ANKARA ÜNİVERSİTESİ ECZACILIK FAKÜLTESİ …journal.pharmacy.ankara.edu.tr/wp-content/uploads/sites/...Ozgen et al. J. Fac. Pharm. Ankara, 42(1): 1-13, 2018 2 the plant did not

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ANKARA ÜNİVERSİTESİ

ECZACILIK FAKÜLTESİ DERGİSİ

JOURNAL OF FACULTY OF PHARMACY

OF

ANKARA UNIVERSITY

Cilt / Vol : 42

Sayı / No : 1

Yıl / Year : 2018

eISSN: 2564-6524

ANKARA ÜNİVERSİTESİ

ECZACILIK FAKÜLTESİ DERGİSİ

JOURNAL OF FACULTY OF PHARMACY

OF

ANKARA UNIVERSITY

Cilt / Vol : 42

Sayı / No : 1

Yıl / Year : 2018

ANKARA ÜNİVERSİTESİ ECZACILIK FAKÜLTESİ DERGİSİ

(Ankara Ecz. Fak. Derg.) eISSN: 2564-6524

Sahibi : Prof. Dr. Gülbin ÖZÇELİKAY

Editör : Prof. Dr. İlkay YILDIZ

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Ankara Üniversitesi Eczacılık Fakültesi Dergisi (Ankara Ecz. Fak. Derg.) Ankara Üniversitesi Eczacılık

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JOURNAL OF FACULTY OF PHARMACY OF ANKARA UNIVERSITY

(J. Fac. Pharm. Ankara) eISSN: 2564-6524

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Prof. Dr. Fügen AKTAN Ankara University, Ankara, TURKEY

Prof. Dr. Nurten ALTANLAR Ankara University, Ankara, TURKEY Prof. Dr. Nuray ARI Ankara University, Ankara, TURKEY

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Prof. Dr. Hakan GÖKER Ankara University, Ankara, TURKEY

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Prof. Dr. Zühre ŞENTÜRK Yuzuncu Yil University, Van, TURKEY

Prof. Dr. Istvan TOTH University of Queensland, AUSTRALIA

Prof. Dr. Fikriye URAS Marmara University, Istanbul, TURKEY Prof. Dr. Selen YEĞENOĞLU Hacettepe University, Ankara, TURKEY

Journal of Faculty of Pharmacy of Ankara University (J. Fac. Pharm. Ankara) is official scientific journal

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İÇİNDEKİLER / CONTENTS 42(1), 2018

Özgün Makaleler / Original Articles Sayfa / Page

Ufuk ÖZGEN, Sıla Özlem ŞENER, Merve BADEM, Hatice SEÇİNTİ, Seda Damla HATİPOĞLU, Ahmet Ceyhan GÖREN, Cavit KAZAZ, Erhan PALASKA - Evaluation of HPLC, phytochemical, anticholinesterase and antioxidant profiles of the aerial parts of Asperula taurina subsp. caucasica - Asperula taurina subsp. caucasica'nın toprak üstü kısımlarının YBSK, fitokimyasal, antikolinesteraz ve antioksidan profillerinin değerlendirilmesi

1

Lyudmila KUCHERENKO, Igor BELENICHEV, Ivan MAZUR, Olga KHROMYLOVA, Natalia PARNIUK - Influence of the fixed

combination of glycine with thiotriazoline on energy metabolism parameters in brain in conditions of experimental cerebral ischemia - Glisin ile tiyotriazolin sabit kombinasyonunun

deneysel serebral iskemi şartlarında beyin enerji metabolizması göstergelerine etkisi

14

Arezoo VIEW, Aras RAFIEE - Upregulation of MIR-17 and MIR-221 by

benomyl, carbaryl, malathion and diazinon pesticides in mice blood - Fare kanında MIR-17 ve MIR-221'in benomil, karbaril, malatiyon ve diazinon pestisitleri ile upregülasyonu

22

Anastasiia DONCHENKO, Svitlana VASYUK - Spectrophotometric

determination of metoprolol tartrate in pure and dosage forms - Saf ve dozaj formlarında metoprolol tartaratın spektrofotometrik

tayini

33

Roman SHCHERBYNA, Volodymyr PARCHENKO, Volodymyr MARTYNYSHYN, Vasyl HUNCHAK - Evaluation of acute and subacute toxicity of oil liniment based on 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazol-3-yl)methyl)morpholine - 4-((5-(Desiltiyo)-4-metil-4H-1,2,4-triazol-3-il)metil)morfolin esaslı yağ merhemi akut ve subakut toksisite parametreleri tayini

43

J. Fac. Pharm. Ankara / Ankara Ecz. Fak. Derg., 42(1): 1-13, 2018 Doi: 10.1501/Eczfak_0000000597

ORIGINAL ARTICLE / ÖZGÜN MAKALE

EVALUATION OF HPLC, PHYTOCHEMICAL,

ANTICHOLINESTERASE AND ANTIOXIDANT PROFILES OF THE

AERIAL PARTS OF ASPERULA TAURINA SUBSP. CAUCASICA

ASPERULA TAURINA SUBSP. CAUCASICA'NIN TOPRAK ÜSTÜ

KISIMLARININ YBSK, FİTOKİMYASAL, ANTİKOLİNESTERAZ VE

ANTİOKSİDAN PROFİLLERİNİN DEĞERLENDİRİLMESİ

Ufuk ÖZGEN1*, Sıla Özlem ŞENER1, Merve BADEM1, Hatice SEÇİNTİ2, Seda Damla

HATİPOĞLU3, Ahmet Ceyhan GÖREN3, Cavit KAZAZ2, Erhan PALASKA4

1Department of Pharmacognosy, Faculty of Pharmacy, Karadeniz Technical University,

61080 Trabzon, Turkey 2Department of Chemistry, Faculty of Science, Atatürk University, 25240 Erzurum, Turkey

3TUBITAK UME, National Metrology Institute, Chemistry Group Laboratories, 41470

Gebze-Kocaeli, Turkey 4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100

Ankara, Turkey

ABSTRACT

Objective: In this study, we aimed to evaluate the HPLC, phytochemical, anticholinesterase

and antioxidant profiles of the aerial parts of Asperula taurina subsp. caucasica.

Material and Method: The fingerprint of the phenolic compounds of the methanolic extract of

the plant was obtained using RP-HPLC method. The method was also validated in terms of detection limits, quantification limits, linearity, accuracy, precision and selectivity. The phenolic contents of A.

taurina subsp. caucasica were detected as proto-catechuic acid, p-OH benzoic acid and benzoic acid.

In the phytochemical studies, quercetin 3-O-β-galactoside was isolated from the ethyl acetate subfraction of A. taurina subsp. caucasica using by several chromatographic methods. The structure

of the pure compound was elucidated by means of spectral analysis (1H NMR, 13C NMR, and ESI-MS). Anticholinesterase and antioxidant activity studies were performed on quercetin 3-O-β-

galactoside and the methanolic extract of the plant.

Result and Discussion: While quercetin 3-O-β-galactoside shown moderate inhibitory activity against butyrylcholinesterase at 200 μg/ml, quercetin3-O-β-galactoside and the metanolic exract of

* Corresponding Author / Sorumlu Yazar: Ufuk ÖZGEN


Submitted/Gönderilme: 18.07.2017 Accepted/Kabul: 06.11.2017

Ozgen et al. J. Fac. Pharm. Ankara, 42(1): 1-13, 2018

2

the plant did not show acetylcholinesterase inhibitory activity. Quercetin 3-O-β-galactoside shown DPPH free radical scavenging activity at 50 and 100 µg/ml, moderate lipid peroxidation inhibitory

activity at 25, 50 and 100 µg/ml; the methanolic extract of the plant moderate lipid peroxidation

inhibitory activity at 25, 50 and 100 µg/ml. In conclusion, A. taurina subsp. caucasica and quercetin 3-O-β-galactoside could be important and valuable sources for protecting our body health, especially

Alzheimer’s disease.

Keywords: anticholinesterase activity; antioxidant activity; Asperula taurina subsp.

caucasica; fingerprint; HPLC; quercetin 3-O-β-galactoside; Rubiaceae

ÖZ

Amaç: Bu çalışmada, Asperula taurina subsp. caucasica'nın toprak üstü kısımlarının YBSK, fitokimyasal, antikolinesteraz ve antioksidan profillerinin değerlendirilmesi amaçlanmıştır.

Gereç ve Yöntem: Bitkinin metanolik ekstresinin fenolik bileşiklerinin parmak izi

kromatogramı, geliştirilen RP-YBSK yöntemi kullanılarak elde edilmiştir. YBSK yöntemi saptama limitleri, nicelik sınırları, doğrusallık, doğruluk, hassaslık ve seçicilik açısından valide edilmiştir. A.

taurina subsp. caucasica fenolik içeriği, protokatekuik asit, p-OH benzoik asit ve benzoik asit olarak tespit edilmiştir. Fitokimyasal çalışmalarda ise, çeşitli kromatografik yöntemler kullanılarak A.

taurina subsp. caucasica etil asetat alt fraksiyonundan kersetin 3-O-β-galaktozit izole edilmiştir. Saf

bileşiğin yapısı, spektrum analizi (1H NMR, 13C NMR ve ESI-MS) yardımıyla aydınlatılmıştır. Kersetin 3-O-β-galaktozit ve bitkinin metanolik ekstresi üzerinde antikolinesteraz ve antioksidan

aktivite çalışmaları yapılmıştır.

Sonuç ve Tartışma: Kersetin 3-O-β-galaktozit, butirilkolinesteraz'a karşı 200 μg/ml'de orta

düzeyde inhibe edici aktivite gösterirken, kersetin 3-O-β-galaktozit ve bitkinin metanolik ekstresi asetilkolinesteraz inhibitör etki göstermemiştir. Kersetin 3-O-β-galaktozit, 50 ve 100 µg/ml'de DPPH

serbest radikal süpürücü aktivite, 25, 50 ve 100 µg/ml'de orta derecede lipit peroksidasyon inhibisyon

aktivitesi gösterirken; bitkinin metanolik ekstresi 25, 50 ve 100 µg/ml'de orta düzeyde lipit peroksidasyonu önleyici aktivite göstermiştir. Sonuç olarak, A. taurina subsp. caucasica ve kersetin

3-O-β-galaktozit, özellikle Alzheimer hastalığından korunmada önemli ve değerli bir doğal ürün

kaynağı olabilir.

Anahtar kelimeler: antikolinesteraz aktivite; antioksidan aktivite; Asperula taurina subsp.

caucasica; kersetin 3-O-β-galaktozit; parmak izi; Rubiaceae; YBSK

INTRODUCTION

The Rubiaceae family is represented by about 500 genera and 6000 species all over the world [1].

Species belong to Rubiaceae contain quinonic compounds [2-4], iridoids [5], coumarins [6], triterpenes

[7] and flavonoids [8]. The genus Asperula (Rubiaceae) has about 200 known species in the world [1]

and has 40 species (52 taxa) in Turkey, and 27 taxa of which are endemic [9]. Asperula taurina L. subsp.

caucasica (Pobed.) Ehrend grows in Northeast Turkey [10].

Some Asperula species have been used in folk medicine as a diuretic, tonic and antidiarrheal in

Turkey [11]. In our previous studies, we isolated β-sitosterol, mollugin, 1-hydroxy-2-methyl-9,10-

anthraquinone, 1,3-dihydroxy-2-methoxymethyl-9,10-anthraquinone, 1,3-dihydroxy-2-carboxy-9,10-

anthraquinone (munjistin), 2-carbomethoxy-3-prenyl-1,4-naphthohydroquinone 1,4-di-O-β-glucoside,

and lucidin 3-O-β-primeveroside from the underground parts of A. taurina subsp. caucasica [12]. Other

J. Fac. Pharm. Ankara, 42(1): 1-13, 2018 Ozgen et al. 3

phytochemical studies have shown that Asperula species also contains iridoid glycosides

(involucratosides A-C, adoxoside), flavone glycosides (apigenin 7-O-β-glucopyranoside, luteolin 7-O-

β-glucopyranoside, apigenin 7-O-rutinoside, lilacifloroside, quercetin, kaempferol, quercetin 3-O-β-

glucopyranosyl-(1→2)-β-galactopyranoside, quercetin 3-O-β-glucopyranosyl-(1→2)-

arabinopyranoside) and phenolic acid derivatives (chlorogenic acid and ferulic acid 4-O-β-

glucopyranoside) [13, 14]. Some previous studies have shown that some Asperula species have

antihypoxic and potent sedative, antioxidant activity [15, 16].

Polyphenols, which include phenolic acids and flavonoids, act as free radical scavengers and have

shown beneficial health-promoting effects in chronic and degenerative diseases such as Alzheimer [17].

Because of this reason, RP-HPLC method were generated and validated to detect phenolic contents.

A survey of the literature revealed that there have been no phytochemical, anticholinesterase

activity and antioxidant activity studies dealing with aerial parts of A. taurina subsp. caucasica. In the

present study, the phytochemical studies have comprised the isolation and structure elucidation of the

major compound, and RP-HPLC studies with regard to phenolic contents. Also, anticholinesterase

activity and antioxidant activity studies were performed on methanolic extracts of the aerial parts of A.

taurina subsp. caucasica, and quercetin 3-O-β-galactoside isolated from the plant.

MATERIAL AND METHOD

Plant Material, Extraction and Isolation Procedure

Plant material

The aerial parts of A. taurina L. subsp. caucasica (Pobed.) Ehrend. (Syn.: A. caucasica Pobed.)

were collected from Ormanüstü village; from forests and scrub, dry open places (Maçka district, 625 m,

August 2000, Trabzon province, Turkey). Voucher specimen of A. taurina subsp. caucasica was

deposited at the Herbarium of Ankara University Faculty of Pharmacy (AEF 19791). A. taurina subsp.

caucasica was identified by Dr. Ufuk Özgen.

Extraction and isolation studies on the aerial parts of A. taurina subsp. caucasica

The air-dried and powdered aerial parts (220 g) of A. taurina subsp. caucasica were extracted

with methanol (2000 ml x 3) under reflux for 3 h for each extraction at 40 °C. The combined methanol

extracts were evaporated to dryness (30 g, yield 10.4%) under reduced pressure at 40 °C. The methanol

extract was suspended with 200 ml of H2O:MeOH (9:1). It was partitioned against chloroform (200 ml

x 3) and ethyl acetate (EtOAc) (200 ml x 3), respectively. The chloroform and EtOAc subfractions were

evaporated at reduced pressure at 40 °C, and were 15.3 g and 1.2 g, respectively. The aqueous phase

was evaporated to give a residue (12.9 g).


4

The EtOAc extract (1.2 g) was subjected to Sephadex LH-20 column chromatography with

MeOH. Fractions 2-3 (225 mg) gave compound 1 (16 mg).

