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Journal of Ethnopharmacology

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Identiﬁcation of the main active antidepressant constituents in a traditional Turkish medicinal plant, Centaurea kurdica Reichardt
Fatma Tuğçe Gürağaç Derelia, Mert Ilhanb, Esra Küpeli Akkola,∗
a Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler, 06330, Ankara, Turkey
b Department of Pharmacognosy, Faculty of Pharmacy, Van Yüzüncü Yıl University, Tuşba, 65080, Van, Turkey

A R T I C L E I N F O

Keywords: Centaurea kurdica Antidepressant Asteraceae BALB/C
A B S T R A C T

Ethnopharmacological relevance: In Turkish folk medicine, infusions and decoctions prepared from the ﬂowers, fruits and aerial parts of Centaurea kurdica Reichardt (Asteraceae) are used as sedative and antidepressant-like eﬀects of various sedative plants have been identiﬁed in many studies. The present study was designed to evaluate the antidepressant activity of this plant.
Materials and methods: n-Hexane, ethyl acetate (EtOAc), and methanol (MeOH) extracts were prepared from the branches with leaves and also ﬂowers of the plant. Antidepressant potentials of these extracts were researched by using the forced swimming test, tail suspension test, and antagonism of tetrabenazine-induced ptosis, hy- pothermia, and suppression of locomotor activity.
Results: After determination of high antidepressant potentials of MeOH extract prepared from ﬂowers and n- hexane extract prepared from branches with leaves, isolation studies were carried out on these two extracts and the main active components were determined as β-amyrin, mixture of β-sitosterol and stigmasterol and costu- nolide for the branches with leaves and quercitrin, isoquercetin and naringenin-7-O-glucopyranoside for the ﬂowers.
Conclusions: As a result of the mechanistic and toxicity studies planned on this plant, it is thought that C. kurdica
may be a glimmer of hope for depressed patients.

1. Introduction

Depression derives from the verb “deprimo”, which means “being under pressure” in Latin and in medical terminology this term is deﬁned as “emotional depression, lack of interest and energy” (Terziivanova et al., 2018). Although its etiology is not yet fully elucidated, depression
is a complex disease associated with psychosocial, biological, and ge- netic risk factors that are closely interacted each other (Fig. 1) (Yemez and Alptekin, 1998).Suicide risk and concomitant health problems are mainly considered in the treatment of depression and there are three types of options that can be applied sometimes in combination; electroconvulsive therapy (ECT), psychotherapy and pharmacotherapy (Keller, 2003). Psy- chotherapy, which is the ﬁrst choice in the treatment of depression, aims to save the patient from low self-esteem and social isolation. Clinical studies have shown that the combination of psychotherapy with pharmacotherapy is more eﬀective than administering both treatments alone (Thase et al., 1997). There are various antidepressant drugs used in the pharmacotherapy of depression which have diﬀerentmechanisms such as selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), tricyclic anti- depressants (TCAs), monoamine oxidase inhibitors (MAOIs). Depres- sion continues to be one of the important health problems waiting for a solution because of the delay in the eﬀectiveness of the therapeutics used for treatment, negative side eﬀect proﬁles of them and the risk of recurrence of the disease at the end of the treatment (Polyakova et al., 2015). Because of recurrent depressive attacks, the patient’s belief in treatment is broken. Therefore, many studies have been carried out to develop alternative therapeutics with higher eﬃcacy and lower side- eﬀect proﬁle for the treatment of depression (Zis and Goodwin, 1979). The genus Centaurea (Asteraceae) is commonly found in Turkey with 180 species, 109 of which are endemic (Wagenitz, 1975; Duran and Duman, 2002). This genus has been used in traditional folk medi- cine because of its antipyretic, anti-inﬂammatory, antidiabetic, anti- bacterial, diuretic, antidiarrhetic, digestive, stomachic, choleretic, co- lagog, antirheumatic, astringent, hypotensive and cytotoxic properties (Farrag et al., 1993; Gurkan et al., 1998; Kaij-A-Kamb et al., 1992; Orallo et al., 1998). Several biological activities of this genus and its

∗ Corresponding author.
E-mail address: [email protected] (E. Küpeli Akkol).

https://doi.org/10.1016/j.jep.2019.112373

Received 23 July 2019; Received in revised form 30 October 2019; Accepted 31 October 2019
0378-8741/©2019PublishedbyElsevierB.V.

ImagePleasecitethisarticleas:FatmaTuğçeGürağaçDereli,MertIlhanandEsraKüpeliAkkol,JournalofEthnopharmacology,

https://doi.org/10.1016/j.jep.2019.112373

Risk factors for depression.
components including anti-inﬂammatory, analgesic, antipyretic, anti- microbial, antiviral, antiulserogenic, antidiabetic, antioxidant, wound healing, hepatoprotective, anticancer activities have been so far re- ported (Kaij-A-Kamb et al., 1992). Centaurea genus is also especially known for containing several secondary metabolites like ﬂavonoids and sesquiterpene lactone derivative compounds (Flamini et al., 2001; Koukoulitsa et al., 2002, 2005; Saroglou et al., 2005; Shoeb et al., 2007; Yayli et al., 2006).
In addition to Turkey, Centaurea species are commonly used in folk medicine in worldwide. For instance, the ﬂowers of C. cyanus have been used as a diuretic, eye-wash and for the treatment of cystitis, coughs, nervous and gastric diseases, uterine bleeding in Russian folk medicine. Furthermore, the plant is used to treat minor ocular inﬂammation, fever, gynecological problems, digestive complaints, wounds and der- matological complaints in European traditional medicine (Bruneton, 1995; Hansel et al., 1992; Kern et al., 1972; Pieroni et al., 2004; Shikov et al., 2014).
In the present work, the antidepressant activity of the Centaurea kurdica Reichardt which is one of the endemic Centaurea species dis- tributed in the Eastern and Southeastern Anatolia Regions of Turkey (Wagenitz, 1975) has been attempted to elucidate and main active chemical constituents of the plant were investigated by bioassay-guided fractionation process. The infusions and decoctions prepared from the
ﬂowers, fruits and aerial parts of the plant known as “pamuk dikeni, papaz tacı” are used as sedative and also for the treatment of kidney
diseases, rheumatism and wound in Turkey (Akgul et al., 2018; Cakilcioglu and Turkoglu, 2010; Hayta et al., 2014; Öztürk and Altay, 2017; Polat et al., 2013).activity of C. kurdica, verifying the traditional knowledge and de- Used Parts Solvents Used for Extraction Yields of Extracts (%)
termining the compound(s) responsible for the activity were aimed in
this study. Branches with leave s Aqueous
n-Hexane 12.8
6.5
EtOAc 13.2
MeOH 17.1
Flowers Aqueous 14.4
n-Hexane 15.3
EtOAc 9.1
MeOH 8.8

