132) and DPPH was dissolved in EtOH (200 mL) to
obtain a 10ꢀ4 M solution. L-Ascorbic acid (Vitamin C) (Sigma) and 3-ter-but-4-
hydroxyanisole (BHA) (Fluka, 20021) were used as standards at a concentration of
0.01% (w/v). The mass ratios of the solutions were prepared as extract
(DPPH ¼ 5.5 : 1) and standard (DPPH 0.5 : 1). For the control, 1 : 1/MeOH : DPPH
solution (AB) was used. Extract : EtOH/1 : 1 solution was used to prevent absorbance
mistakes during measurement of the coloured extracts. The samples were incubated
for 15 min in the dark at 30ꢁC and from each sample 200 mL was added to a 96-well
plate (PS Microplate 96 well, Greiner). A decrease in absorbance at 517 nm was
measured against ethanol using a spectrophotometer (ꢄ Quant Universal Microplate
Spectrophotometer, KCJunior). Ethanol was used as a blank in spectrophotometric
assay; a blank sample containing the same amount of ethanol and DPPH was
prepared and measured daily. They were stored in a flask covered with aluminium
foil and kept in the dark at 4ꢁC between consecutive measurements (Koleva, van
Beek, Linssen, Groot, & Evstatieva, 2002). All values were performed in triplicate.
The radical scavenging activities of the tested samples are expressed as percentage
inhibition of DPPH and they were calculated according to the following formula
(Yen & Duh, 1994):
Percentage inhibition ¼ 1 ꢀ ½ðAB ꢀ AAÞ=ABꢄ ꢂ 100
where AB and AA are the absorbance values of the test and of the blank samples,
respectively (Table 3).
3.5.2. Antimicrobial activity
Antimicrobial activity was examined by the minimum inhibition concentration
(MIC) values. In this technique, the MIC values of the extracts were developed by
National Committee of Clinical Laboratory Standards (NCCLS, 2003) and Atlas,
Parks and Brown (1995). In vitro antimicrobial activity studies were carried out
against different bacteria, which were obtained from the Microbiology Department
Culture Collection of the Faculty of Science, Ege University. The bacteria strains
were inoculated on Mueller–Hinton broth (Difco) and incubated for 24 h at
37 ꢅ 0.1ꢁC. The inocula of the bacterial strains were prepared from 24 h broth
cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. This
test was carried out using a series of test tubes containing the concentrations from 0.5
to 40 mg mLꢀ1 for C. paphlagonica extracts. The 96-well plates were prepared by
dispensing into each well 100 mL of broth containing the inoculum
(1 ꢂ 105 CFU mLꢀ1). The extract prepared at a concentration of 40 mg mLꢀ1 was
added into the first well. Then, its serial dilutions were transferred into consecutive
wells. The last well, containing Mueller–Hinton broth without compound and the
inoculum on each strip, was used as negative control. The plate was covered with a
sterile plate sealer. The plates were incubated at 37ꢁC for 24 h. Gentamycin (Sigma)
and clotrimazole (Sigma) were used as positive controls for bacteria strains. All
assays were performed in duplicate (Table 2).

Natural Product Research
1345
Acknowledgements
The authors are grateful to Prof. Dr H. Sumbul and Asst. Prof. R.S. Gokturk for collection
¨
¨
¨
¨
¨
˘
and identification of the plant, and Prof. Dr N. Arda and Assoc. Prof. Dr U.K. Yavasoglu for
discussing the antioxidant and antimicrobial activity results. We thank our Faculty for NMR
spectra and the Biology Department of our Faculty for ESI-MS analysis, respectively. We are
also thankful to TUBITAK (107T028) for financial support.
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