Chemicals and Instruments

Electric eel AChE, horse serum BChE, acetylthiocholine iodide, butyrylthiocholine chloride,

DTNB [5,50-dithio-bis(2-nitrobenzoic) acid], 100 mM sodium phosphate buffer (pH 8.0), galanthamine,

Sephadex LH-20 (Sigma-Aldrich) and silica gel 60 (0.063-0.2 mm Merck 7734, 0.040-0.063 mm Merck

9385 and LiChroprep RP-18 25-40 µm Merck 9303) for column chromatography; silica gel 60 F254

(Merck 5554) for TLC were used. TLC spots were detected with a UV lamp and spraying 1%

Vanillin/H2SO4. DPPH, BHA, BHT, α-tocopherol, β-carotene and linoleic acid were used for

antioxidant activity studies.

1H NMR and 13C NMR spectra were recorded with a Varian Mercury plus spectrometer at 400

(100) MHz. 96-well microplate reader (SpectraMax PC340, Molecular Devices, USA) was used for

antioxidant and anticholinesterase activity. Softmax PRO v5.2 software was used for anticholinesterase

activity studies.

HPLC analyse was practiced using a Shimadzu liquid chromatograph (Shimadzu Corporation,

LC 20 AT, Kyoto, Japan) and C18 column (Zorbax, 4,6 mm x 150 mm, 5 μm particle size) for 10

phenolic compounds (gallic acid, protocatechuic acid, protocatecualdehyde, p-hydroxy benzoic acid,

chlorogenic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid and benzoic acid) (Sigma-

Aldrich).

Anticholinesterase Activity Assay

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were

measured by slightly modifying the spectrophotometric method developed by Ellman et al. [18]. The

measurements and calculations were evaluated by using Softmax PRO v5.2 software. Percentage of

inhibition of AChE or BChE was determined by comparison of reaction rates of samples relative to

blank sample (ethanol in phosphate buffer, pH 8) using the formula (E-S) / E × 100, where E is the

activity of enzyme without test sample, and S is the activity of enzyme with test sample. The experiments

were carried out in triplicate. Galanthamine was used as reference compound.

DPPH free radical-scavenging assay

The free radical-scavenging activity of the methanol extract of A. taurina subsp. caucasica was

determined by the DPPH۬ assay described by Blois (1958) with slight modification [19, 20, 21]. BHA,

BHT and α-tocopherol were used as standard compounds.

The ability to scavenge the DPPH radical was calculated by using the following equation:

DPPH۬ Scavenging Effect (%) = Acontrol - Asample / Acontrol × 100


Determination of the antioxidant activity by the β-carotene bleaching method

The antioxidant activity of the samples was evaluated, using the β-carotene-linoleic acid test

system by Miller (1971) with slight modifications [16]. BHT and BHA were used as standard

compounds. The bleaching rate (R) of β-carotene was calculated according to the following equation:

R= ln a/b/t where, ln = natural log, a = absorbance at time zero, b = absorbance at time t (120 min).

The antioxidant activity (AA) was calculated in terms of percent inhibition relative to the control,

using the following equation:

AA = Rcontrol- Rsample/Rcontrol ×100

HPLC Analysis

Preparing of Standard Solutions

In this study, 10 phenolic compounds, gallic acid, protocatechuic acid, protocatecualdehyde, p-

hydroxy benzoic acid, chlorogenic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid and

benzoic acid were used as standards. Previously, a stock solution including each standard (100 ppm)

was prepared and filtered through 0.45 µm membranes. To make calibration curve, the stock solutions

of mixed standards were diluted in the concentrations range of 5-100 ppm.

Preparing of Sample Solutions

The aerial parts of the plant were extracted in methanol for 12 h at room temperature and the

solvent was removed under vacuum. The extract was redissolved in HPLC grade methanol (10 mg/ml)

and filtered through 0.45 µm membranes.

HPLC Conditions

Chromatographic analysis was performed using a Shimadzu liquid chromatograph. A C18

column (4,6 mm x 150 mm, 5 μm) was used with a gradient elution of 100% HPLC-grade methanol

(Solvent A) and 2% (v/v, adjust to pH 2,85) acetic acid in HPLC-grade water (Solvent B) as mobile

phase at a flow rate of 1.5 ml/min, injection volume 20 µl for the method. The method was studied with

diode array detector at wavelengths between 240 and 320 nm. The method was run with the following

gradient elution program: 0,01 min 20% A, 80% B; 4 min 30% A, 70% B; 7 min 40% A, 60% B; 10

min 45% A, 55% B; 12 min 50% A, 50% B; 16 min 60% A, 40% B; 17 min 80% A, 20% B. Mixed

standards diluted in the concentrations range of 5-100 ppm were performed five repetitive. The method

was run 17 minutes to identify the concentrations of 10 phenolic compounds in the plant.

Method Validation

The validation of the method was evaluated for detection limits, quantification limits, linearity,

accuracy, precision and selectivity. LOD and LOQ were calculated to assess the detection limits and


6

quantification limits of the method using signal-to-noise ratios. Linearity was determined by means of

calibration curves including five concentrations of standards and five repetitive data. Accuracy was

verified adding known amounts of the phenolic standards to a preparation of the plant extract. Precision

was evaluated by measurement of intra-day and inter-day precision. The selectivity of the method was

appraised by comparing the chromatograms of the phenolic standards.

RESULT AND DISCUSSION

Compound isolated from A. taurina subsp. caucasica

Yellow powder. 1H NMR (400 MHz, CD3OD) δ: 7.84 (1H, d, H-2′, J = 2.0 Hz), 7.58 (1H, dd, H-

6′, J = 8.4 Hz, J = 2.2 Hz), 6.86 (1H, d, H-5′, J = 8.4 Hz), 6.40 (1H, d, H-8, J = 1.8 Hz), 6.20 (1H, d, H-

6, J = 1.8 Hz), 5.20 (1H, d, H-1′′, J = 7.7 Hz), 3.85-3.30 (5H, sugar protons). 13C NMR (100 MHz,

CD3OD) δ: 178.3 (C-4), 164.9 (C-7), 161.8 (C-5), 157.6 (C-2), 157.3 (C-9), 148.8 (C-4′), 144.6 (C-3′),

134.6 (C-3), 121.8 (C-6′), 121.7 (C-1′), 116.6 (C-2′), 114.9 (C-5′), 104.4 (C-10), 104.2 (C-1′′), 98.7 (C-

6), 93.5 (C-8), 76.0 (C-5′′), 73.9 (C-3′′), 72.0 (C-2′′), 68.8 (C-4′′), 60.8 (C-6′′). 1H NMR and 13C NMR

data are in agreement with data given in the literature for quercetin 3-O-β-galactoside (Figure 1) [22].

O

O

O

OH

OH

OH

HO1'

2

2

2'3'

4'

5'

6'

345

6

7

89

10

O

HO

OH

HO

HO

1''2'' 3''

4''5''6''

Figure 1. Quercetin 3-O-β-galactoside

The results of antioxidant and anticholinesterase activity studies

The results of antioxidant and anticholinesterase activity studies of quercetin 3-O-β-galactoside

and the methanolic extract of the aerial parts of A. taurina subsp. caucasica have been shown in Table

1-4.

Table 1. The results of the DPPH free radical scavenging activity

Sample DPPH Free Radical Scavenging Activity Inhibition (%)

10 25 50 100

(µg/ml)

A1 28,51±1,32 52,88±2,62 77,12±1,69 80,51±0,48

Asp 4,71±1,81 19,37±1,37 25,81±0,97 39,79±1,97

α-TOC 32,92±0,26 77,35±0,20 80,38±0,46 81,18±0,87

BHT 38,80±1,01 58,68±1,31 76,78±1,08 81,10±0,43

BHA 57,05±0,48 77,01±0,30 80,79±0,83 81,20±0,54


A1 = Quercetin 3-O-β-galactoside, Asp = The methanol extract of A. taurina subsp. caucasica, α-TOC = α-Tocopherol,

BHT = Butylatedhydroxytoluene, BHA = Butylatedhydroxyanisole

Table 2. The results of the lipid peroxidation inhibitory activity

A1 = Quercetin 3-O-β-galactoside, Asp = The methanol extract of A. taurina subsp. caucasica, α-TOC = α-Tocopherol,

BHT = Butylatedhydroxytoluene, BHA = Butylatedhydroxyanisole

Quercetin 3-O-β-galactoside showed important DPPH free radical scavenging activity at 50 and

100 µg/ml; moderate lipid peroxidation inhibitory activity at 25, 50 and 100 µg/ml, and moderate

inhibitory activity against butyrylcholinesterase at 200 μg/ ml. The methanol extract of A. taurina subsp.

caucasica have shown moderate lipid peroxidation inhibitory activity at 25, 50 and 100 µg/ ml. None

of the samples has shown acetylcholinesterase inhibitory activity.

Table 3. The results of the anticholinesterase activity (AChE) assays

A1 = Quercetin 3-O-β-galactoside, Asp = The methanol extract of A. taurina subsp. caucasica,

Table 4. The results of the anticholinesterase activity (BChE) assays

A1 = Quercetin 3-O-β-galactoside, Asp = The methanol extract of A. taurina subsp. caucasica,

Sample Lipid Peroxidation Inhibitory Activity Inhibition (%)

10 25 50 100

(µg/ml)

A1 - 34,02±3,81 54,61±0,02 63,28±5,65

Asp 11,38±2,39 33,27±1,56 55,45±5,20 68,22±3,04

α-TOC 77,66±0,36 79,27±2,38 84,63±0,04 87,99±0,12

BHT 58,16±2,19 71,87±0,39 75,73±0,36 82,65±0,36

BHA 79,22±3,13 82,34±2,07 85,45±0,08 74,59±0,36

Samples AChE Inhibition (%)

25 50 100 200

(µg/ml)

A1 - - - -

Asp - - - -

Galanthamine 77,62±0,39 78,85±0,08 79,52±0,76 79,65±0,60

Samples BChE Inhibition (%)

25 50 100 200

(µg/ml)

A1 6,87±1,69 14,64±1,37 19,97±0,46 25,21±0,92

Asp - - -

Galanthamine 59,62±0,35 66,65±0,60 69,15±0,42 69,58±0,81


8

The results of HPLC Studies

Method Development

Solvent type, solvent ratio in the mobil phases, flow rate and detection wavelenght were changed

to specify the most useful and quickly separation. The appropiate HPLC conditions were found out

100% HPLC-grade methanol and 2% (v/v, adjust to pH 2,85) acetic acid in HPLC-grade water for

mobile phases, 1,5 ml/min for flow rate and 270 nm for detection wavelenght. The chromatogram of the

phenolic standards was obtained by using these HPLC conditions (Figure 2).

Figure 2. The HPLC chromatogram of the mixture of 10 phenolic standards (1* Gallic acid, 2*

Protocatechuic acid, 3* Protocatecualdehyde, 4* p-OH Benzoic acid, 5* Chlorogenic Acid, 6*

Vanillic Acid, 7* Caffeic Acid, 8* p-Coumaric Acid, 9* Ferulic Acid, 10* Benzoic Acid)

Validation of the Method

The validation of the method were evaluated in terms of detection limits, quantification limits,

linearity, accuracy, precision and selectivity pursuant to ICH guidelines [23].

Determination of limits of detection and quantification

The limits of detection and quantification were determined as signal-to-noise ratios by use of the

values of 3:1 and 10:1, respectively (Table 5).

Determination of Linearity

The mixture solution of the phenolic compounds in the range of 5-100 ppm were analyzed in five

repetitive and at least five concentrations. The peak areas were plotted against each concentration of the

mixture solutions to establish a linear regression equation and to identify value of correlation coefficient

(Table 5).


Table 5. Validation data from calibration curves of phenolic compounds

Determination of Accuracy

The accuracy of the method was verified by addition of standard solutions to sample solution at

three different levels 80, 100 and 120% by triplicate analysis. The recovery tests of all compounds were

detected range of 97-102%.

Determination of Precision

The intra-day and inter-day precision were identified for retention times. Peak areas were

determined for 10 phenolic standards (5 ppm) with repetitive analysis (n= 6). The precision data were

predicated as the relative standard deviation (R.S.D) (Table 6).

Table 6. Precision data of phenolic compounds

Compound No

Retention time Regression equation

Correlation

coefficient

(R)

LOD LOQ

Mean (min)

% RSD

Std ( mg/ml)

1 3,73 0,39 0,015 y = 29361x - 17284 0,9996 0,006 0,020

2 5,91 0,42 0,025 y = 19697x - 2766,8 0,9998 0,009 0,030

3 7,09 0,37 0,026 y = 70768x - 15508 0,9997 0,003 0,008

4 8,18 0,36 0,030 y = 17043x - 16244 0,9997 0,011 0,034

5 8,51 0,33 0,028 y = 17549x + 10845 0,9998 0,011 0,033

6 9,25 0,30 0,028 y = 37378x - 13137 0,9997 0,005 0,016

7 10,11 0,27 0,027 y = 64091x - 9006,5 0,9997 0,003 0,009

8 12,02 0,30 0,036 y = 91761x + 8933,8 0,9998 0,002 0,006

9 12,47 0,26 0,032 y = 46118x - 5106,4 0,9998 0,004 0,012

10 14,96 0,22 0,033 y = 5639x - 679,56 0,9998 0,034 0,105

Compound No

Intra-day R.S.D for RT

(%)

Intra-day R.S.D for Peak Area

(%)

Inter-day R.S.D for RT

(%)

Inter-day R.S.D for Peak

Area (%)

1 0,13 0,35 0,44 0,55

2 0,13 0,53 0,17 0,62

3 0,11 0,33 0,16 0,32

4 0,10 0,38 0,16 0,58

5 0,10 0,43 0,06 0,42

6 0,10 0,44 0,16 0,35

7 0,08 0,43 0,11 0,17

8 0,08 0,39 0,17 0,24

9 0,07 0,31 0,15 0,25

10 0,05 1,00 0,09 0,69


10

Determination of Selectivity

The method selectivity was appraise by the resolution study among standard peaks. Through the

HPLC conditions, all standard peaks were completely separated.

RP-HPLC Analysis of the Methanolic Extract of the Plant

The determination of phenolic compounds found in the plant was carried out using the same RP-

HPLC conditions. Sample peaks were detected by comparing retention time of known phenolic

standards. As a result, three phenolic compounds (proto-catechuic acid, p-OH benzoic acid and benzoic

acid) were identified (Figure 3, Tablo 7).

Table 7. The phenolic contents of A. taurina subsp. caucasica

Compounds

Retention Time

(Mean) Peak area

(Mean) Concentration

(mg/100 g)

2 Protocatechuic acid 5,91 301536 135,67

4 p-OH Benzoic acid 8,18 4779259 2568,84

10 Benzoic Acid 14,96 181560 623,41

Figure 3. The HPLC chromatogram of the plant extract

CONCLUSION

This study is the first one to evaluate the antioxidant and anticholinesterase activity of quercetin

3-O-β-galactoside, and the methanolic extract of the aerial parts of A. taurina subsp. taurina.

Anticholinesterase activity of quercetin 3-O-β-galactoside has been reported for the first time herein.


The substances with cholinesterase inhibitory activity have been used for treating of some diseases such

as mystenia gravis, Alzheimer’s disease. Galanthamine, well known as a medicine used for the treatment

of vascular dementia and Alzheimer’s disease, is used as positive control in anticholinesterase studies.