For aught we know, no report on the antidepressant activity of C. kurdica is available to date. Therefore, investigating the antidepressant

2. Materials and methods

2.1. Plant material

The plant was collected from Artuklu/Mardin on the date of June 22, 2018 with attention to the morphology of the leaves in order not to confuse it with Centaurea sclerolepis Boiss. Because these two similar endemic taxons growing in the same area could be separated only with morphology of their leaves. The leaves are undivided and basal leaves are oblong-ovate with cordate base in C. kurdica, while lower and basal leaves are lyrate or lanceolate in C. sclerolepis (Wagenitz, 1975). The plant material was authenticated by Prof. Dr. Hayri DUMAN from Gazi University, Department of Biology, Faculty of Science and Art, Ankara. A voucher specimen with the code GUEF3474 is kept in the Herbarium of the Faculty of Pharmacy, Gazi University, Ankara, Turkey.

2.2. Extraction, fractionation, isolation and structural identiﬁcation procedures

2.2.1. Extraction of the plant materials
Milled dry branches with leaves and the ﬂowers were separately subjected to extraction at room temperature ﬁrstly with aqua and then n-hexane, ethyl acetate (EtOAc), and methanol (MeOH), successively. After ﬁltration, the extracts were evaporated to dryness by using a ro- tary evaporator. The yield of the each extract was presented as a table (Table 1). Since n-hexane extract prepared from the leaves and MeOH

Table 1
Yields of the extracts prepared from C.kurdica.
Eﬀects of the aqueous extracts prepared from C. kurdica in the antidepressant activity tests.
Forced Swimming Test
Material Dose (mg/kg. Duration of immobility (s) Variation (%) F (DFn, DFd), p
p.o.) (Mean ± S.E.M.)
Control
Branches with leaves –
100 205.13 ± 22.54
190.91 ± 20.66 –
−6.93 F (4, 25) = 10.29
p < 0.0001
extract
Flowers extract 187.15 ± 20.32 −8.77
Imipramine HCl 30 102.87 ± 9.98** −49.85
50 85.41 ± 7.64*** −58.36
Tail Suspension Test
Material Dose (mg/kg. Duration of immobility (s) Variation (%) F (4, 25) = 14.10
p.o.) (Mean ± S.E.M.) p < 0.0001
Control
Branches with leaves –
100 215.37 ± 27.01
198.91 ± 21.13 –
−7.64
extract
Flowers extract 201.18 ± 21.95 −6.59
Imipramine HCl 30 82.81 ± 7.82*** −61.55
50 71.26 ± 6.91*** −66.91
Antagonism of tetrabenazine-induced ptosis, hypothermia and suppression of locomotor activity
Material Dose (mg/kg. Ptosis mean score (Mean ± S.E.M.) Locomotor activity Mean decrease in rectal F (3, 20) = 3.008
p.o.) (%) temperature (°C) p = 0.0544
(Mean ± S.E.M.)
Control – 3.83 ± 1.27 0.00 5.12 ± 0.43
Branches with leaves 100 3.50 ± 1.19 16.70 4.81 ± 0.40
extract
Flowers extract 3.67 ± 1.21 0.00 4.43 ± 0.39
Fluoxetine HCl 25 0.00 ± 0.00*** 100.00*** 0.30 ± 0.03***
*: p < 0.05; **: p < 0.01; ***: p < 0.001; S.E.M.: Standard Error of the Mean.

extract prepared from the ﬂowers were found active in bioactivity ex- periments, it was decided to continue fractionation process on these two extracts.

2.2.2. Fractionation of the branches with leaves n-hexane extract
n-Hexane extract of the branches with leaves (12 g) was subjected to silicagel column chromatography to obtain 183 fractions which were combined as follows after thin-layer chromatography (TLC) control
using n-hexane:CHCl3 (5:5) as the mobile phase: Fr. (1–42), Fr. (43–78), Fr. (79–105), Fr. (106–139) and Fr. (140–183). The active fractions Fr. (43–78) and Fr. (140–183) were applied to silicagel column to obtain pure compounds.

2.2.3. Fractionation of the ﬂowers MeOH extract
The separation of the MeOH extract of the ﬂowers (26 g) was per- formed on RP-18 column vacuum liquid chromatography to obtain 28 fractions which were combined as follows after thin-layer chromato- graphy (TLC) control using EtOAc:CHCl3:MeOH:H2O (15:8:4:1) as the mobile phase: Fr. (1–4), Fr. (5–8), Fr. (9–23) and Fr. (24–28). The active
fraction Fr. (9–23) was applied to silicagel column to obtain pure
compounds.

2.2.4. Structure determination of the compounds
Nuclear magnetic resonance (1H and 13C NMR) and mass spectro- scopy (MS) techniques were used for the structural elucidation of the compounds. While NMR spectra were recorded on a Bruker Ascend spectrometer, mass analyses were performed by using Agilent G6550A Q-TOF mass spectrometer and Waters LCT Premier XE UPLC/MS-TOF spectrometer. A total of 6 compounds were isolated from both active extracts and the isolates were identiﬁed as β-Amyrin, the mixture of stigmasterol and β-sitosterol, costunolide, quercitrin (quercetin-3-O- rhamnopyranoside), isoquercetin (quercetin-3-O-glucopyranoside) and naringenin-7-O-glucopyranoside by comparing their spectroscopic data
with related literatures (Dos Santos et al., 2014; Ferrari et al., 2005; Masoko and Nemudzivhadi, 2015; Pierre and Moses, 2015; Ragab et al., 2010; Sunil et al., 2014; Zhang et al., 2014).

2.3. Experimental animals

Male BALB/c mice weighing 25–30 g were purchased from Laboratory Experimental Animals, Kobay (Ankara, Turkey). All of the animals were kept under laboratory conditions (12 h light-12 h dark
light period at 21–24 °C temperature and 40–45% humidity conditions) for at least 3 days before starting the experiment in order to adapt to the
environment. During this waiting period, all of the animals were fed with standard pellet chow and tap water. The experiment was approved by the Experimental Animal Ethics Committee of Kobay (Protocol number: 256). Six animals were used for each of the control and test groups. BALB/c mice were preferred in experiments because this strain is more susceptible to acute stress stimuli when faced with new con- ditions and has a higher level of fear than other strains and the reason for preferring male mice in experiments was to prevent mood changes caused by estrous cycle of female mice (Lassalle et al., 1994; Tannenbaum and Anisman, 2003).

2.4. Preparation of test samples

The prepared extracts were suspended in 0.5% carboxymethyl cel- lulose (CMC) solution with the help of the ultrasonic bath and these test samples were administered to the test group animals by oral gavage at the dose of 100 mg/kg. The antidepressant activity evaluation was done with a single dose in accordance with the ethical guidelines for the use of animals in research. Since there is no information about the appli- cation dose of C. kurdica in the literature, 100 mg/kg dose was preferred in practice. In order to determine the activity of the test materials, only 0.5% CMC solution was administered orally to the animals in the

Eﬀects of the extracts and fractions prepared with organic solvents from C. kurdica in the forced swimming test.
Eﬀects of the Extracts
Material Dose (mg/kg. p.o.) Duration of immobility (s) (Mean ± S.E.M.) Variation (%) F (DFn, DFd), p
Control
Branches with leaves
n-Hexane extract –
100 210.17 ± 25.59
118.54 ± 13.04** –
−43.60 F (8, 45) = 3.845
p = 0.0016
EtOAc extract 131.18 ± 15.04 −37.58
MeOH extract 142.69 ± 20.93 −32.11
Flowers n-Hexane extract 145.37 ± 21.12 −30.83
EtOAc extract 132.41 ± 15.05 −37.00
MeOH extract 120.23 ± 13.09** −42.80
Imipramine HCl 30 115.76 ± 10.48** −44.92
50 91.27 ± 8.63*** −56.57
Eﬀects of the Fractions Obtained from Active n-Hexane Extract
Material Dose (mg/kg. p.o.) Duration of immobility (s) Variation (%) F (DFn, DFd), p
(Mean ± S.E.M.)
Control Fr. (1–42)
Fr. (43–78)
Fr. (79–105)
Fr. (106–139)
Fr. (140–183)
Imipramine HCl –
100

30 208.67 ± 23.46
131.40 ± 15.07
119.67 ± 13.06** 143.91 ± 21.05 152.86 ± 23.01
117.29 ± 12.99**
107.16 ± 9.86** –
−37.03
−42.65
−31.03
−26.75
−43.79
−48.65 F (7, 40) = 4.707
p = 0.0006
50 88.63 ± 8.43*** −57.53
Eﬀects of the Fractions Obtained from Active MeOH Extract
Material Dose (mg/kg. p.o.) Duration of immobility (s) Variation (%) F (DFn, DFd), p
(Mean ± S.E.M.)
Control Fr. (1–4)
Fr. (5–8)
Fr. (9–23)
Fr. (24–28)
Imipramine HCl –
100

30 212.69 ± 26.03
135.41 ± 17.91
138.37 ± 19.03
119.21 ± 13.04**
146.83 ± 21.20
113.86 ± 10.22** –
−36.33
−34.94
−43.95
−30.97
−46.47 F (6, 35) = 4.738
p = 0.0013
50 91.15 ± 8.87*** −57.14
*: p < 0.05; **: p < 0.01; ***: p < 0.001; S.E.M.: Standard Error of the Mean.
control group.

2.5. Reference substances

Tricyclic antidepressant imipramine HCl (30 and 50 mg/kg) and selective serotonin reuptake inhibitor ﬂuoxetine HCl (25 mg/kg) were preferred as reference materials.

2.6. In vivo analysis of antidepressant activity in three diﬀerent models

In this study, in addition to the forced swimming test, tail suspen- sion and antagonism of tetrabenazine-induced ptosis, hypothermia, and suppression of locomotor activity were used.

2.6.1. Forced swimming test
The most commonly used preclinical in vivo antidepressant activity model is forced swimming test because it is fast, economical and reli- able and allows simultaneous investigation of the antidepressant ac- tivity of many compounds (Petit-Demouliere et al., 2005). In this model, it is thought that the inactivity of mice placed separately in
transparent glass cylinders (10 cm diameter by 25 cm height containing 10 cm of water at 21–24 °C) reﬂects the behavioral despair seen in depressive people. Acute antidepressant activity of test sample was assessed by comparing the immobility time of the last 4 min of the 6 min test period according to a modiﬁed method described by Cryan
et al. and Porsolt et al. (Cryan et al., 2005; Porsolt et al., 1977).

2.6.2. Tail suspension test
The tail suspension test is based on the same theoretical basis as the
forced swimming test, which is based on the determination of the period of inactivity of rodents hanging from the tail tips during a single 6-min test session. The inactivity observed in animals is thought to reﬂect the behavioral despair seen in depressed people (Steru et al., 1985).

2.6.3. Antagonism of tetrabenazine-induced ptosis, hypothermia, and suppression of locomotor activity
Tetrabenazine leads to depletion of presynaptic monoamine neu- rotransmitters in the central nervous system and causing several changes as ptosis, hypothermia and akinesia. The acute antagonism of tetrabenazine-induced ptosis, hypothermia, and suppression of loco- motor activity is based on the observation of the durations of im- mobilization, ptosis scores and changes in rectal temperatures of mice.
In this experiment mice with rectal temperatures of 36–38 °C were used
(Pettibone et al., 1984). 60 min after oral administration of suitable materials for the animals in the control and experimental groups, tet- rabenazine which was dissolved in 0.1 M aqueous tartaric acid solution and adjusted to pH 6 with 10% NaOH administered intraperitoneally at 32 mg/kg 30 min after administration, the animals were placed one by one in the center of a 20 cm diameter disc and their locomotor activity percentages, ptosis scores and rectal temperature changes were de- termined. Mice walking to the edge of the disc and looking to the edge, rotating 180° in place, or moving the head 90° in one direction were not considered akinetic. The following formula was used to calculate the percentage of animals showing locomotor activity:
Locomotor activity (%) = (Number of non-akinetic mice X 100) / Number of mice in group