Quercetin 3-O-β-galactoside having similar effect with galanthamine may be suggested to reduce the

progression of Alzheimer’s disease (AD) and neuronal degeneration. Initial studies have indicated that

phenolic compounds may have preventive effects on the development of dementia or AD. According to

approach, we investigated the phenolic contents with RP-HPLC profiles. While the methanolic extract

of the plant include rich phenolic contents, the cholinesterase inhibitory activity of the extract was not

observed. Quercetin 3-O-β-galactoside isolated methanolic extract of the plant has shown moderate

butyrylcholinesterase inhibitory activity. As is seen, pure compounds may show more strong activity in

comparison with total extract. In conclusion, quercetin 3-O-β-galactoside is an important natural

compound for protecting our body and brain health.

ACKNOWLEDGEMENT

Sıla Özlem Şener and Merve Badem would like to acknowledge the scholarship during their

postgraduate program provided by the Turkish Scientific and Technical Research Council (TUBITAK).

Also, authors would like to thank Prof. Dr. Hasan Seçen for structure elucidation of the compounds.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

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INFLUENCE OF THE FIXED COMBINATION OF GLYCINE WITH

THIOTRIAZOLINE ON ENERGY METABOLISM PARAMETERS IN

BRAIN IN CONDITIONS OF EXPERIMENTAL CEREBRAL

ISCHEMIA

GLİSİN İLE TİYOTRİAZOLİN SABİT KOMBİNASYONUNUN DENEYSEL

SEREBRAL İSKEMİ ŞARTLARINDA BEYİN ENERJİ METABOLİZMASI

GÖSTERGELERİNE ETKİSİ

Lyudmila KUCHERENKO1,2, Igor BELENICHEV1, Ivan MAZUR1,2, Olga

KHROMYLOVA1*, Natalia PARNIUK1

1Zaporozhye State Medical University, Head of the Department of Pharmaceutical Chemistry,

69035, Zaporozhye, Ukraine 2SPA Farmatron

ABSTRACT

Objective: The perspective direction of primary neuroprotection in cerebral ischemia is the correction

of the imbalance of excitatory and inhibitory neurotransmitter systems by activating natural inhibitory

processes. There is evidence of the ability of anti-oxidant thiotriazoline to potentiate the therapeutic effect of

neuro-metabolic cerebroprotectors. Therefore, it is interesting to create a new combined drug based on glycine

and thiotriazoline. The purpose of this study is to investigate the effect of glycine, as well as its combination

with thiotriazoline, on the parameters of hydrocarbon-energy processes and oxidative metabolism under the

conditions of simulation of acute cerebrovascular disorder (ACVD).

Material and Method: To create ACVD, a classic model consisting of simultaneous ligation of common

carotid arteries was used in 50 Wistar male rats. All drugs were administered intraperitonally for four days

starting with anesthesia recovery of rat groups. The content of adenyl nucleotides, pyruvate, lactate, malate,

isocitrate and activities of succinate dehydrogenase, cytochrome C-oxidase, glutamate decarboxylase, GABA-

transferase were determined in the homogenates of brain cortex by biochemical methods.

Result and Discussion: Our results showed that the combination of glycine with thiotriazoline is better

than such reference drugs like pyracetam and glycine, according to degree of effect on the indicators of energy

metabolism of the brain, indicating the relevance of further study of the proposed combination.

* Corresponding Author / Sorumlu Yazar: Olga KHROMYLOVA



J. Fac. Pharm. Ankara, 42(1): 14-21, 2018 Kucherenko et al. 15

Keywords: glycine; neuroprotective action; stroke; thiotriazoline; transmitter amino acids

ÖZ

Amaç: Serebral iskemide primer nörokoruma uyarıcı ve inhibitor nörotransmiter sistemleri arasındaki

bozulan dengenin doğal inhibitor yolakların aktivasyonu ile düzeltilmesi yaklaşımına dayanır. Antioksidan bir

madde olan tiyotriazolinin nöro-metabolik serebral koruyucuların terapötik etkilerini arttırdığına ilişkin

kanıtlar bulunmaktadır. Bu nedenle glisin ve tiyotriazolini birlikte içeren yeni bir ilaç kombinasyonu oluşturmak

ilginç olabilir. Bu çalışmada glisinin, ve tiyotriazolin ile kombine kullanımının akut serebrovasküler bozukluk

(ASVB) oluşturulmuş rat modelinde hidrokarbon-enerji prosesleri ve oksidatif metabolizma parametreleri

üzerindeki etkilerinin araştırılması amaçlanmıştır.

Gereç ve Yöntem: ASVB, klasik yöntem olan ana karotis arterlerin simültane ligasyonu ile

gerçekleştirilmiş ve deneylerde 50 erkek Wistar rat kullanılmıştır. Uygulamalara gruplardaki ratlar anesteziden

çıktıktan hemen sonra intraperitonal olarak başlanmış ve 4 gün boyunca devam edilmiştir. Beyin kortekslerinde

adenil nükleotidler, piruvat, laktat, malat ve isositrat düzeyleri, suksinat dehidrogenaz, sitokrom C-oksidaz,

glutamat dekarboksilaz ve GABA-transferaz aktiviteleri biyokimyasal yöntemlerle saptanmıştır.

Sonuç ve Tartışma: Glisin ve tiyotriazolinin birlikte kullanımının, beyin enerji metabolizması

göstergeleri üzerine referans ilaçlar olan pirasetam ve glisinden daha etkili olması, bu kombinasyon üzerinde

araştırmaların devam etmesi gerekliliğini göstermektedir.

Anahtar kelimeler: amino asit transmitterler; glisin; inme; nörokoruyucu etkinlik; tiyotriazolin

INTRODUCTION

Cerebrovascular diseases are widespread throughout the world and are among the most

dangerous for the population. High indicators of mortality and disability of patients have caused great

interest in this pathology over the past decades. Brain strokes often cause death, complete or partial

disability, and significant decrease in the quality of life of patients [1]. From this perspective, it is

extremely important to prevent death of nerve cells, protect them from damage in ischemia, restoration

of impaired blood flow with pathological changes in blood circulation [2, 3].

The promising direction of primary neuroprotection in cerebral ischemia is the correction of

imbalance of excitatory and inhibitory neurotransmitter systems by activating natural inhibitory

processes [4]. In this regard, the natural inhibitory neurotransmitter glycine and its role in the

mechanisms of acute cerebral ischemia [5] are attracting attention. Traditionally, glycine was thought

to exhibit neurotransmitter properties at the spinal cord. GABA and glycine are equivalent

neurotransmitters that provide protective inhibition of the central nervous system. Glycine is also a

coagonist of glutamate NMDA receptors and is required for their normal functioning in

submicromolecular concentrations. There is evidence of the ability of antioxidant thiotriazoline to

potentiate the therapeutic effect of neurometabolic cerebroprotectors [6]. Therefore, it is interesting to

create a new combined drug based on glycine and thiotriazoline. This work is an integral part of the

joint integrated work of the Department of Pharmaceutical Chemistry of the Zaporizhzhya State Medical

University and TOV Scientific-Production Association “Farmatron” regarding the creation of new drugs

based on combinations of derivatives of 1,2,4-triazoles, which lasts more than 20 years [7, 8]. The

Kucherenko et al. J. Fac. Pharm. Ankara, 42(1): 14-21, 2018

16

purpose of this study is to investigate the effect of glycine, as well as its combination with thiotriazoline,

on the parameters of hydrocarbon-energy processes and oxidative metabolism under the conditions of

simulation of acute cerebrovascular disorder (ACVD).

MATERIAL AND METHOD

50 “Wistar” male rats weighing 180-200 g were used in experiments from the kennel of the

Institute of Pharmacology and Toxicology of the Academy of Medical Sciences of Ukraine. All

manipulations were carried out in accordance with the “European Convention for the Protection of

Vertebrate Animals Used for Experiments or for Other Scientific Purposes”. The protocols of

experimental studies and their results are approved by decision of the Commission on Bioethics of

Zaporozhye State Medical University (Record No. 33 as of October 26, 2016).

To create an ACVD, a classic model consisting of simultaneous ligation of common carotid

arteries was used. The operation was performed with ethaminal-sodium anesthesia (40 mg/kg). Through

the incision on neck, the right and left carotid arteries were found and segregated, placed ligatures under

them and ligated [9].

All animals were divided into 5 experimental groups (10 animals in each group): the first - intact

(falsely operated rats, which, after anesthesia, which common carotid arteries were segregated without

carrying out their ligation); the second one - rats with ACVD (control); third - rats with ACVD, which

were received glycine every day for 4 days at a dose of 200 mg/kg; the fourth - rats with ACVD which

were received glycine every day for 4 days in combination with thiotriazoline (4:1) at a dose of 200

mg/kg (in terms of glycine), the fifth - rats with ACVD, which were received pyracetam in dose of 500

mg/kg. All drugs were administered intraperitoneally every day, starting with anesthesia recovery of

rats.

On the fourth day of the experiment, the animals were withdrawn from the experiment under

the ethaminal-sodium anesthesia (40 mg/kg). Blood was quickly removed from brain, separated from

the meninges and the studied pieces were placed in liquid nitrogen. It was then ground in liquid nitrogen

to a powdered state and homogenized in a 10-fold volume of medium at (2°C) containing (in mmol):

sucrose-250, tris-HCl-buffer-20, EDTA-1 (pH 7,4) [10]. At a temperature (+4°C), a mitochondrial

fraction was isolated by differential centrifugation at a Sigma 3-30k (Germany) reefer centrifuge. To

purify the mitochondrial fraction from large cell fragments, centrifugation was carried out within 7

minutes at 1000g, and then the supernatant was re-centrifuged within 20 minutes at 17000g. The

supernatant was drained and stored at -80°C.

The content of pyruvate, lactate, malate, isocitrate, activities of succinate dehydrogenase,

cytochrome C-oxidase, glutamate decarboxylase, GABA-transferase were determined in the


homogenates of cortex by biochemical methods. The brain quickly removed, cerebral cortex was

isolated, which homogenized in liquid nitrogen. Protein-free extract was obtained by adding an accurate

weigth of brain tissue morselized in liquid nitrogen to chloric acid (0.6M) followed by 5.0M potassium

neutralization by carbonate [10]. Determination of the content of adenyl nucleotides, glycine, glutamate

and γ-aminobutyric acid was carried out by chromatographic methods [10]. The method is based on the

separation of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine

monophosphate (AMP) in the dioxane-isopropanol-water-ammonia system on a thin sorbent layer

followed by quantitation by direct spectrophotometry at 260 nm. Isopropanol, dioxane, ammonia

(Sigma, U.S.A.) and Merck sheets were used in experiments. 0.2 ml of protein-free tissue extract is

applied to the starting line of sheet and chromatographed in a dioxane-isopropanol-water-ammonia

(4:2:4:1) system. ATP, ADP, AMP are identified in the ultraviolet in a 365-nm UVS chromatographic

layer. The samples are eluted in 4.0 ml of 0.1 N HCl and measured in spectrophotometer at 260 nm

(Libra spectrophotometer, U.K.). The ATP, ADP and AMP (μmol/g of tissue) content is calculated from

the calibration curve, corrected to a tissue weight. The obtained results were processed using the MS

Excell computer program; probability of reliability was determined using the Student’s T-test.


ACVD modeling leads to persistent disorders of energy metabolism. Reduction of energy

resources of the brain occurred against the background of discortization of Krebs cycle reactions, as

evidenced by a decrease in the level of malate, isocitrate, inhibitory activity of succinate dehydrogenase

and cytochrome-c-oxidase (CHO). Compensatory activation of glycolysis was observed, as evidenced

by an increase in lactate in brain tissues. These changes occurred against the background of the detected

activation of the GABA system, which was expressed in the increase of glutamine decarboxylase and

GABA-T, and a decrease in the content of glutamate and GABA in the brain tissues of experimental

animals. In parallel, a decrease in the level of glycine was registered. In addition, there was an inhibition

of energy transport and utilization, as shown by the decrease in ATP activity and mitochondrial creatine

phosphokinase activity (m-CPK). Similar changes in the state of the GABA-ergic system in the creatine

phosphokinase activity occur as a compensatory activation of additional shunt of energy creation under

inhibition of Krebs cycle. Thus, the inhibition of the oxidation of -ketoglutarate results in the activation

of the gamma-aminobutyric acid and the conversion of glutamate to GABA, and then when the GABA-

T is activated to amber semialdehyde, which, being converted into succinate, is oxidized in the Krebs

cycle. However, the inhibition of the Krebs cycle on the site of isocyte succinate and the suppression of

succinate reductase show inhibition of the succinate oxidase pathway of supply of protons to the

respiratory chain and the inability to use succinate, which is additionally formed in the Roberts shunt. It

is likely that the GABA amber semialdehyde turns into -hydroxy-butyric acid, which has a stronger


18

inhibitory effect than GABA and glycine, the deficit of which we have discovered, and is able to limit

the harmful effects of harmful effect of excitatory aminoacids in cerebral ischemia. Thus, inhibition of

oxidative production of energy, its transport and utilization, activation of compensatory ways of

formation of ATP-glycolysis and Roberts shunt, which, however, do not fully satisfy the brain's need

for energy and cause development of lactic acidosis and deficiency of inhibitory amino acids (GABA

and glycine), is observed in the process of ACVD.

Glycine had a positive effect on the oxidative metabolism of the brain in ACVD, which was

shown in the increase of the level of ATP and ADP. Administration of glycine contributed to the

utilization of energy (increased ATP activity in the brain of animals receiving glycine). It reduced the

activity of anaerobic glycolysis and limited the development of lactic acidosis. Glycine increased the

oxide production of energy by means of normalization on the site of isocitrate-succinate in the Krebs

cycle.

The use of a fixed combination of glycine with thiotriazoline in animals with ACVD resulted in

significant activation of oxidative energy production in the dicarboxylic region of the Krebs cycle, as

evidenced by an increase in malate and an increase in the activity of succinate dehydrogenase. At the

same time there was an increase in the activity of cytochrome-C-oxidase and the level of isocitrate,

which ensured the increase of ATP production. In parallel, there was an increase in the level of ADP

and a decrease in the level of AMF (Table 1and Table 2).

Table 1. The content of adenine nucleotides in the cerebral cortex of rats on the 4th day of ischemia

Animal group

АТP

μm/g of

tissue

АDP

μm/g of tissue

АМP

μm/g of

tissue

m-CPK

μmol/mg

protein/min

АТPase

activity

Intact animals 2,85±0,05 0,47±0,01 0,13±0,02 1,876±0,021 21,47±0,78

Animals with

ACVD (control) 1,00±0,08 0,27±0,01 0,21±0,01 0,621±0,012 16,44±0,65

Animals with

ACVD + glycine 2,11±0,01* 0,33±0,01* 0,15±0,03* 0,724±0,022 19,22±0,23*

Animals with

ACVD + glycine +

thiotriazoline

2,79±0,01*1 0,44±0,02*1+ 0,13±0,01*1 2,132±0,011*1+ 25,07±0,12*1+

Animals with

ACVD+ piracetam 1,67±0,04 0,3±0,01 0,18±0,03 0,685±0,02 18,55±0,2

Нereinafter: * р <0,05 as related to control; *1 р <0,05 as related to piracetam group; *+ р <0,05 as related to glycine group.