Eﬀects of the extracts and fractions prepared with organic solvents from C. kurdica in the tail suspension test.
Eﬀects of the Extracts
Material Dose (mg/kg. p.o.) Duration of immobility (s) Variation (%) F (DFn, DFd), p
(Mean ± S.E.M.)
Control
Branches with leaves
n-Hexane extract –
100 203.50 ± 22.13
92.83 ± 9.26*** –
−54.38 F (8, 45) = 8.160
p < 0.0001
EtOAc extract 111.99 ± 13.87 −44.97
MeOH extract 176.10 ± 21.14 −13.46
Flowers n-Hexane extract 123.31 ± 15.49 −39.41
EtOAc extract 150.46 ± 20.11 −26.06
MeOH extract 100.17 ± 10.03** −50.78
Imipramine HCl 30 88.11 ± 8.01*** −56.70
50 75.37 ± 7.32*** −62.96
Eﬀects of the Fractions Obtained from Active n-Hexane Extract
Material Dose (mg/kg. p.o.) Duration of immobility (s) Variation (%) F (DFn, DFd), p
(Mean ± S.E.M.)
Control Fr. (1–42)
Fr. (43–78)
Fr. (79–105)
Fr. (106–139)
Fr. (140–183)
Imipramine HCl –
100
30 211.12 ± 26.02
152.47 ± 22.98
118.90 ± 13.01** 139.33 ± 19.01 141.17 ± 19.89
120.39 ± 13.12**
91.26 ± 8.89** –
−27.78
−43.68
−34.00
−33.13
−42.98
−56.77 F (7, 40) = 5.350
p = 0.0002
50 80.72 ± 7.45*** −61.77
Eﬀects of the Fractions Obtained from Active MeOH Extract
Material Dose (mg/kg. p.o.) Duration of immobility (s) Variation (%) F (DFn, DFd), p
(Mean ± S.E.M.)
Control Fr. (1–4)
Fr. (5–8)
Fr. (9–23)
Fr. (24–28)
Imipramine HCl –
100
30 215.41 ± 27.02
135.86 ± 17.93
147.67 ± 21.22
111.53 ± 10.11**
139.32 ± 19.00
95.81 ± 9.12** –
−36.93
−31.45
−48.22
−35.32
−55.52 F (6, 35) = 6.257
p = 0.0002
50 83.72 ± 7.98*** −61.13
*: p < 0.05; **: p < 0.01; ***: p < 0.001; S.E.M.: Standard Error of the Mean.

The ptosis scoring was done according to the following scale: 0: Eyes open.
1: One quarter oﬀ. 2: Half closed.
3: Three quarters oﬀ. 4: Fully enclosed.
60 min after the administration of tetrabenazine, the rectal tem- perature of each mouse was measured again with a clinical digital thermometer (BIO-TK9882 Rodent Thermometer) to record the change.

2.7. Statistical analysis of the data

The results of the animal experiments were expressed as mean ± standard error of the mean. Statistical diﬀerences between the data obtained from treatment and control groups were evaluated using one- way analysis of variance (ANOVA) and Student–Newman–Keuls post hoc tests. P value less than 0.05 was considered signiﬁcant (*p < 0.05;
**p < 0.01; ***p < 0.001).

3. Results and discussion

In Turkey and many other countries, indigenious people have a wealth of knowledge about the plants used against central nervous system diseases. In our study, the antidepressant activity of Centaurea kurdica Reichardt, which is found to be used against central nervous system disorders in the ethnobotanical studies conducted in Turkey, was evaluated in various screening tests using for the assessment of antidepressant-like eﬀects (Cakilcioglu and Turkoglu, 2010). A variety of in vitro, ex vivo and in vivo methods can be utilized to determine
eﬃcacy against depression. However, mechanistic in vivo experimental models are generally preferred to determine the activity against de- pression whose mechanism is not fully understood.

Due to the fact that aqueous extracts of the plant prepared according to public use did not show remarkable activity in any experimental model (Table 2), it was decided to evaluate the antidepressant activity of the extracts prepared with organic solvents.
In the forced swimming test, n-hexane extract prepared from branches and leaves of the plant and MeOH extract prepared from ﬂowers reduced the immobility time in mice by 43.60% and 42.80%, respectively, and these alterations were found to be statistically meaningful (Table 3). Active n-hexane extract was fractionated by silicagel column chromatography to determine the compound(s) responsible for the activity and Fr. (1–42), Fr. (43–78), Fr. (79–105), Fr. (106–139) and Fr. (140–183) were obtained. Fr. (43–78) and Fr. (140–183) within these fractions decreased the inactivity time in mice 42.65% and43.79% respectively, and these results were found to be statistically signiﬁcant (Table 3). In order to determine the compound(s) re- sponsible for the activity, MeOH extract was fractionated by RP-18 si- lica gel column chromatography and four main fractions Fr. (1–4), Fr.
(5–8), Fr. (9–23) and Fr. (24–28) were obtained. The results reported in
Table 3 demonstrated that Fr. (9–23) which reduced immobility time in mice by 43.95% was the most eﬀective among the other fractions.

In the tail suspension test, n-hexane extract prepared from the branches and leaves of the plant and MeOH extract prepared from the ﬂowers reduced the immobility time in mice by 54.38% and 50.78%, respectively, and these results were statistically distinct from the others (Table 4). Among the fractions obtained from n-hexane extract by silica
gel column chromatography Fr. (43–78) and Fr. (140–183) showed a

Eﬀects of the extracts and fractions prepared with organic solvents from C. kurdica in the antagonism of tetrabenazine-induced ptosis, hypothermia and suppression of locomotor activity.
Eﬀects of the Extracts
Material Dose (mg/ Ptosis mean score Locomotor activity Mean decrease in rectal temperature F (DFn, DFd), p
kg. p.o.) (Mean ± S.E.M.) (%) (°C)
(Mean ± S.E.M.)
Control – 3.83 ± 1.29 0.00 5.01 ± 0.43 F (7, 40) = 4.180
Branches with n-Hexane 100 0.08 ± 0.01*** 83.3 0.45 ± 0.04** p = 0.0015
leaves extract
EtOAc extract 1.75 ± 0.52 66.7 1.25 ± 0.09
MeOH extract 3.58 ± 1.18 33.3 2.37 ± 0.19
Flowers n-Hexane 2.08 ± 0.72 16.7 1.72 ± 0.16
extract
EtOAc extract 3.50 ± 1.17 16.7 3.10 ± 0.26
MeOH extract 0.17 ± 0.02** 66.7 0.57 ± 0.05**
Fluoxetine HCl 25 0.00 ± 0.00*** 100.00 0.24 ± 0.02***

Eﬀects of the Fractions Obtained from Active n-Hexane Extract
Material Dose (mg/ Ptosis mean score Locomotor activity Mean decrease in rectal temperature F (DFn, DFd), p
kg. p.o.) (Mean ± S.E.M.) (%) (°C)
(Mean ± S.E.M.)
Control – 3.67 ± 1.21 0.00 5.20 ± 0.46 F (6, 35) = 4.311
Fr. (1–42) 100 2.08 ± 0.72 33.3 3.32 ± 0.29 p = 0.0023
Fr. (43–78) 0.17 ± 0.02** 66.7 0.62 ± 0.06**
Fr. (79–105) 2.25 ± 0.86 33.3 3.55 ± 0.31
Fr. (106–139) 2.08 ± 0.72 16.7 2.75 ± 0.25
Fr. (140–183)
Fluoxetine HCl
25 0.08 ± 0.01***
0.00 ± 0.00*** 83.3
100.00*** 0.56 ± 0.05**
0.31 ± 0.03***
Eﬀects of the Fractions Obtained from Active MeOH Extract
Material Dose (mg/ Ptosis mean score Locomotor activity Mean decrease in rectal temperature F (DFn, DFd), p
kg. p.o.) (Mean ± S.E.M.) (%) (°C)
(Mean ± S.E.M.)
Control – 3.92 ± 1.30 0.00 4.73 ± 0.39 F (5, 30) = 3.524
Fr. (1–4) 100 2.25 ± 0.86 33.3 3.27 ± 0.28 p = 0.0126
Fr. (5–8) 3.67 ± 1.21 66.7 2.55 ± 0.22
Fr. (9–23) 0.17 ± 0.02** 83.3 0.20 ± 0.02***
Fr. (24–28)
Fluoxetine HCl
25 2.42 ± 0.94
0.00 ± 0.00*** 66.7
100.00*** 1.46 ± 0.09
0.17 ± 0.01***
*: p < 0.05; **: p < 0.01; ***: p < 0.001; S.E.M.: Standard Error of the Meanstatistically signiﬁcant antidepressant activity by reducing the in- activity time in mice by 43.68% and 42.98%, respectively (Table 4). The MeOH extract was fractionated by RP-18 silica gel column chro- matography for the purpose of determining the compound(s) re- sponsible for the activity and as observed in Table 4, Fr. (9–23) showed
a statistically signiﬁcant activity by reducing the immobility time by
48.22%.In the antagonism of tetrabenazine-induced ptosis, hypothermia, and suppression of locomotor activity, it was determined that n-hexane extract prepared from the branches and leaves of the plant and MeOH extract prepared from ﬂowers increased the locomotor activities of mice compared to the control group by 83.3% and 66.7%, respectively. The extracts also decreased the ptosis scores of the mice to 0.08 and
0.17 and the amount of the reduction in the rectal temperature of mice as a result of administration of these fractions were found as 0.45 and
0.57 °C, respectively (Table 5). Fr.(43–78) and Fr. (140–183) from the
n-hexane extract fractionated to determine the compound(s) re- sponsible for the activity increased the locomotor activity of mice by 66.7% and 83.3%, respectively. These fractions decreased the ptosis scores of the mice to 0.17 and 0.08 and the amount of the reduction in the rectal temperature of mice as a result of administration of these fractions 0.62 and 0.56 °C, respectively (Table 5). Fr. (9–23) obtained
from the MeOH extract increased the locomotor activity of mice by
83.3%.

This fraction decreased the score of ptosis to 0.17 and the amount of the reduction in the rectal temperature of mice as a con- sequence of administration of Fr. (9–23) was found as 0.20 °C. has beenfound to show a signiﬁcant antidepressant activity compared to the control group (Table 5).
The molecular formula of the isolated compounds from the active fractions were elucidated in the light of mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy data. Molecular weight measurements and hydrogen-carbon analysis are the parameters used to determine the formulas of molecules.30 resonances observed in 13C-NMR spectrum of the CK-1 com- pound isolated as a white powder suggest that the compound was tri- terpenoid. The presence of two resonances at δ 145.4 and δ 121.8 ppm
indicated an intra-ring double bond. The presence of 8 methyl signals
was determined in 1H-NMR spectrum. When the literature ﬁndings and the NMR data of the compound (Table 6) were compared, it was con- cluded that the molecular formula C30H50O and the compound was β-
amyrin (Fig. 2a.) (Sunil et al., 2014).

In the 13C-NMR spectrum of the CK-2 compound isolated as a white
powder, it was concluded that the substance was a mixture because most of the resonances were double. Resonances at δ 140.9 and δ
121.9 ppm in 13C-NMR spectrum indicated the double bond in the ring,
at δ 138.5 and 129.4 ppm suggested the presence of a double bond in the side chain. The signals observed in the 1H-NMR spectrum at δ
3.51 ppm and δ 3.54 ppm indicated the presence of protons in C-3 po- sition, at δ 5.34 ppm and δ 5.35 ppm indicated the presence of protons in C-6 position; at δ 5.15 ppm and δ 5.03 ppm indicated the presence of oleﬁnic protons in C-22. and C-23 positions. When the NMR data were compared with the literature, it was found that CK-2 was a mixture of β-
Table 6
1H and 13C NMR data of CK-1 [β-Amyrin] (CDCl3, 1H: 400 MHz, 13C:
100 MHz).
molecular formula of the compound as C21H21O11 through the ap- pearance of a positive ion peak at 449.1094 m/z due to a protonated molecular ion. When the results were compared with the literature ﬁndings, the compound was identiﬁed as quercitrin (quercetin-3-O- rhamnopyranoside) (Fig. 2e.) (Dos Santos et al., 2014; Zhang et al., 2014).
The CK-5 compound was obtained as a yellow powder and the

C/H δC (ppm) δH (ppm)/J (Hz)
1 (CH2) 38.4 –
2 (CH2) 22.8 –
3 (CH–OH) 81.1 4.50 (1H, dd, J = 8.1; 7.9 Hz)
4 (C) 37.9 q
5 (CH2) 55.4 –
6 (CH2) 18.4 –
7 (C) 32.7 q
8 (CH) 40.0 –
9 (C) 47.4 q
10 (CH) 37.0 –
11 (CH2) 23.7 –
12 (CH) 121.8 5.18 (1H, t, J = 3.7 Hz)
13 (C) 145.4 q
14 (C) 41.9 q
15 (CH2) 27.1 –
16 (CH2) 26.3 –
17 (C) 32.6 q
18 (CH) 47.7 –
19 (CH2) 46.9 –
20 (C) 31.2 q
21 (CH2) 34.9 –
22 (CH2) 37.3 –
23 (CH3) 28.2 0.82–0.87 (3H, s)
24 (CH3) 16.8 0.82–0.87 (3H, s)
25 (CH3) 15.7 0.82–0.87 (3H, s)
26 (CH3) 17.0 0.97 (3H.s)
27 (CH3) 26.1 –
28 (CH3) 28.5 0.82–0.87 (3H, s)
29 (CH3) 33.5 0.82–0.87 (3H, s)
30 (CH3) 23.8 0.82–0.87 (3H, s)