Glycine in combination with thiotriazoline suppresses anaerobic activity of glycolysis (lowered

lactate levels), reduced the “flow” of inhibitory amino acids in the compensatory and energy-less

beneficial Roberts shunt (Table 2). Also, the level of glutamate, GABA and glycine was increased

against the background of decreasing the activity of glutamate carboxylase and GABA-T (GABA-

transferase). An increase in the level of inhibitory amino acids under the action of the combination is


likely to limit the action of the excitatory aminoacids of the brain and, thus, aggravating the total

neuroprotective effect of the drug. A fixed combination of glycine with thiotriazoline had a positive

effect on the oxidative energy production in the brain of rats with ACVD, and intensified transport and

energy utilization, as evidenced by the corresponding increase in m-CPK activity and ATP activity in a

series of animals which were administered glycine with thiotriazoline (Table 3).

Table 2. The content of carbohydrate-energy metabolism parameters in the cerebral cortex of rats on

the 4th day of ischemia


Table 3. Content of indicators of GABA-ergic system in the cerebral cortex of rats on the 4th day of

ischemia

Animal group GBA, μm/g

of tissue

Glycine,

μm/g of tissue

Glutamate, μm/g

of tissue

Glutamic acid

decarboxylase,

μm/ mg /h

GABA-Т

μm/ mg /h

Intact animals 3,87 ± 0,12 6,42 ± 0,21 14,72 ± 0,3 14,16±0,7 12,7±0,1

Animals with ACVD

(control) 1,12 ± 0,04 2,33 ± 0,22 5,02 ± 0,05 18,05±0,1 24,1±0,3

Animals with ACVD +

glycine 3,00 ± 0,07* 6,51 ± 0,34* 11,00 ± 0,10* 15,22±0,5* 16,1±0,4*

Animals with ACVD +

glycine + thiotriazoline 3,85 ±0,15* 7,78± 0,33*+1 14,21 ± 0,11*1+ 15,10±0,7* 15,2±0,7*1

Animals with ACVD+

piracetam 2,65±0,06 5,1±0,2 9,7 ± 0,11 16,8±0,35 20,5±0,55


Animal group

Pyruvate,

μm/g of

tissue

Lactic acid,

μm/g of

tissue

Malate,

μm/g of

tissue

Isocitrate,

μm/g of

tissue

Succinate

dehydrogenase,

μm/mg/min

Cytochrome-

c-oxidase,

μm/mg/min

Intact animals 0,46±0,01 2,32±0,06 0,31±0,02 0,52±0,07 6,44±0,10 3,44±0,11

Animals with

ACVD

(control)

0,22±0,01 8,52±0,11 0,11±0,05 0,20±0,03 2,88±0,17 1,00±0,07

Animals with

ACVD +

glycine

0,34±0,02* 5,22±0,21* 0,18±0,06* 0,33±0,01* 5,22±0,12* 2,77±0,10*+

Animals with

ACVD +

glycine +

thiotriazoline

0,44±0,01*1+ 3,85±0,12*1+ 0,47±0,03*1+ 0,57±0,03*1+ 7,89±0,33*1 3,95±0,22*1

Animals with

ACVD+

piracetam

0,3±0,02 5,8±0,15 0,16±0,05 0,28±0,03 4,85±0,15 2,2±0,15


20

The apparent neuroprotective effect of the combination of glycine and thiotriazoline, in our

opinion, is explained by the mutually intensifying effect of these drugs. Thus, thiotriazoline, which is

an effective scavenger of active forms of oxygen, limits the oxidative modification of protein structures

of receptors including NMDA Red/Oxi-dependent way; prevent the formation of energy deficiency,

oxidative stress [6]. Glycine, due to its connection with the glycine sites of the NMDA receptors, ensures

the normal functioning of the entire receptor- ionform complex, preventing its excessive activation and

thereby limiting glutamate excitotoxicity and possibly increasing the action of magnesium ions [4]. It

was found that the administration of a fixed combination of glycine with thiotriazoline to animals with

ACVD resulted in a significant activation of the oxidative energy production in the dicarboxylic region

of the Krebs cycle. It was found that the administration of the combination of glycine with thiotriazoline

inhibited the activity of anaerobic glycolysis, which leads to a decrease in lactic acidosis. The

combination had a positive effect on oxidative energy production in the brain of rats with ACVD, and

intensified transport and energy utilization. It was found that the administration of fixed combination of

glycine and thiotriazoline to animals with ACVD resulted in the normalization of GABA-shunt and

restored the concentration of inhibitory transmembrane amino acids, which increases the total

neuroprotective effect of the drug. The combination of glycine with thiotriazoline was better than such

reference drugs like piracetam and glycine by degree of influence on the parameters of energy

metabolism of the brain, indicating the prospect of further research of the proposed combination

indicating the relevance of further study of the proposed combination.

REFERENCES

1. Gusev, Е.I., Skvortsova, V.I. (2001). Brain ischemia, Medicine, Moscow, p.328.

2. Mazur, I.А., Chekman, I.S., Belenichev, I.F. (2007). Metabolitotropic drugs, Pechatnyi Dom,

Zaporozhye, p.309.

3. Galenko-Yaroshevskiy, P.А., Chekman, I.S., Gorchakova, N.А. (2001). Essays on the

pharmacology of metabolic therapy, Medicine, Moscow, p.240.

4. Khizhnyak, А.А., Kursov, S.V. (2003). Participation of excitatory amino acid transmitters in the

mechanisms of neurodestruction and perspective methods of pathogenetic correction. Pain

Anesthetization and Acute treatment, 1, 43 – 46.

5. Astakhov, А. (2004). Glycised-KMP: just amino acid or multipurpose anti stress medication?

Ukraine Drugs, 1, 35 – 36.

6. Gorchakova, N.А., Belenichev, I.F., Mazur, I.А. (2007). Mechanism of antioxidant and anti-

ischemic action of thiotriazoline. Medicine Pharmacy News, 2(206), 11-21.

7. Belenichev, І.F., Gorbachova, S.V., Golovkin, V.V., Bukhtiyarova, N.V. (2006). Influence of the

composition “Magnelong”, glycine, emoxipine and piracetam on the development of oxidative


stress in the brain of rats with acute cerebrovascular disorders (ischemic stroke). Medical

Chemistry, 3, 107-110.

8. Mazur, I.A., Chekman, I. S., Belenichev, I.F., Gorchakova, N.А. (2011). Development of drugs

based on fixed combinations with antioxidants - a promising area of modern pharmacology.

Pharmacology and Drug Toxicology, 5, 199–200.

9. Kozhemyakin, Y.М., Khromov, О.S., Filonenko, М.А., Sayftedinovna, G.А. (2002). Scientific

and methodical recommendations for the maintenance of laboratory animals and work with them.

Avitsena Kiev, p.156.

10. Voronina, Т.А., Seredinin, S.B. (2002). Manual on experimental (preclinical) study of new

pharmacological substances, Information and analytical portal of the Ministry of Health of the

Russian Federation ZAO “Remedium”, p.320.



UPREGULATION OF MIR-17 AND MIR-221 BY BENOMYL,

CARBARYL, MALATHION AND DIAZINON PESTICIDES IN MICE

BLOOD

FARE KANINDA MIR-17 VE MIR-221'İN BENOMİL, KARBARİL,

MALATİYON VE DİAZİNON PESTİSİTLERİ İLE UPREGÜLASYONU

Arezoo VIEW, Aras RAFIEE*

Department of Biology, Central Tehran Branch, Islamic Azad University, Tehran, IRAN

ABSTRACT

Objective: Increasing evidence demonstrate that the expression of miRNAs is affected by several known

toxicants and environmental contaminants. To evaluate the toxicity effect of the pesticides including benomyl,

carbaryl, malathion, diazinon on male Balb/c mice, expression profile of two oncogenic miRNAs were analysed

by real-time PCR.

Material and Method: The 72 male mice were divided into 6 groups (n = 6 per group), including control

(0 mg/kg), malathion (30 mg/kg), carbaryl (20 mg/kg), benomyl (30 mg/kg), diazinon (20 mg/kg) and mixture

of all pesticides. Mice were intragastrically gavaged for 60 days, then sacrificed on the 30(th) and 60(th) day.

The levels of oncogenic mir-17 and mir-221 in the serum were measured.

Result and Discussion: The results showed that compared with the normal controls, mir-17 and mir-

221 were overexpressed in all treatment groups during 2 months. The expression level of miR-17 and mir-221

after 60 days were 9.2-17.7 fold and 1.9-4 fold higher than the first month respectively. The lowest increase

was 1.9-fold, belongs to mir-221, which is still enough for easy diagnosis. These results provide new insights

into the negative pesticide’s carcinogenic probability via dysregulation of two oncogenic miRNAs. Our results

suggest that due to positive association between mir-17 and mir-221 levels and the risk of toxicity, these miRNAs

might be a useful biomarker in malignancy prediction and have a diagnostic value.

Keywords: Dysregulation; miRNA; oncogene; pesticides; toxicity

ÖZ

Amaç: Artan kanıtlar miRNA'ların ekspresyonunun bazı bilinen toksik maddeler ve çevresel kirleticiler

tarafından etkilendiğini göstermektedir. Pestisitlerin toksisite etkisini değerlendirmek üzere erkek Balb/c

farelerinde benomil, karbaril, malatiyon, diazinonun onkojenik miRNA ekspresyona etkisi gerçek zamanlı PCR

ile analiz edildi.

* Corresponding Author / Sorumlu Yazar: Aras RAFIEE



J. Fac. Pharm. Ankara, 42(1): 22-32, 2018 View and Rafiee 23

Gereç ve Yöntem: 72 erkek fare 6 gruba ayrıldı: kontrol (0 mg/kg), malatiyon (30 mg/kg), karbaril (20

mg/kg), benomil (30 mg/kg) ve diazinon (20 mg/kg). Fareler 60 gün boyunca intragastrik yoldan sonda ile

beslendi, daha sonra 30. ve 60. gününde öldürüldü. Serumda onkojenik mir-17 ve mir-221 düzeyleri ölçüldü.

Sonuç ve Tartışma: Sonuçlar normal kontrollerle karşılaştırıldığında, mir-17 ve mir-221 tüm tedavi

gruplarında 2 ay boyunca aşırı eksprese edildiği görüldü. Mir-17 ve mir-221 ekspresyon düzeyi ilk aya göre 60

gün sonra sırasıyla 9,2-17,7 kat ve 1,9-4 kat daha yüksekti. En düşük artış 1,9 kat ile mir-221'e aittir ki, hala

kolay teşhis için yeterlidir. Bu sonuçlar iki onkojenik miRNA’nın disregülasyonuyla pestisitlerin negatif

karsinojenik olasılığına yeni bilgiler sağlamaktadır. Sonuçlarımız mir-17 ve mir-221 seviyeleri ve toksisite riski

arasındaki pozitif ilişki nedeniyle, bu miRNA'ların malignite tahmininde yararlı bir biyobelirteç olabileceğini

ve diyagnostik değeri olduğunu göstermektedir.

Anahtar kelimeler: Disregülasyon; miRNA; onkojen; pestisitler; toksisite

INTRODUCTION

There are many different types of pesticides that are meant to control specific pests. The most

important types are classified as four groups. Fungicides used to control fungi, herbicides remove

unwanted weeds, trees or grasses [1], insecticides used to control insects and other arthropods and

rodenticides that kills rodents like mice, rats, and gophers [2-4]. Another classification of pesticides

includes organophosphate (OP), organochlorine (OC), and carbamate (CB) compounds. These families

have special tense because of water pollution, soil contamination and persistent in the environment [5].

Pesticide exposure can happen in many ways such as eating, drinking, touching or breathing anything

that bear pesticide residue [6].

Most pesticides are intrinsically toxic and cause potential hazard to human health. Cancer,

endocrine disruption, reproductive and sexual dysfunction [7] and dermatitis are among the health

effects [8]. Carbaryl is a carbamate insecticide which can inhibit acetylcholinesterase, and associate

with lower birth weight in rats and mice [9].

Early accurate diagnosis of diseases like cancer increases the chances for successful treatment.

The improvement of genomic technologies and the ability to evaluate the toxicant risks are valuable in

therapeutic targets. Several preclinical and clinical trials have been approached for miRNA-based

therapeutics [10]. Also microRNAs (miRNAs) are a class of endogenous noncoding RNAs with 18 to

25 nucleotides in length that play an important regulatory role in developmental and physiological

mechanisms in human body [11]. Dysregulation of miRNAs is correlated with toxicogenomics, disease

aetiology and the effect of toxicants. Circulating miRNAs are useful in diagnostics as biomarkers in the

evaluation of toxicant risks [12, 13].

MiR-222/221 cluster is a typical up-regulated miRNA in human cancer[14]. Another example of

miRNA overexpression with oncogenic effect is miR-17–92 cluster that is highly overexpressed in

different types of cancers, suggesting a mechanism of involvement in human tumorigenesis [15, 16].

http://npic.orst.edu/pest/weeds.html

View and Rafiee J. Fac. Pharm. Ankara, 42(1): 22-32, 2018

24

In this study we aimed at investigating the expression levels of two oncogenic miRNAs (mir-17

and mir-221) in blood samples of mice treated with four pesticides including benomyl, carbaryl,

diazinon and malathion.

MATERIAL AND METHOD

Chemicals and treatment of mice

All pesticides (benomyl, carbaryl, diazinon and malathion) were dissolved in corn oil and

administered intragastrically to female mice daily for 60 days. All of the mice had the average weight

of 35 gr. For this study 72 BALB/c mice were classified into 6 groups, including control which received

normal saline (9%), the second group of mice received 30 mg/kg malathion, the third group received 20

mg/kg carbaryl, the forth group received 30 mg/kg benomyl, the fifth group received 20 mg/kg diazinon

and the last group received mixture of all pesticides. (Formulated product by Iranian companies were

57% emulsifiable concentration for malathion, 85% wettable powder for carbaryl, 85% wettable powder

for benomyl and 60% emulsifiable concentration for diazinon).

At week 4 post gavage, 6 mice from each group (36 mice altogether) as described above, were

sacrificed and their sera were separated for RNA extraction and cDNA synthesis. cDNA kept at -20° C.

At week 8 post gavage, again 6 mice from each group (the rest of 36 mice) were sacrificed. Their blood

was separated for RNA extraction. The sera were collected by centrifugation at 5500 r/min for 10 min.

RNA extraction and cDNA synthesis

Total RNA was isolated from serum samples using RNXTM reagent (Cinnagen, Iran) following

the manufacturer’s instructions. Two steps including chloroform for removing proteins and isopropanol

for RNA precipitation were performed respectively. RNA purity was determined with a Nanodrop 1000

Spectrophotometer (Thermo Fisher Scientific, USA). The miRNAs assessed in the present study

included mir-17 (Ensembl:ENSMUSG00000065508; miRBase:MI0000687) and mir-221

(Ensembl:ENSMUSG00000065422; miRBase:MI0000709).

Briefly, the input RNA was polyadenylated using 10 µl of RNA in a final volume of 20 μl

including 2 μl of 10x poly(A) polymerase buffer, 0.2 μl of 5 U/ µl Poly A polymerase, 1µl of 10 mM

rATP and 6/8 µl DEPC water. The mixture was incubated at 37°C for 30 min follow by enzyme

inactivation at 65°C for 20 minutes.

cDNA synthesis was performed using BONmiR miRNA 1st-Strand cDNA synthesis kit following

the manufacturer’s protocol. To brief a report, 10 μl of polyadenylated RNA was reverse-transcribed

to cDNA using RT enzyme (BONmiR, Iran) and a BON-RT universal primer (BONmiR, Iran). The

http://www.ensembl.org/id/ENSMUSG00000065508

http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000687

http://www.ensembl.org/id/ENSMUSG00000065422

http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000709


following reaction conditions were used: 55 °C for 5 min, 25 °C for 15 min, 42 °C for 30 min and

95 °C for 5 min.

Quantitative real-time PCR for miR-17 and miR-221 expression in the BALB/c blood

SYBR green gene expression assay was carried out for mir-17 and mir-221 to evaluate their

different expressions in control and treated mice. Real-time PCR analysis was performed in the Bioneer

thermocycler with 20 μL volume reaction containing 1 μL cDNA, 0.5 μL miRNA-specific forward

primer (BonMir), 0.5 μl universal reverse primer, 6.5 μl 2× miRNA QPCR master mix and nuclease-

free, PCR-grade H2O up to 13 μl. The reactions were incubated in 96-well plates at 95°C for 20 secs,

following by 40 cycles (95°C for 5 secs, 60°C for 30 sec). miR-93 (reference gene) was measured by

the same method and used for normalization. The relative levels of each miRNA in mice blood,

normalized to miR-93 and relative to the expression in control, was calculated using RQ = 2−ΔΔCT

equation, in which ΔΔCT = (CT miRNA − CT mir-93) test − (CT miRNA − CT mir-93) control and CT is the threshold

cycle to detect fluorescence.

Statistical analysis

Data analysis was performed using SPSS software and Graphpad Prism (Prism 7.0 Graphpad

Software Inc., La Jolla, USA). Comparisons between groups were done using parametric unpaired t-test

to measure the statistical difference of expression levels between 5 groups that were not normally

distributed followed by one-way Anova analysis of variance. The data were expressed as the means ±

SEM. P-values <0.05 were considered statistically significant.


Epigenetic alterations such as DNA methylation reprogramming, altered histone modification,

maternal effects and X chromosome inactivation are the consequences of applying a number of toxicant

pesticides [17, 18]. For instance, rat-liver epithelial cell lines treated with arsenic showed decrease in S-

adenosyl-methionine (SAM) levels and DNA methyltrasferase activity. Toxic materials such as

pesticides can modify gene expression in organisms expose to them. One of the most commonly genetic

hallmark is miRNA. Dysregulation in miRNA expression can affect the protein expression in cells,

leading negative biological effects. Due to such power of miRNA in changing the expression of proteins,

measuring RNA profiling data could be a suitable tool to prognosticate the effective capacity of

pesticide’s toxicity [19].

For the purpose of keeping homeostasis, miRNAs play a key role as a mediator between cellular

response and extracellular signals [20].


26

For example, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is an environmental pollutant that

could increase tumor suppressing miRNAs and decreased oncogenic miRNAs in the liver and brain of

mouse [21]. Also exposure to metal-rich particulate matter (PM) and bisphenol A (BPA) unregulated

miR-222 and miR-638 [22, 23].

In the present study, we evaluated the effects exerted by four different pesticides and a mixture

of them on the expression of two oncogenic miRNAs in the blood of BALA/c mice. We chose four

pesticides (benomyl, malathion, diazinon and carbaryl) that have high consumption in agriculture and

were also detected as probably non-carcinogen compounds [24-26]. The precise dose of lowest observed

effect level (LOEL) of our used pesticides for BALA/c mice is not yet being determined. So based on

the overall results of articles (lower than LOEL) and LD50 similarity that was almost being among

benomyl-malathion together and diazinon- carbaryl together, we choose the gavage amount of 30 mg

for benomyl and malathion and 20mg for diazinon and carbaryl.

Different expression of mir-17 in mice treated with pesticides corresponding to healthy mice

The expression of oncogenic mir-17 was analyzed using quantitative real-time PCR. The results

showed that compared with the normal controls, mir-17 was overexpressed in all treatments during 2

months (Figure 1).

The level of mir-17 in mice was significantly upregulated by ≥ 25.13-fold (day 30th) and 233.33-

fold (day 40th) respectively after treatment with all pesticides in comparison with the control group. Also

mir-17 was upregulated by ≥ 7.21-fold and 122.72-fold respectively after 30 and 60 days’ treatment with

diazinon. At the time of using carbaryl, mir-17 was overexpressed by ≥ 10.94-fold and 152.2-fold after

30 and 60 days respectively. In return to malathion, mir-17 was increased about ≥ 4.48-fold (after 30

days) and ≥79.53-fold (after 60 days). In return to benomyl, mir-17 was increased about ≥ 1.12-fold

(after 30 days) and ≥17.06-fold (after 60 days). All the results were significant at P< 0.05 (Figure 1.a).

Next, we comparison the expression level of miR-17 on the 60(th)/30(th) day for each treatment group.

The results have shown in figure 3.1.B indicated that mir-17 was extremely higher (at least 9.2-fold to

the most 17.7-fold) after 60 days.

Different expression of mir-221 in treated mice corresponding to healthy mice

The change in the expression of oncogenic mir-221 was confirmed by real-time PCR. The results

showed that mir-221 was overexpressed in all treatments compared to healthy mice during 2 months

(Figure 2). Compared to the non-treatment group, oncogenic mir-221 expression in mix pesticide’s

treatment group was upregulated by ≥ 476.88-fold and 4715-fold after 30 and 60 days respectively. Also

compared to the non-treatment group, the diazinon- treated group showed increased


Figure 1. The expression level of mir-17.

a: The expression level of mir-17was remained significantly higher in comparison with the control group versus control

(*P < 0.05). b: The expression level of mir-17 was extremely higher after 60 days in comparison with day 30th. The folds are

written on the arrows connected the months of every pesticide together.


28

mir-221 expression by ≥ 150.48-fold and 2056-fold after 30 and 60 days respectively. The mir-221

expression was also found increased in carbaryl-teatment group about ≥ 215.54-fold (after 30 days) and

≥3577-fold (after 60 days). At the time of using malathion, mir-221 was overexpressed by ≥ 114.32-fold

and 1610-fold after 30 and 60 days respectively. In return to benomyl, mir-221 was increased about ≥

184.68-fold (after 30 days) and ≥1877-fold (after 60 days). All the results were significant at P< 0.05.

Next, we determined the expression level of miR-221 on the 60(th)/30(th) day for each treatment group.

The results have shown in figure 3.2.B indicated that mir-221 was at least 1.92-fold higher after 60 days

in comparison with 30 days.

The current study shows up-regulation of mi-17 and mir-221 levels in treatment mice compared

to non-treatment controls. As mir-17 and mir-221 are both considered as an oncogene [27, 28] increased

expression of these two miRNAs represents their properties of dysregulation in facing with pesticide’s

toxicity. Although both miRNAs had significant dysregulation but most applied changes were made on

mir-17. The lowest increase was 1.9-fold, belongs to mir-221, which is still enough for easy diagnosis.

It is true that the rise of mir-17 and mir-221 reflects the toxicity of these materials, but may also be

suspected of being carcinogen compounds.

Among four pesticides, benomyl and carbaryl was attracted our attention. Although their LD50

was lower than diazinon, but mir-17 and mir-221 of the serum had the greatest change in expression

against them. This can be due to benomyl and carbaryl ability on changing the expression of oncogenic

miRNAs. This property can also increase the pesticide’s carcinogenic probability. It may also be

expected that potential targets of mir-17 and mir-221 could contain genes encoding oncogenic proteins

that increase their carcinogenic impact. Taken together, these findings indicate that there is a positive

association between mir-17 and mir-221 levels and the risk of malignancy, contributing towards

improved predictive human toxicity. This result suggests that both miRNAs might be a useful biomarker

and have a diagnostic value. However, the detailed mechanism will require further investigations.


Figure 2. The expression level of mir-221.

a: Compared to the non-treatment group, the expression level of mir-221 was remained significantly higher after 2 months

versus control (*P < 0.05). b: The expression of mir-221 varied significantly among the treatment groups (*P < 0.05). mir-221

levels were significantly increased in second month. The folds are written on the arrows connected the months of every pesticide

together


30

ACKNOWLEDGEMENT

This study was a part of a master thesis. We thank F.Riazi-rad, R.Jazayeri and S.Sarabi for their

assistance. We are also thankful to Mr H.Khodayari for his technical assistance.

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MicroRNAs as early toxicity signatures of doxorubicin in human-induced pluripotent stem cell-

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27-33.



SPECTROPHOTOMETRIC DETERMINATION OF METOPROLOL

TARTRATE IN PURE AND DOSAGE FORMS

SAF VE DOZAJ FORMLARINDA METOPROLOL TARTARATIN

SPEKTROFOTOMETRİK TAYİNİ

Anastasiia DONCHENKO*, Svitlana VASYUK

Zaporizhzhia State Medical University, Pharmaceutical Faculty, Analytical Chemistry Department,

Zaporizhzhia, Ukraine

ABSTRACT

Objective: A new spectrophotometric method has been developed for the determination of metoprolol

tartrate in pure and dosage forms.

Material and Method: This method is based on the reaction between metoprolol tartrate and 2,3-

dichloro-1,4-naphthoquinone in dimethylformamide (DMF) medium to form the colored reaction product with

maximum absorption at 493 nm. Optimum conditions to carry out the reaction such as concentration of reagent,

temperature and heating time were carefully studied and optimized. Beer’s law was performed at the

concentration range of 18.00-28.00 mg/100 ml. The proposed method is valid according to the validation

requirements of the State Pharmacopoeia of Ukraine.

Result and Discussion: The results of the study show that the procedure is accurate, simple and relevant

for application at the quality control laboratories for dosage forms.

Keywords: 2,3-dichloro-1,4-naphthoquinone; metoprolol tartrate; spectrophotometry; validation

studies

ÖZ

Amaç: Metoprolol tartaratın belirlenmesi için saf ve farmasötik ilaçlarında yeni spektrofotometrik bir

yöntem geliştirildi.

Gereç ve Yöntem: Bu yöntem 493 nm’de maksimum emilim ile boyalı reaksiyon ürünün oluşturulması

için dimetilformamid (DMF) ortamında metoprolol tartarat ile 2,3-dikloro-1,4-naftokinon arasında olan

reaksiyona dayanmaktadır. Reaksiyonun yapılması için reaktif konsantrasyonu, sıcaklık ve ısıtma süresi gibi

* Corresponding Author / Sorumlu Yazar: Anastasiia DONCHENKO



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Donchenko and Vasyuk J. Fac. Pharm. Ankara, 42(1): 33-42, 2018

34

optimal koşullar dikkatli bir şekilde incelendi ve optimize edildi. Beer kanunu 18.00-28.00 mg/100 ml

konsantrasyon aralığında gerçekleştirildi. Önerilen yöntem Ukrayna Devlet Farmakopesi onaylama taleplerine

göre geçerlidir.

Sonuç ve Tartışma: Çalışma sonuçları, işlemin ilaç formları için kalite kontrol laboratuvarlarında

uygulama açısından doğru, basit ve güncel olduğunu göstermektedir.

Anahtar kelimeler: 2,3-dikloro-1,4-naftokinon; metaprolol tartarat; spektrofotometri; validasyon

çalışmaları

INTRODUCTION

One of the most common noninfectious diseases in many countries of the world is cardiovascular

diseases. During the last years mortality from circulatory system diseases have significantly declined,

but they remain the main cause of sudden death in Ukraine [1]. Specialists carry out the development of

new drugs for improving the quality of treatment. Also significant attention is paid to the improvement

of existing treatment regimens with drugs that have proven effective not only in large randomized trials

but also in the daily practice of doctors. β-adrenergic blockers can be attributed to them. These drugs

are prescribed for treatment of arterial hypertension, ischemic heart disease, chronic heart failure and

various arrhythmias (supraventricular tachycardia, atrial fibrillation, ventricular extrasystole and

others). In this case, an advantage is given to such blockers of β-adrenergic receptors, which have a long

half-life, the favorable balance of lipophilic and hydrophilic properties and high cardioses selectivity.

One of such drugs is metoprolol tartrate [2].

Metoprolol tartrate is a selective blocker of β1-adrenergic receptors which used to treat high blood

pressure (hypertension) and congestive heart failure [3]. Chemically it is bis[(2RS)-1-[4-(2-

methoxyethyl)phenoxy]-3-[(1-methylethyl)-amino]propan-2-ol] (2R,3R)-2,3-dihydroxybutanedioate

with the molecular formula C34H56N2O12 (Figure 1). European and British Pharmacopoeia recommend

titrimetric method with potentiometric fixation end-point for the assay of metoprolol tartrate [4, 5].

According to the literature data, spectrophotometry [6, 7], HPLC [8], spectrofluorimetry [9] are the most

widely used techniques for the determination of metoprolol tartrate. These methods are highly sensitive,

selective, cost-effective and available to quality control laboratories for dosage forms. However, there

is a need to find new analytical reagents. In this issue, derivatives of quinone are promising, namely 2,3-

dichloro-1,4-naphthoquinone.

J. Fac. Pharm. Ankara, 42(1): 33-42, 2018 Donchenko and Vasyuk 35

Figure 1. The chemical structure of metoprolol tartrate

Therefore, the purpose of this work was to develop and validate spectrophotometric method based

on reaction with 2,3-dichloro-1,4-napthoquinone for the determination of metoprolol tartrate in pure and

dosage forms.

MATERIAL AND METHOD

All chemicals and reagents used were of analytical or pharmaceutical grade.

The research objects: tablets “Metoprolol tartrate” 50 mg (“Farmak” PJSC, Ukraine, series No.

20617), tablets “Metoprolol” 50 mg (“Kievmedpreparat” PJSC, Ukraine, series No. 176802).

Materials and reagents: pure metoprolol tartrate substance (Sun Pharmaceutical, series AH-9-

46930), 2,3-dichloro-1,4-napthoquinone (Sigma–Aldrich Corporation, USA, D67200),

dimethylformamide (DMF) (BASF, China, series 20150611).

Apparatus: Analytic Jena UV-visible spectrophotometer model Specord 200 with 1 cm matched

quartz cells, Kern electronic scales ABT-120-5DM, water bath (Memmert WNB 7-45).

Reagents and solutions

2,3-dichloro-1,4-naphthoquinone 4%: It was prepared by dissolving 4 g of 2,3-dichloro-1,4-

naphthoquinone in 100 ml of DMF.

Working standard solution 0.23%: It was prepared by dissolving 0.2300 g of pure metoprolol

tartrate in 100 ml of DMF.