molecular formula of the compound was determined to be C H O
21 20 12

sitosterol (C29H50O) (Fig. 2b.) and stigmasterol (C29H48O) (Fig. 2c.) (Masoko and Nemudzivhadi, 2015)The compound CK-3 was obtained as a white powder and the 1H- NMR spectrum of the compound showed that the signals of methylene protons outside the ring were at δ 6.26 ppm (1H, d, J = 3.9 Hz) and δ
5.52 ppm (1H, d)., J = 3.5 Hz); methyl signals were at δ 1.42 ppm (3H,
s) and δ 1.70 ppm (3H, s). The double bond methyn proton was ob- served at δ 4.85 ppm (1H, dd, J = 11.6–4.2 Hz), the double bond proton adjacent to the lactone ring was at δ 4.74 ppm (1H, d, J = 10.0 Hz), the lactone proton was at δ 4.57 ppm (1H, t, J = 10.0 Hz) and proton in H-7 position was at δ 2.58 ppm (1H, t, J = 9.5; 9.5 Hz). The presence of 15 resonances at 13C-NMR spectrum and the resonance at δ 170.6 ppm suggested that the compound could be sesquiterpene lactone.

Furthermore, according to ESI-MS data, the molecular weight of the compound was determined as 233.1533 m/z [M + H] +. When the NMR data (Table 7) were compared with the literature, it was de- termined that the compound was costunolide (C15H21O2) (Fig. 2d.).
NMR spectra of CK-4 compound which was isolated as yellow powder were evaluated and the molecular formula of the compound was determined as C21H20O11. The resonances observed in the aromatic region in the 1H-NMR spectrum of the compound and the presence of 21 carbons in the 13C-NMR spectrum suggested that the compound could be ﬂavonoid. In the 1H-NMR spectrum of the compound, the
resonances detected at δ 7.34 ppm (1H, d, J = 2.1 Hz), δ 7.31 ppm (1H, dd, J = 8.3; 2.1 Hz) and δ 6.91 ppm (1H, d, J = 8.3 Hz) in the aromatic region, the presence of the ABX system and the presence of two doublets at δ 6.37 ppm (1H, d, J = 2.1 Hz) and δ 6.20 ppm (1H, d, J = 2.1 Hz) suggested that this ﬂavonoid was a quercetin derivative. The presence of anomeric proton at δ 5.35 ppm (1H, d, J = 1.7 Hz) and the presence of a doublet at δ 0.94 ppm (at J = 6.1 Hz) suggested the presence of α-L-rhamnopyranoside in the structure. Thus, the presence
of quercetin and α-L-rhamnopyranoside in the structure was conﬁrmed by the NMR data (Table 8). Moreover ESI-MS spectra indicated the
by evaluating the NMR spectra. The resonances observed in the aro- matic region in the 1H-NMR spectrum of the compound and the pre- sence of 21 carbons in the 13C-NMR spectrum suggested that the compound could be ﬂavonoidal. In the 1H-NMR spectrum of the com- pound, the resonances detected at δ 7.58 ppm (1H, dd, J = 8.2; 2.1 Hz),δ 7.57 ppm (1H, d, J = 2.1 Hz) and δ 6.84 ppm (1H, d, J = 8.2 Hz) in
the aromatic region, the presence of the ABX system and the presence of two doublets at δ 6.40 ppm (1H, d, J = 2.0 Hz) and δ 6.20 ppm (1H, d, J = 2.0 Hz) suggested that this ﬂavonoid was a quercetin derivative. The presence of anomeric proton at δ 5.46 ppm (1H, d, J = 7.3 Hz) suggested the presence of β-D-glucopyranoside in the structure. Thus,
the presence of quercetin and β-D-glucopyranoside in the structure was conﬁrmed by the NMR data (Table 9). The molecular ion peak of CK-5 in positive ion mode was m/z = 465.1047 showing the molecular for- mula C21H21O12. According to the NMR and mass data, CK-5 was identiﬁed as isoquercetin (Fig. 2f.) (Dos Santos et al., 2014; Zhang et al., 2014).

The presence of AB system was determined by proton signals seen in δ 7.31 (2H, d, J = 8.6 Hz) and δ 6.80 (2H, d, J = 8.6 Hz) ppm in the 1H- NMR spectrum of colourless CK-6 compound. Furthermore, the pre- sence of a doublet signal at δ 5.46 ppm (1H, d, J = 7.4 Hz) indicated the
presence of β-D-glucopyranoside in the structure. The presence of 15 carbons in 13C-NMR spectrum, except for β-D-glucopyranoside re- sonances, suggested that the structure was a ﬂavonoidal compound. Resonances at δ 196.4, δ 78.5 and δ 40.1 ppm showed that the com- pound was ﬂavanon. In addition, according to ESI-MS data, the mole-cular weight of the compound was determined to be 435.1288 m/z [M
+ H]+ (C21H23O10). When the NMR data (Table 10) were compared with the literature ﬁndings, the compound was found to be nar- ingenin-7-O-glucopyranoside (C21H22O10) (Fig. 2g.) (Ragab et al., 2010).
To summarize, the above data indicated that isolation studies per- formed by us were ﬁnalized with the isolation of pentacyclic triterpene β-amyrin, the steroidal mixture of β-sitosterol and stigmasterol and sesquiterpene lactone costunolide from the n-hexane extract prepared from the branches and leaves of C. kurdica and also ﬂavonoidal com- pounds quercitrin, isoquercetin and naringenin-7-O- glucopyranoside from the methanolic extract prepared from the ﬂowers of the plant.