Procedure for calibration graph

The aliquots of the working standard solution containing 18.00-28.00 mg of metoprolol tartrate

were transferred into a series of test tubes. 1 ml of 4% 2,3-dichloro-1,4-naphthoquinone was added. The

resulting reaction mixture was heated on the water bath at 95°C for 10 min. After cooling, the contents

of the tubes were quantitatively transferred into a series of 10 ml calibrated flasks and diluted to volume

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36

with DMF. The absorption was measured on the background of a compensating solution which did not

contain the test substance at a wavelength of 493 nm.

Procedure for dosage forms

Twenty tablets were weighed and powdered. An accurately weighed quantity of the powdered

tablets (for tablets “Metoprolol” – 0.3230 g, for tablets “Metoprolol tartrate” – 0.3450 g) was transferred

into a 25 ml calibrated flask and diluted to volume with DMF. Obtained solutions were mixed and

filtered. First portions of filtrate were discarded, since the first portions of the filtrate were cloudy. The

aliquots of the obtained solution were analyzed according to the procedure for calibration graph.


In the present work, we investigated the development of spectrophotometric determination of

metoprolol tartrate in pure and dosage forms. It was experimentally established that pure metoprolol

tartrate reacts with 2,3-dichloro-1,4-naphthoquinone in DMF medium to formation the reaction product

with maximum absorption at 493 nm. The absorption spectrum of product is recorded in Figure 2.

Figure 2. Absorption spectrum of metoprolol tartrate (0.23 %) against DMF (- - -); 2,3-dichloro-1,4-

naphthoquinone (4%) against DMF (····); reaction product of metoprolol tartrate with 2,3-dichloro-1,4-

naphthoquinone against reagent blank (──)

Optimum conditions to carry out the reaction between metoprolol tartrate and 2,3-dichloro-1,4-

naphthoquinone has been established during the process of development this procedure. The influence

of various parameters such as nature of the solvent, concentration of reagent, temperature, time of

heating was investigated.

The choice of the solvent for this reaction was based on the metoprolol tartrate and 2,3-dichloro-

1,4-naphthoquinone solubility data. In the result, DMF became the optimal solvent for this reaction.


Necessary quantity of reagent was determined experimentally by reaction product maximum

yield, i.e. by maximum value of absorbance. To this end, the reaction of the substance of metoprolol

tartrate with the reagent at concentration of 1-5% was investigated. The absorption maximum was

attained at 2,3-dichloro-1,4-naphthoquinone concentration of 4% (Figure 3).

Figure 3. Effect of reagent concentration on the reaction of metoprolol tartrate with 2,3-dichloro-1,4-

naphthoquinone. Metoprolol tartrate (0.1%): 1 ml, reagent: 1 ml, temperature: 95°С, reaction time:10 min.

The effect of temperature and heating time on formation of the reaction product was studied too

(Figure 4, 5). The highest absorption was obtained after heating at 95°С for 10 min.

Figure 4. Effect of temperature on the reaction of metoprolol tartrate with 2,3-dichloro-1,4-naphthoquinone.

Metoprolol tartrate (0.1%): 1 ml, reagent (4%): 1 ml, reaction time:10 min.


38

Figure 5. Effect of heating time on the reaction of metoprolol tartrate with 2,3-dichloro-1,4-

naphthoquinone. Metoprolol tartrate (0.1%): 1 ml, reagent (4%): 1 ml, temperature: 95°С.

Validation of the proposed method

All of the validation characteristics for the proposed method were determined according to

requirements of the State Pharmacopoeia of Ukraine. The following parameters were considered:

specificity, linearity, accuracy, precision and robustness.

Specificity

During the study of the specificity of the method, the contribution of auxiliary substances included

in the dosage forms in the total absorption of the solution was determined. For this purpose, the

absorption of placebo solutions and working standard solution of metoprolol tartrate was measured. It

has been found that this contribution is insignificant for the investigated dosage forms. The resulting

spectrum is shown in Figure 6.

Figure 6. Absorption spectrum of placebo of tablets “Metoprolol tartrate” (- - -); placebo of tablets

“Metoprolol” (····); reaction product of metoprolol tartrate (0.23%) with 2,3-dichloro-1,4-naphthoquinone (──)


Linearity

To determine the linearity, 9 measurements of the absorption of the working standard solution of

metoprolol tartrate were performed in the range of concentrations in which the obedience of a Beer’s

law was observed, namely 18.00-28.00 mg/100 ml. The calibration graph of the absorption from

metoprolol tartrate concentration was plotted according to the obtained data. It is given in Figure 7.

Figure 7. Calibration curve of metoprolol tartrate at 493 nm

The linearity of proposed method was evaluated by the linear regression analysis, which was

calculated by the least square method. Received values are given in the Table 1.

Table 1. Optical specifications and basic parameters of linear dependency

Molar absorption coefficient, ε 1278.4

Sendel’s coefficient, WS 0.5356

Identification limit, Сmin (g/ml) 26.78

Equation of linear regression Y = bX + а

Slope, b±(Sb) 0.0178±(0.0003)

Intercept term, а±(Sa) 0.0115±(0.0080)

Residual standard deviation, Sx,o 0.1741

Сorrelation coefficient, r 0.9987

Precision

The precision of the proposed method for each dosage form was determined at the level of

repeatability. For this purpose, 9 parallel measurements were performed. Of the three weights, three

solutions were prepared, each with three parallel measurements under optimum conditions. In parallel,


40

the absorption of the blank solution was determined and the content of the test substance was calculated.

The obtained data are given in the Table 2.

Table 2. Precision determination results for metoprolol tartrate dosage forms

Dosage form Content

Metrological characteristics

Х S RSD rx,Δ %AsΔ

Tablets “Metoprolol tartrate” 0.050 g 0.0499 3.8·10-4 0.768 1.42 3.20

Tablets “Metoprolol” 0.050 g 0.0501 3.4·10-4 0.693 1.28 3.20

Х - mean, g; S – standard deviation; RSD – relative standard deviation; x,rΔ – relative confidence interval;

%AsΔ – critical value for repeatability of results

Accuracy

Accuracy was established by standard addition method. In the experiment, to three equal samples

of the appropriate dosage form were added a known amount of working standard solution of metoprolol

tartrate (n = 9). Then the absorption of the solutions obtained was measured. The accuracy of the method

was evaluated as the ratio added/found. The results of the determinations are correct, if there is no

meaningful systematic mistake, i.e. the true value of the determined amount is getting in a setting

confidence interval. The obtained data are given in Table 3.

Table 3. Accuracy determination results for metoprolol tartrate dosage forms

Dosage form Taken

mg/100 ml

Additive

mg/100ml ∆Z RSD ∆ Z | 100−Z |

Tablets “Metoprolol

tartrate”

18.64 4.60

100.40 0.497 2.77 0.40 18.64 6.90

18.64 9.20

Tablets “Metoprolol”

18.47 4.60

100.03 0.345 1.92 0.03 18.47 6.90

18.47 9.20

∆Z – mean, %; RSD – relative standard deviation; ∆ Z – sided confidence interval; | 100−Z | – systematic error

Robustness

During the test of robustness, the influence of time on the stability of the tested solutions was

investigated. For this purpose, the absorption of the analyzed solution of the appropriate dosage form

(A1 – for tablets “Metoprolol tartrate”, A2 – for tablets “Metoprolol”) and working standard solution of

metoprolol tartrate (A0) was measured every 5 minutes for 30 minutes (Table 4).

Table 4. Robustness determination results


t, min 0 5 10 15 20 25 30 Mean RSD,% t% maxδ,%

A0 0.4290 0.4295 0.4298 0.4300 0.4307 0.4314 0.4310 0.4302 0.199 0.38

0.51 A1 0.4277 0.4281 0.4286 0.4290 0.4299 0.4300 0.4298 0.4290 0.214 0.41

A2 0.4301 0.4306 0.4310 0.4312 0.4321 0.4328 0.4331 0.4316 0.261 0.51

RSD,% – relative standard deviation; t% – confidence interval; maxδ,% – critical value of the systematic error

According to the table, t%≤ maxδ,%, i.e., the test solutions are stable for at least 30 minutes.

CONCLUSION

Simple and reproducible spectrophotometric method has been developed for quantitative

determination of metoprolol tartrate in pure and dosage forms. The proposed method is valid according

to the validation requirements of the State Pharmacopoeia of Ukraine. The results of the study show that

the method is precise, simple and relevant for application at the quality control laboratories for dosage

forms.

REFERENCES

1. Kovalenko, V., & Dorohoy, A. (2016). Sertsevo-sudynni khvoroby: medychno-sotsialʹne

znachennya ta stratehiya rozvytku kardiolohiyi v Ukrayini [Cardiovascular disease: medical and

social significance and the strategy of cardiology development in Ukraine]. Ukrayins'kyy

kardiolohichnyy zhurnal, 4(3), 5-14.

2. Buchhorn, R., & Borst, M. (2017). The History and Future of Beta Blockers in Heart Failure

Treatment in Children and Adults. Medical Research Archives, 5(9), 1-11.

3. Mashkovskiy, M. (2012). Lekarstvennye sredstva [Medicine remedies]. Moscow: Novaya Volna.

4. European pharmacopoeia (2011). (7th ed.). Strasbourg: Council of Europe.

5. British Pharmacopoeia (2009). London: The Stationery Office.

6. Nabil A. F., A., & Eman. M., S. (2015). Spectrophotometric determination of metoprolol in

pharmaceutical formulation by charge transfer complexation. International Journal Of Chemical

Studies, 3(2), 24-29.

7. Cesme, M., Tarinc, D., & Golcu, A. (2011). Spectrophotometric Determination of Metoprolol

Tartrate in Pharmaceutical Dosage Forms on Complex Formation with Cu(II). Pharmaceuticals,

4(12), 964-975. http://dx.doi.org/10.3390/ph4070964

8. Hussain, S., R.Munjewar, R., & Farooqui, M. (2012). Development and Validation of a

Simultaneous HPLC Method for Quantification of Amlodipine Besylate and Metoprolol Tartrate in


42

Tablets. Journal Of Pharmascitech, 1(2), 1-5.

9. Bavili-Tabrizi, A., Bahrami, F., & Badrouj, H. (2017). A Very Simple and Sensitive

Spectrofluorimetric Method Based on the Oxidation with Cerium (IV) for the Determination of

Four Different Drugs in Their Pharmaceutical Formulations. Pharmaceutical Sciences, 23(1), 50-

58. http://dx.doi.org/10.15171/ps.2017.08

10. State Pharmacopoeia of Ukraine (2015). (2th ed). Kharkiv: Ukrainian Scientific Pharmacopoeial

Center for Quality of Medicines.

http://dx.doi.org/10.15171/ps.2017.08

Ankara Ecz. Fak. Derg. / J. Fac. Pharm. Ankara, 42(1): 43-52, 2018 Doi: 10.1501/Eczfak_0000000601

ÖZGÜN MAKALE / ORIGINAL ARTICLE

EVALUATION OF ACUTE AND SUBACUTE TOXICITY OF OIL

LINIMENT BASED ON 4-((5-(DECYLTHIO)-4-METHYL-4H-1,2,4-

TRIAZOL-3-YL)METHYL)MORPHOLINE

4-((5-(DESİLTİYO)-4-METİL-4H-1,2,4-TRİAZOL-3-İL)METİL)MORFOLİN ESASLI

YAĞ MERHEMİ AKUT VE SUBAKUT TOKSİSİTE PARAMETRELERİ TAYİNİ

Roman SHCHERBYNA1* , Volodymyr PARCHENKO1, Volodymyr MARTYNYSHYN2,

Vasyl HUNCHAK2

1Zaporizhzhya State Medical University, Faculty of Pharmacy, Department of Toxicological and

Inorganic Chemistry, Zaporizhzhya, Ukraine

2Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies, Faculty of

Veterinary Medicine, Department of Pharmacology and Toxicology, Lviv, Ukraine

ABSTRACT

Objective: It has now been demonstrated that compound 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazol-

3-yl)methyl)morpholine shows antifungal and antimicrobial activities. This study was aimed to estimate

toxicity values of novel oil liniment, which is based on mentioned compound and has antifungal effect.

Material and Method: Research was held in accordance with guidelines “Toxicological screening

of new substances for animal safety products” and “Preclinical esearch of veterinary drugs”. Toxicity level

(amount of toxic doses) and benchmark doses for subacute study were assessed in conditions of acute study.

Experiments were conducted on male wistar rats using karber method.

Result and Discussion: Findings in the research of the acute effect have shown that studied substance

belongs to the group of low-toxic compounds in conditions of intragastric administration. All rats survived

and completed subacute study, and daily administration of oil liniment in duration of 14 days did not cause

possible changes in body and organ weight among animals in experimental groups. It was demonstrated that

prolonged exposure to the liniment caused possible increase of ALP and LDH on the background of possible

cholesterol decrease, which may be the evidence of cholestatic liver disease, and enhancement of

permeability of the cell membranes, which may be highlighted by destructive changes in liver.

* Sorumlu Yazar / Corresponding Author: Roman Shcherbyna


Gönderilme/Submitted: 02.07.2018 Kabul/Accepted: 01.08.2018

Shcherbyna et al. J. Fac. Pharm. Ankara, 42(1): 43-52, 2018

44

Keywords: 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazol-3-yl)methyl)morpholine; rat; toxicity.

ÖZ

Amaç: 4-((5-(desiltiyo)-4-metil-4H-1,2,4-triazol-3-il)metil)morfolin maddesinin antifungal

ve antimikrobiyal özelliklere sahip olduğu gösterilmiştir. Bu araştırmanın amacı, bahsi geçen

bileşen esaslı antifungal etkiye sahip yağ merhemi toksisitesini tespit etmektir.

Gereç ve Yöntem: Araştırma, ‘“Toxicological screening of new substances for animal

safety products” and “Preclinical research of veterinary drugs‘da yer alan yönergelere göre

gerçekleştirilmiştir. Akut çalışma koşullarında toksisite düzeyi (toksik doz miktarı) ve subakut

çalışma için kıyaslama dozları değerlendirilmiştir. Araştırmalar, Wistar sıçanları üzerinde karber

yöntemi ile gerçekleştirilmiştir. Sonuç ve Tartışma: Akut etkinin araştırılmasında bulgular, çalışılan maddenin intragastrik

uygulama koşullarında düşük toksik bileşikler grubuna ait olduğunu göstermiştir. Subakut çalışmalar tamamlandı ve tüm sıçanlar sağ kurtuldu. 14 gün boyunca günlük olarak yağ liniment

uygulaması deney grupları içindeki hayvanlar ve organ ağırlığı arasında olası değişikliklere neden

olmadı. Çalışma merheme uzun süreli maruz kalmanın, kolesterol düzeyinin azalmasına neden olabilecek ALP ve LDH'nin artışına neden olduğu ve karaciğerde yıkıcı değişikliklere sebep

olabilecek hücre zarlarının geçirgenliğinin artışına sebep olabileceğini göstermiştir.

Anahtar kelimeler: 4-((5-(desiltiyo)-4-metil-4H-1,2,4-triazol-3-il)metil)morfolin; toksisite;

sıçan

INTRODUCTION

In last decades chemistry of triazole compounds has been greatly developed and is well studied

[1-3]. Many of the medications, which contain compounds with 1,2,4-triazole ring, were discovered

and applied in practice [3-6]. In that way, pharmaceuticals like vorazole [7] and anastrozole [6]

demonstrate antitumor activity, trazodone, alprazolam show antidepressant effects [4, 8]. Drug

substances based on itraconazole, fluconazole, voriconazole, posaconazole, albaconazole,

ravuconazole, isavuconazole, efinaconazole have been introduced into medical practice due to their

antifungal activity [1].

In our previous studies, it was demonstrated that 4-R-5-R1-1,2,4-triazole-3-thiol derivatives

exhibit wide spectrum of pharmacological [9-11] and biological activities [12-14]. Thus, one of alkyl

derivatives of 4-R-5-(morpholinomethylene)-4H-1,2,4-triazole-3-thiols, compound 4-((5-

(decylthio)-4-methyl-4H-1,2,4-triazol-3-yl) methyl) morpholine shows antifungal and antimicrobial

effects. In so doing, taking into account previous results, the aim of this work was to estimate toxicity

of oil liniment, which contains 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazol-3-yl)methyl)morpholine

[15] using laboratory animals in conditions of single administration (“acute toxicity”) and prolonged

exposure (“subacute toxicity”).

J. Fac. Pharm. Ankara, 42(1): 43-52, 2018 Shcherbyna et al.

45

MATERIAL AND METHOD

The object of the study was a liniment prepared in a form of oily solution of 4-((5-(decylthio)-

4-methyl-4H-1,2,4-triazol-3-yl) methyl) morpholine (Figure 1) using conventional technique with

load of solvent in order of ascending viscosity or density [15].

Toxicity level (amount of toxic doses) and benchmark doses for subacute study were assessed

in conditions of acute study. Experiments were conducted on male Wistar rats using Karber method.

Research was held in accordance with guidelines “Toxicological screening of new substances

for animal safety products” [16] and “Preclinical research of veterinary drugs” [17].

Figure 1. The structure of 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazol-3-yl) methyl) morpholine

Animal ethics

The study was approved by the Committee on Animal Use and Ethical Review, Stepan

Gzhytskyi National University of Veterinary Medicine and Biotechnology Lviv according to policy

of European Convention for the protection of vertebrate animals (Protocol №5, 26 October 2016).

The study of acute toxicity

Parameters estimated in conditions of acute experiment were: a) toxic level (amount of toxic

doses); b) benchmark doses for subacute study.

Acute toxicity has been investigated performing intragastric administration of oil liniment on

rats, which were 2-3 months of age and weighed 190-200 g. For this purpose, in preliminary study

animals were divided into three groups following the principle of analogues. Oil liniment was

administrated to each group in doses of 5000, 10000 and 25000 mg/kg respectively. In a detailed

study, animals were divided into six groups of six animals in each group. Substance was

administrated to animals in group I in dose of 5000 mg/kg, group II – 10000 mg/kg, group III –


46

15000 mg/kg, group IV – 20000 mg/kg, group V – 25000 mg/kg of body weight. LD50 was

determined using Karber method.

After administration of oil liniment, animals were further monitored during 14 days. In the

experiment, the following markers were taken into account: appearance, behavior of animals, hair

condition and state of visible mucous membranes, appetite, respiration rhythm and rate, time of onset

and profile of intoxication, its severity, course, time of animals’ death or their recovery [18].

The study of subacute toxicity of oil liniment

During the study of subacute toxicity, team relied upon the results obtained in the study of

acute toxicity. Substance of research was administrated intragastrically on daily basis during 14 days.

Subacute toxicity was studied on rats of 2-3 months of age and with body weight of 190-210

g. In the experiment, animals were divided into three groups in accordance with the principle of

analogues; each group had five rats. Water was administrated to animals in the reference group.

Animals in group I received oil liniment in amount of 1/50 LD50, group II – 1/20 LD50, group III –

1/10 LD50. Clinical condition and animals’ behavior were monitored during the study.

On the next day after administration was ceased, brief ether anesthesia was applied and animals

were decapitated, team collected blood samples, studied hematology and biochemistry following

generally accepted procedures, and applied necropsy to evaluate organ weight indexes, comparing

results with the reference group.

For hematologic studies, blood samples stabilized with EDTA were used, for biochemical

studies – the blood serum. The following blood counts were investigated: amount of hemoglobin,

red cells, white cells, hematocrit; values of mentioned counts were obtained using hematology

analyzer Mythic-18. Levels, which were assessed in blood serum samples: total protein using IRF-

22 refractometer, enzyme activity (AST, ALT, LDH), total bilirubin, creatinine, and urine using

biochemical analyzer HumaLyzer 3000 coupled with Human standard kit [16-19].

Obtained results were processed statistically; averages and confidence intervals were

evaluated with significance level of р>0.05 and considering Student’s t-test.


47

RESULTS AND DISCUSSION

Results for the study of acute toxicity

The results for the study of acute toxicity are shown in Table 1.

Table 1. Values of acute toxicity of oil liniment applied on rats using intragastric administration.

Dose of oil liniment,

mg/kg

5000 10000 15000 20000 25000

Survived 6 5 3 2 0

Died 0 1 3 4 6

z 0,5 2,0 3,5 5,0

d 5000 5000 5000 5000

Σ(zd) 2500 10000 17500 25000

LD50 was obtained using expression:

LD50= LD100 – Σ (z d)/ m,

here: LD100 – a dose, which caused death to all animals;

Σ – the sum symbol;

z – a half of all animals, which died from two next doses;

d – difference between two next doses;

m – number of animals in each group per amount of dose

Applying the expression, LD50 of oil liniment was:

LD50 = 25000 - [(2500+10000+17500+25000) : 6] = 15833,2 mg/kg

Hence, according to toxicity classification, in accordance with UCS 85.2-37-736:2011, the

substance of research belongs to IV Toxicity Class, or practically non-toxic substances, if

administrated intragastrically [18].


48

Results for the study of subacute toxicity of oil liniment

During the study of subacute toxicity none of laboratory rats had died. Table 2 contains

evaluated organ weight indexes.

Table 2. Rats’ organ weight indexes evaluated on 15-th day of the subacute study

(M±m, n=5)

Internal

organs

Group of animals

Reference Group І Group ІІ Group ІІІ

Liver, mg 35,3±1,4 34,0±0,8 34,8±1,9 38,4±1,7

Spleen, mg 5,0±0,7 4,9±0,3 5,0±0,4 4,9±1,0

Heart, mg 4,2±0,2 4,2±0,2 4,3±0,2 4,5±0,5

Lungs, mg 7,9±0,3 12,5±1,8 10,4±1,2 10,7±1,6

Kidneys, mg 6,9±0,2 6,7±0,2 7,1±0,3 6,8±0,3

Body weight,

mg

207,2±6,0 209,7±3,6 199,6±9,0 217,3±9,6

The results obtained after hematologic tests are shown in Table 3.

Table 3. Hematologic blood parameters of rats evaluated on 15-th day of the subacute study of the

oil liniment (M±m, n=5)

Counts Reference Group І Group ІІ Group ІІІ

Hemoglobin, g/L 140,3±4,57 145,6±2,81 152,9±4,83 153,1±2,47

Red cells, Т/L 6,0±0,13 6,35±0,09 6,6±0,23 6,76±0,48

White cells, g/L 11,75±1,58 10,78±0,29 10,63±0,87 10,3±1,37

Hematocrit, % 37,3±1,37 38,55±0,97 40,05±1,31 40,7±0,96

Leukogram

Neutrophils 22,0±3,37 21,0±1,91 21,0±1,91 24,0±2,31

Lymphocytes 69,5±3,40 71,0±3,51 69,0±1,29 64,7±3,71

Monocytes 6,5±0,5 6,5±0,96 8,0±0,82 7,33±0,67

Eosinophils 4,0±0,0 3,0±1,0 2,67±0,67 4,0±2,0

МСН, pg 23,4±0,66 22,9±0,67 23,2±0,37 22,8±1,24

МСНС, g/dL 37,7±0,19 37,7±0,29 38,15±0,30 37,6±0,47

МСV, µm3 62,1±1,86 60,75±2,06 60,65±0,48 60,66±2,69


49

The results obtained after biochemical tests are shown in Table 4.

Table 4. Biochemical blood parameters of rats evaluated on 15-th day of the subacute study of the

oil liniment (M±m, n=5)

Counts Reference Group І Group ІІ Group ІІІ

Total protein,

g/L

72,6±0,54 75,5±4,28 69,4±1,47 78,27±0,14***

Glucose,

mmole/L

5,65±0,75 6,1±0,21 5,38±0,34 6,23±0,59

Creatinine,

µmole/L

71,1±3,58 65,13±1,08 66,03±3,89 66,5±2,66

Total bilirubin,

mmole/L

1,18±0,32 0,58±0,15 1,65±0,62 2,2±0,57

Total

cholesterol,

mmole/L

1,07±0,03 0,98±0,09 0,88±0,03*** 1,53±0,17*

Lactate

dehydrogenase

(LDH), u/L

2647,8±

132,63

4776,7±

354,66**

4630,0±

561,3*

3563,7±

149,3**

AST, u/L 206,98±10,75 243,9±8,04 200,73±9,43 189,5±2,17

ALT, u/L 62,1±3,71 63,2±3,85 54,8±5,39 60,9±2,56

ALP, u/L 266,78±9,20 254,18±15,2 388,75±35,07** 363,7±43,44*

Note: * - р<0,05, ** - р<0,01,*** - р<0,001

Results in Table 2 show that intragastric administration of oil liniment during 14 days did not

cause possible changes in body weight and weight indexes of liver, spleen, heart and lungs of animals

in groups I, II and III.

Hematologic tests determined that administration of oil liniment to animals in groups I, II and

III did not cause significant changes in hemoglobin amounts, counts of red cells, white cells,

hematocrit, levels of mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin

concentration (MCHC), mean corpuscular volume (MCV) (Table 3).

Test for morphological composition of white cells in peripheral blood showed, that after rats

in groups I, II and III received oil liniment absolute lymphocyte, monocyte, and neutrophil counts

did not change significantly and were comparable to values obtained for the reference group (Table

3).

Results in Table 4 contain data obtained during biochemical tests, which highlight that total

protein in animals in groups I and II did not change significantly, while amount of this value was

increased by 7,8 % (р<0,001) in animals in group III, in relation to the reference group.

Test for total cholesterol in blood serum, taken on 15-th day of the experiment, showed that


50

this value was possibly down by 17,8 % (р<0,001) in animals in group II, while the same value was

possibly up by 42,9 % (р<0,05) in animals in group III, in relation to measured levels in reference

group (lipid storage disorder) (Table 4).

Assessment of LDH activity in blood serum after administration of oil liniment showed that

amount of LDH raised by 80 (р<0,01), 74,9 (р<0,05) and 34,6 % (р<0,01) in groups I, II and III

respectively, in relation to the reference group.

Assessment of ALP activity in conditions of prolonged exposure showed no significant

changes of this value among animals in group I, although possible increase of ALP levels was seen

in animals of group II and III by 45,7 (р<0,01) and 36,3 % (р<0,05) respectively, in relation to the

reference group.

Moreover, it is worth noting that during administration of oil liniment to animals in groups I,

II and III levels of creatinine, urine, glucose, total bilirubin, activities of AST and ALT did not differ

significantly, compared to values in the reference group.

Autopsy of animals in group I revealed small flatulence, and presence of liquid fecal matter in

a bowel. During autopsy of animals in groups II and III flatulence, and liquid yellow colored fecal

matter with air bubbles and sharp odor were observed. Liver of animals in all groups was colored

dark-red in some spots.

Interpreting obtained data, it must be underlined that prolonged exposure of oil liniment caused

liver disease in animals, which was highlighted by cholestasia (increase of ALP) on the background

of permeability enhancement of cell membranes, which may be evidenced by destructive changes in

liver (increase of LDH activity in blood serum).

CONCLUSION

This work has revealed that oil liniment formulation of 4-((5-(decylthio)-4-methyl-4H-1,2,4-

triazol-3-yl)methyl)morpholine belongs to Toxicity Class IV, i. e. practically non-toxic substances. It

was demonstrated, that prolonged exposure of oil liniment caused possible increase of ALP and LDH

in the background of possible decrease of total cholesterol, which may indicate on cholestatic liver

disease, and increase of membrane permeability, which may be evidenced by destructive changes in

liver.


51

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7. Jakič, M., Vogler, A., Rižner, T.L. (2016). Pharmacological treatment of endometriosis: review

of current and new options for treatment. Slovenian Medical Journal, 85(7-8), 410-426.

8. Jarema, M., Dudek, D., Landowski, J., Heitzman, J., Rabe-Jabłońska, J., Rybakowski, J. (2011).

Trazodon -the antidepressant: mechanism of action and its position in the treatment of

depression. Psychiatria polska, 45(4), 611-625.

9. Shcherbyna, R.О., Panasenko, O.I., Knysh, Y.G., Fotina, H.A., Vashchyk, Y.V., Fotina, T.I.

(2016). The study of antimicrobial activity of 2-((4-R-3-(morpholinomethylene)-4H-1, 2, 4-

triazole-5-yl) thio) acetic acid salts. Zaporozhye medical journal, (4), 97-100.

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acetate (PKR-177) influence on biochemical indices in rats blood under hepatitis initiated by

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V.V. (2001) Preclinical research of medicinal products: Methodical recommendations,

Avicenna, Kiev, p.528.

53

Yayım Koşulları

1. Ankara Üniversitesi Eczacılık Fakültesi Dergisi (Ankara Ecz. Fak. Derg. - J. Fac. Pharm. Ankara)

yılda üç kez (Ocak-Mayıs-Eylül) yayımlanır.

2. Dergiye Eczacılığın her alanında daha önce hiç bir yerde yayınlanmamış, Türkçe veya İngilizce

olarak hazırlanmış makaleler kabul edilir. Deneylerde, insan için “the Declaration of Helsinki” ve

hayvan için “European Community Guidlines”’a bağlı kalınmalıdır.

3. Yayın Komisyonuna gelen makaleler en az 2 danışmana gönderilir.

4. Makaleler yayına kabul ediliş sırasına göre yayınlanır.

5. Danışmanlar tarafından önerilen düzeltmelerin yapılması için yazar/ yazarlara geri gönderilen

makaleler, düzeltilip yayınlanmak üzere 3 ay içinde tekrar yayın kuruluna gönderilmezse, yeni

başvuru olarak işlem görür. Makale yayımlanmadan önce yazarların yayımcıya makalenin

“Copyright Transfer Form’unu doldurarak telif hakkını göndermesi gerekmektedir.