Current literature data suggested that only three phytochemical studies have been performed on C. kurdica so far and fatty acids, ﬂa- vonoids and sesquiterpene lactone derivative compounds have been isolated from the plant (Aktumsek et al., 2011; Appendino and Ozen, 1993; Degirmenci et al., 2015). In the study conducted by Degirmenci et al. (2015) hesperidine, naringin, eupatorin, quercetin dihydrate and crisin were isolated from chloroform extract prepared from aerial parts of the plant (Degirmenci et al., 2015). In another study carried out by Appendino and Ozen (1993) sesquiterpene lactone derivative com- pounds reynosine, costunolide and 8-desoxysalonitenolide were iso- lated from the chloroform extract prepared from the aerial parts of the plant. The volatile oils of diﬀerent Centaurea species growing in ﬂora of Turkey analyzed by Aktümsek et al. and the fatty acid contents of the oils were found to be rich in palmitic, linoleic and linolenic acid (Aktumsek et al., 2011).
Steroidal compounds have a wide variety of biological activities mediated by functional groups surrounding the tetracyclic nucleus, which are essentially present in their structure. Recent studies on these compounds have demonstrated that their antidepressant activity. It was determined in a study designed by Zhao et al. (2016) sterols and Chemical structures of isolated compounds from the Centaurea kurdica.

Table 7
1H and 13C NMR data of CK-3 [Costunolide] (CDCl3, 1H: 600 MHz, 13C: 150 MHz).
Table 8
1H and 13C NMR data of CK-4 [Quercitrin] (CD3OD, 1H: 400 MHz, 13C:
100 MHz).

C/H δC (ppm) δH (ppm)/J (Hz) C/H δC (ppm) δH (ppm)/J (Hz)
1 (CH) 127.4 4.85 (1H, dd, J = 11.6–4.2 Hz) 2 (C) 158.5 q
2 (CH2) 26.3 – 3 (C) 136.2 q
3 (CH2) 39.6 – 4 (C]O) 179.7 –
4 (C) 141.6 q 5 (C–OH) 163.2 –
5 (CH) 127.2 4.74 (1H, d, J = 10 Hz) 6 (CH) 99.8 6.20 (1H, d, J = 2.1 Hz)
6 (CH) 82.1 4.57 (1H, t, J = 10 Hz) 7 (C–OH) 165.9 –
7 (CH) 50.6 2.31 (1H, d) 8 (CH) 94.7 6.37 (1H, d, J = 2.1 Hz)
8 (CH2) 28.2 – 9 (C) 159.3 q
9 (CH2) 41.1 – 10 (C) 105.9 q
10 (C) 137.1 q 1' (C) 122.9 q
11 (C) 140.3 q 2' (CH) 116.4 7.34 (1H, d, J = 2.1 Hz)
12 (C]O) 170.6 – 3' (C–OH) 146.4 –
13 (CH2) 119.8 6.26 (1H, d, J = 3.9 Hz). 5.52 (1H. d, J = 3.5 Hz) 4' (C–OH) 149.8 –
14 (CH3) 16.3 1.42 (3H, s) 5′(CH) 116.9 6.91 (1H, d, J = 8.3 Hz)
15 (CH3) 17.5 1.70 (3H, s) 6′(CH) 123.0 7.32 (1H, dd, J = 8.3; 2.1 Hz)
1''(CH) 103.5 5.36 (1H, d, J = 1.7 Hz)
q: quaterner.

especially β-sitosterol provide antidepressant activity by increasing serotonin and noradrenaline levels in the central nervous system. In a

2''(CH–OH) 71.9
3''(CH–OH) 72.1
4''(CH–OH) 73.3 4.3–3.3
5''(CH) 72.0
6''(CH3) 17.7 0.95 (3H, d, J = 6.1 Hz)

study evaluating the activity of sea weed, known as Sargassum fusiforme against depression, it was found that steroidal compound fucosterol isolated from the ethanolic extract prepared from sea weed showed antidepressant activity by increasing serotonin, noradrenaline and BDNF levels in the central nervous system (Zhen et al., 2015). In a si- milar study, it was reported that the steroidal compound “sar-
ingosterol” isolated from the ethanolic extract prepared from Sargassum fusiforme showed antidepressant eﬀect in the forced swimming and tail
q: quaterner.suspension tests (Jin et al., 2017).

In various studies which antidepressant activity of ﬂavonoidal compounds were evaluated, it was determined that these constituents exhibited activity by reversing the distortions in serotonergic, nora- drenergic and dopaminergic neurotransmission systems or regulating

Table 9
1H and 13C NMR data of CK-5 [Isoquercetin] (DMSO, 1H: 400 MHz, 13C:
100 MHz).

C/H δC (ppm) δH (ppm)/J (Hz)
2 (C) 156.3 q
3 (C) 133.3 q
4 (C]O) 177.4 –
5 (C–OH) 161.2 –
6 (CH) 98.6 6.20 (1H, d, J = 2 Hz)
7 (C–OH) 164.1 –
8 (CH) 93.5 6.40 (1H, d, J = 2 Hz)
9 (C) 156.2 q
10 (C) 104.0 q
1' (C) 121.6 q
2' (CH) 115.2 7.58 (1H, d, J = 2.1 Hz)
3' (C–OH) 144.8 –
4' (C–OH) 148.5 –
5' (CH) 121.2 7.59 (1H, dd, J = 8.2; 2.1 Hz)
6' (CH) 116.2 6.84 (1H, dd, J = 8.2 Hz)
1'' (CH) 100.8 5.46 (1H, d, J = 7.3 Hz)
2'' (CH–OH) 76.5
3'' (CH–OH) 74.1
4'' (CH–OH) 69.9 4.3–3
5'' (CH) 77.6
6'' (CH2–OH) 61.0 5.28. 5.05 (2H)
q: quaterner.

Table 10
1H and 13C NMR data of CK-6 [Naringenin-7-O-glucopyranoside] (DMSO,
1H: 400 MHz, 13C: 100 MHz).activity of the aqueous extract of Gossypium hirsutum L. seeds was linked to the eﬀect of the cell protection mechanism of glucoside 3-O-apiosyl (1 → 2) - [rhamnosyl (1 → 6)] - glucoside (Li et al., 2000). In various studies, antidepressant activity of naringenin has been demonstrated by various mechanisms such as regulation of monoamine neurotransmis- sion, modulation of noradrenergic and serotonergic nervous systems, increasing hippocampal glucocorticoidal and monoaminergic receptor levels, activation of BDNF signaling in the hippocampus, and lowering serum corticosterol levels (Yi et al., 2010, 2012; 2014). In a study conducted by Machado et al. (2008), it was reported that the rutin isolated from the ethanolic extract of aerial parts of Schinus molle L. showed antidepressant activity by increasing serotonin and nora- drenaline levels in the synaptic range (Machado et al., 2008).

In addition to the antimicrobial, antioxidant, anticancer and anti-
arrhythmic activities of terpenic compounds, there are various studies on their antidepressant activities (Brahmkshatriya and Brahmkshatriya, 2013). In a study evaluating the antidepressant eﬃcacy of α and β- amyrin mixture isolated from the resin of Protium heptaphyllum (Aubl.) Marchand root cortex, it was found that these pentacyclic triterpenes interacted with GABA-A receptors to provide antidepressant activity (Aragao et al., 2006). Similarly, sesquiterpene lactones have been shown to have antidepressant activity and this eﬀect is caused by in- teraction with serotonergic, noradrenergic, dopaminergic and GABAergic systems (Gürağaç Dereli et al., 2018; Tolardo et al., 2010). The
C/H δC (ppm) δH (ppm)/J (Hz)
2 (CH) 78.5 5.07 (1H, d)
3 (CH2) 42.0 2.70 (1H, dd) 2.66 (1H, d)
4 (C]O) 196.4 –
5 (C–OH) 163.5 12.15 (1H)
6 (CH) 95.0 6.20 (1H, d, J = 2 Hz)
7 (C) 166.7 q
8 (CH) 95.8 6.40 (1H, d, J = 2 Hz)
9 (C) 157.8 q
10 (C) 101.8 q
1' (C) 128.9 q
2' (CH) 128.3 6.80 (1H, d, J = 8.2 Hz)
3' (CH) 115.2 7.31 (1H, d, J = 8.2 Hz)
4' (C–OH) 157.8 12.15 (1H)
5' (CH) 115.2 7.31 (1H, d, J = 8.2 Hz)
6' (CH) 128.3 6.80 (1H, d, J = 8.2 Hz)
1'' (CH) 99.9 5.45 (1H, d, J = 7.3 Hz)
2'' (CH–OH) 77.2
3'' (CH–OH) 76.5
4'' (CH–OH) 73.1 4.3–3
5'' (CH) 69.6
6'' (CH2–OH) 60.6 5.07 (2H)

antidepressant activity of the methanolic extract prepared from the ﬂowers of Anthemis wiedemanniana Fisch.&Mey. was reported to be due to the isolated sesquiterpene lactone compounds named tatridine A and tanacin (1-epi-tatridine B) (Gürağaç Dereli et al., 2018). Sesquiterpene

lactone type compounds podoandin and 13-hydroxy-8,9-dehy-
q: quaterner.

gene expressions at receptors of these neurotransmitters (Bjorkholm and Monteggia, 2016; Meng et al., 1996; Shen et al., 2004). According to the results of the study conducted by Küpeli Akkol et al. (2019), naringenin apigenin, myricetin and rosmarinic acid which were iso- lated from Micromeria myrtifolia Boiss.&Hohen demonstrated signiﬁcant activity in the forced swimming and tetrabenazine-induced ptosis models, however only rosmarinic acid showed statistically remarkable activity in the tail suspension test (Küpeli Akkol et al., 2019). Anti- depressant activity of kaempferol and quercetin derivatives isolated from ethanolic extract prepared from Opuntia ﬁcus indica (L.) Mill was evaluated in a study and the results of the study showed that these compounds reduced the immobility time of the experimental animals in the forced swimming and tail suspension tests (Park et al., 2010). It was found that quercetin, which suppresses the microglial neuroin- ﬂammatory response, showed antidepressant activity in olfactory bul- bectomy rat model (Rinwa and Kumar, 2013). The antidepressantdrosizucanolide have been reported to be responsible for the anti- depressant activity of ethanolic extract prepared from the leaves of Hedyosmum brasiliense Miq. (Tolardo et al., 2010). Bilobalite is a ses- quiterpene trilactone compound isolated from Ginkgo biloba L. exhibits neuroprotective eﬀect by regulating GABAergic neurotransmission (Ahlemeyer and Krieglstein, 2003; Defeudis, 2002; Kiewert et al., 2007).

This paper describes the isolation and structural determination of β- amyrin, costunolide and the mixture of β-sitosterol and stigmasterol from the n-hexane extract prepared from the branches and leaves of C. kurdica and also quercitrin, isoquercetin and naringenin-7-O- gluco- pyranoside from the methanolic extract prepared from the ﬂowers of the plant. This is the ﬁrst report on the presence of these isolated compounds from Centaurea kurdica, except costunolide (Aktumsek et al., 2011; Appendino and Ozen, 1993; Degirmenci et al., 2015) and the present study demonstrated that terpenic, ﬂavonoidal and sesqui- terpene lactone-derived compounds play important roles for in vivo antidepressant activity of the plant. It is planned to elucidate the me- chanism of action of these compounds isolated from the active fractions for a new antidepressant drug which may be developed from C. kurdica in the future.

4. Conclusions
In this study, the andtidepressant eﬀect of C. kurdica was evaluated. According to our results, the n-hexane extract prepared from branches with leaves displayed the best activity in forced swimming test and also the MeOH extract prepared from ﬂowers showed similar activity with the n-hexane extract. Furthermore, Frs. (43–78) and (140–183) ob-
tained from n-hexane extract had statistically signiﬁcant activity and Fr.
(9–23) obtained from the MeOH extract showed prominent activity in forced swimming test. In the tail suspension test, similar to the forced swimming test, the n-hexane extract obtained from branches with
leaves, the MeOH extract obtained from the ﬂowers, Frs. (43–78) and (140–183) obtained from n-hexane extract, Fr. (9–23) obtained from
the MeOH extract showed statistically signiﬁcant activity. Additionally, we found the similar results in the antagonism of tetrabenazine-induced ptosis, hypothermia and suppression of locomotor activity. According to the isolation studies, β-amyrin, the mixture of β-sitosterol and stig- masterol and costunolide were isolated from the n-hexane extract pre- pared from the branches with leaves and quercitrin, isoquercetin and naringenin-7-O- glucopyranoside were isolated from the MeOH extract prepared from the ﬂowers. As the limit of the study in the area of ethnopharmacology, single dose study has been performed to reduce the number of experimental animals in accordance with the ethical guidelines for the use of animals in research. In the future studies, it is planned to determine the activity of diﬀerent doses of isolated com- pounds thought to be responsible for the activity.

Authors’ contributions
FTGD: Isolation and Biological Activity Studies ([email protected]) MI: Isolation and Biological Activity Studies ([email protected]) EKA: Biological Activity Studies ([email protected])

Declaration of competing interest
None.

Acknowledgements
This study was supported by the Scientiﬁc Research Project of Gazi University, Grant No: 02/2018-04.

Appendix A. Supplementary data
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jep.2019.112373.

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