6. Yayımlarda intihal olup olmadığı kontrol edilmelidir.

7. Dergimize aşağıdaki makale türleri kabul edilir:

a) Araştırma makalesi: Türkçe veya ingilizce hazırlanmış, şekiller ve tablolar dahil tamamı en çok

20 A4 kağıdı sayfası olan, orjinal araştırmaların bulgu ve sonuçlarını açıklayan makalelerdir.

b) Derleme: Türkçe veya ingilizce hazırlanmış, şekil ve tablolar dahil tamamı en çok 25 A4 kağıdı

sayfası olan, yeterli sayıda bilimsel makale taranarak, o güne kadarki gelişmeleri özetleyerek

ortaya koyan ve sonuçlarını yorumlayarak değerlendiren makalelerdir.

c) Önbilgiler: Devam etmekte olan bir çalışmanın bulgularını zaman kaybetmeden duyurmak için

Türkçe veya ingilizce yazılan en çok 5 A4 kağıdı sayfası olan makalelerdir.

Yayım Gönderme

Yazarlar makalelerini http://journal.pharmacy.ankara.edu.tr adresinden online olarak yükleyeceklerdir.


54

Yazım Kuralları

1. Metinler, A4 normunda (21 x 29,7 cm) yazılmış olmalıdır.

2. Bütün tablo ve şekiller metin içindeki yerlerine yazım alanından taşmadan yerleştirilmiş olmalıdır.

3. Metinler A4 normundaki sayfanın sağ ve sol tarafından 2,5 cm., üst ve alt kenarlarından 3 er cm

boşluk bırakılarak (ilk sayfada yukarıdan 5 cm) 1.5 satır aralıkla yazılmalıdır. Yayımı kabul edilen

makaleler doğrudan “Microsoft Word” dosyası halinde online olarak sisteme yüklenecektir (online

submission). Yazı karakteri “Times New Roman” ve 11 punto olmalıdır.

4. Sayfa numaraları makalede belirtilmemelidir.

5. Yazar adı (küçük harf) ve soyadı (büyük harf) koyu olarak başlığın altına üç satır aralık verildikten

sonra altına unvan belirtmeden yazılmalıdır. Birden çok yazar varsa virgülle ayrılıp bir boşluk

bırakılarak yazılmalıdır. Yazarların soyadları üzerine konulacak rakamlarla hemen isimlerin

altındaki satıra kurum adları ve posta adresleri açıkça yazılmalıdır.

6. Başlık sayfasında yayın adı, yazar/yazarların adları ve yazışma yapılacak yazarın açık adresi,

telefon ve faks numaraları, varsa e-mail adresi belirtilmelidir. Sorumlu yazarın soyadının üstüne

(*) işareti konularak belirtilmelidir. Bu kişinin, açık adresi, fax numarası, telefon numarası ve e-

mail adresi başlık sayfasının en altında belirtilmelidir.

7. Tablolar üstlerine, şekiller (formül, grafik, şema, spektrum, kromatogram, fotoğraf v.b.) de altlarına

arabik rakamlarla (Şekil 1., Tablo 2.,) numaralandırılmalıdır. “Tablo”, “Şekil” sözcükleri ile

bunlara ait numaralar koyu yazılmalı ve 11 punto olmalıdır. Şekil/Resim (JPG formatında) makale

içinde yerleşmiş olmalıdır.

8. Tablo adları Tabloların üstüne ve şekil adları da Şekillerin altına birer satır aralıkla ve bunların

genişliğini aşmayacak şekilde 11 punto yazılmalıdır. Tabloya ait açıklama varsa tablonun altına 1

boşluk bırakılarak 9 punto ile yazılmalıdır. Tablo ve Şekiller metin içine yerleştirilirken metin ile

aralarında net ayırımı sağlayacak kadar boşluk bırakılmalıdır.

9. Paragraf başları 5 boşluk içeriden başlamalıdır.

10. Uluslararası kısaltmalar kullanılabilir. Metin içinde mililitre için ml; dakika için dak. olarak

belirtilen şekliyle yazılmalıdır.

11. Makalelerin bölümleri Başlık, Özet, Anahtar kelimeler, Giriş, Materyal – Yöntem, Sonuç ve

Tartışma ve Kaynaklar sırasına uygun olarak hazırlanmalıdır. Derleme makalelerinde Materyal –

Yöntem bölümü bulunmayabilir. Bu bölümler birbirlerinden 2 satır aralık ile ayrılmalıdır. Bu

bölümleri ifade eden başlıklar 12 punto ile koyu olarak büyük harflerle ve sayfanın solundan

başlanarak yazılmalıdır. Bölüm başlıkları ile metin arasında ayrıca aralık bırakılmamalıdır.

a. Başlık: Türkçe ve İngilizce olarak büyük harf ve 14 punto ile başlık koyu ve ikinci başlık beyaz

olarak yazılmalıdır. Başlık metine uygun, kısa, çalışmayı tanıtıcı ve açık ifadeli olmalıdır.

b. Özet: Türkçe ve İngilizce (Abstract) olarak makalelerin başında 200 er kelimeyi geçmeyecek

şekilde 10 punto ile, italik olarak ve çerçeve içinde yazılmalıdır. Yabancı dilde yazılmış

makalelerde mutlaka Türkçe özet bulunmalıdır.

c. Anahtar kelimeler: En fazla 5 sözcükten oluşmalı ve özetlerin hemen altına ilgili dilde

alfabetik ve italik olarak yazılmalıdır.

d. Giriş: Araştırmanın amacı ve konuyla ilgili çalışmaların yer aldığı bölüm olmalıdır.

e. Materyal ve Yöntem: Kullanılan materyal belirtilerek, uygulanan yöntem hakkında gerekli

bilgiler açıkça ifade edilmelidir. Deneylerde hayvan kullanılması durumunda lokal etik

komiteden veya ilgili düzenleyici makamlardan onay alınmalıdır ve bilgilendirilmiş onam

belgelendirilmelidir.

f. Sonuç ve Tartışma: Bulguların verilerek değerlendirildiği bölümdür.

g. Teşekkür: Varsa araştırmayı destekleyen kuruluşa ve katkısı olan kişilere kaynaklardan önce

yer alan bu bölümde kısaca teşekkür edilebilir.

h. Kaynaklar: Kaynak yazım stili Amerikan Psikoloji Derneği’ne (APA) göredir. Metinde, geçiş

sırasına göre köşeli parantez içinde, örneğin: [1,2,…] gibi numaralandırılmalı ve metin sonunda

bu numaralara göre sıralanmalıdır. Kaynaklar aşağıdaki örneklere uygun olarak yazılmalıdır.

55

i. Makale için: Yazarın soyadı, adının baş harfleri, makalenin tam başlığı derginin adı, cilt no,

varsa sayı no (parantez içinde), başlangıç ve bitiş sayfa no, yıl yazar isimlerinden sonra (parantez

içinde) olarak yazılmalıdır. Birden fazla yazar varsa hepsi yazılmalıdır. Makalenin adı yazılırken

ilk kelimenin ilk harfi büyük diğer kelimelerin ilk harfi küçük yazılmalıdır. Kaynaklarda verilen

dergi adları kısaltma yapılmadan açık olarak yazılmalıdır.

Moncada, S., Palmer, R.M.J., Higgs, E.A. (1989). Biosynthesis of nitric oxide from L-arginine.

A pathway for the regulation of cell function and communication. Biochemistry and

Pharmacology, 38, 1709 – 1715.

ii. Elektronik Makale için:

Perneger, T. V. and Giner, F. (1998). Randomized trial of heroin maintenance programme for adults

who fail in convential drug treatments. British Medical Journal, 317. Retrieved August 12, 2005,

from ttp://www.bmj.com/cgi/content/full/317/7150/

iii. Web sitesi için:

Clinical Pharmacology Web site. (2001). Retrieved June 16, 2004, from http://cpip.gsm.com/

iv. Kitap için: Yazarın soyadı, adının baş harfleri, kitabın adı, cilt no (varsa), kitabevi,

yayınlandığı şehir, sayfa no, basıldığı yıl (parantez içinde) yazılmalıdır.

Franke, R. (1984). Theoretical Drug Design Methods, Elsevier, Amsterdam, p.130.

v. Kitap Bölümü için: Yazarın soyadı, adının baş harfleri, bölümün başlığı, editör/editörlerin

soyadı, adının baş harfleri, (Ed./Eds.) ibaresi, kitabın adı, varsa cilt no, kitabevi, yayınlandığı şehir,

sayfa no, basıldığı yıl (parantez içinde) yazılmalıdır.

Weinberg, E.D. (1979). Antifungal Agents. In: M.E. Wolff and S.E. Smith (Eds.), Burger’s

Medicinal Chemistry, (pp. 531-537). New York: John Wiley and Sons.

12. Bileşiklerin karakterizasyonu ayrı bir paragraf ile gösterilmeli ve yeni bileşiklerin saflıkları ve yapı

aydınlatılmaları sağlanmalıdır.

56

Instruction for Authors

1. The Journal of Faculty of Pharmacy of Ankara University (J. Fac. Pharm. Ankara) is published

three times (January-May-September) a year.

2. The Journal of Faculty of Pharmacy of Ankara University publishes articles in every field of

Pharmaceutical Sciences. The manuscript to the journal should not be published previously as a

whole or in part and not be submitted elsewhere. Manuscript should be written in Turkish or

English The experiments used have to be adhered to the Declaration of Helsinki for humans and

European Community Guidlines for animals.

3. All manuscripts will be submitted to a review process by the editors and by qualified at least 2

outside reviewers.

4. Manuscripts are published in order of final acceptance after review and revision.

5. If a manuscript returned to the authors for revision is not received back to the editor within 3

months it will be treated as a new article. When the article is published, the by authors are

considered to transfer all rights of the manuscript to the Publisher.

6. Manuscript will be controlled using plagiarism checker.

7. Manuscripts with the following charactheristics are accepted:

a) Research article: Articles written in English or Turkish in scientific format presenting

original research. Articles should be printed on A4 size papers not exceeding 20 pages

(including tables and figures)

b) Review: An updated comprehensive review of scientific works on a particular subject.

Articles written in English or Turkish should be printed on A4 size papers not exceeding 25

pages (including tables and figures).

c) Rapid communication: Rapid announcement of the results of a continuing research written

in English or Turkish, no longer than 5, A4 size pages.

Submission of Manuscripts

Online submission: http://journal.pharmacy.ankara.edu.tr


57

Preparation of Manuscript

1. Manuscripts should be typed on A4 size papers marked in 21 x 29,7 cm area.

2. All tables and figures should be inserted in the text, not exceeding text margins.

3. Manuscripts should be typed with 1.5 line spacing with a margin of 2,5 cm on left-hand and

right-hand sides, 3 cm on the top (5 cm on the first page) and bottom. Since articles will be

loading online, authors are requested to submit their manuscripts as “Microsoft Word” file. Font

should be “Times New Roman” with 11 pt font size.

4. Page numbers shouldn’t be placed on the pages.

5. Author names (first name with small letters, surname with capital letters, no qualification)

should be written allowing 3 line space from the title of the article. Having more than one author,

the names should be separated with comma and 1 free space. By using number as superscripts,

the institution and mailing address of authors must be indicate on the next line.

6. Title page of the manuscript should include title, authors’ names and full mailing addresses.

Corresponding author should be indicated by an asteriks (*). His/Her marking address, a fax,

telephone numbers and e-mail address should indicate at the bottom of the title page.

7. All tables and figures/images must be cited in the text consecutively. Every table must have a

descriptive title at the top and should be numbered with Arabic numerals (Table 1., Table 2.)

Please submit tables as editable text and not as images. Figures (chemical formulas, graphics,

photographs, chromatographs, spectra etc) should also be numbered with Arabic numerals

(Figure 1., Figure 2.,) Captions should be typed with 11 pt font size. Figures/Images (JPG)

should be embedded in the Manuscript file.

8. An appropriate heading of tables and figures should be used for each and typed with 11 pt font

size at the top of the table, at the bottom of the figure with one line space. If there is an

explanation about the table, it should be written with 1 line space below and should be typed

with 9 pt font size. Between text and figures/tables must be adequate space to distinquish each

of them.

9. In each paragraph, indentation must be done (5 letter space).

10. International abbreviations should be used. In text ‘ml’ should be used for mililiter and ‘min’

should be used for minute to make harmonize for common abbreviation.

11. Manuscripts should be organise as follows: Title page, Abstract, Keywords, Introduction,

Material-Method, Results and Discussion, References. Each section must be separated with 2

line spaces. The section titles must be written with bold capital letters at 12 pt font size. No line

spaces between section headings and text.

a) Title: It should be written in Turkish and English. Font size must be 14 pt as a bold. The title

must be appropriate to the text.

b) Abstract: It should be written in Turkish and English no longer than 200 words, 10 pt, Italic.

Abstract should be written in a border. If manuscript is written in a foreign language, must

include Turkish abstract.

c) Keywords: Up to 5 key words should be provided in alfabetic and italic at the end of the

abstract.

d) Introduction: It should contain a clear statement of the aim and novelty of the study.

e) Materials and methods: It should be described in sufficient detail to allow other works to

dublicate the study. If animals are used, authors must indicate that approvals of the

relevant regulatory authorities or local ethical commitees were obtained and that

appropriate regulatory or local ethical commitee approvals were obtained and that

informed consent was documented.

f) Results and Discussion: The results must be clearly and concisely described with the help

of appropriate illustrative material. The discussion should deal with the interpretation of the

58

results.

g) Acknowledgements: If necessary, this section should be given at the end of the text, before

references.

h) References: The style of references is that of the American Psychological Association

(APA). They should be numbered with Arabic numerals consecutevily in the order in which

they first appear in the paper, for example: [1,2,…] Cited publications should be listed in

numerical order at the end of the paper. If there is more than one author, all the names of the

authors should be written. Examples are given below;

i) Article: Reference to a journal publication (journal names in full, not abbreviated)

Moncada, S., Palmer, R.M.J., Higgs, E.A. (1989). Biosynthesis of nitric oxide from L-

arginine. A pathway for the regulation of cell function and communication, Biochemistry

and Pharmacology, 38, 1709 – 1715.

ii) Electronic Article:

Perneger, T. V. and Giner, F. (1998). Randomized trial of heroin maintenance programme

for adults who fail in convential drug treatments. British Medical Journal, 317. Retrieved

August 12, 2005, from ttp://www.bmj.com/cgi/content/full/317/7150/

iii) Web page:

Clinical Pharmacology Web site. (2001). Retrieved June 16, 2004, from http://cpip.gsm.com/

iv) Book:

Franke, R. (1984). Theoretical Drug Design Methods, Elsevier, Amsterdam, p.130.

v) Chapter in a book:

Weinberg, E.D. (1979). Antifungal Agents. In: M.E. Wolff and S.E. Smith (Eds.), Burger’s

Medicinal Chemistry, (pp. 531-537). New York: John Wiley and Sons.

12. The characterization of compounds should be presented in a seperate paragraph and for all new

compounds, evidence to confirm both identity and purity have to be provided.

ANKARA ÜNİVERSİTESİ ECZACILIK FAKÜLTESİ DERGİSİ

YAYIN SAHİBİNİN ADI : Prof. Dr. Gülbin ÖZÇELİKAY

SORUMLU YAZI İŞLERİ MÜDÜR ADI : Prof. Dr. İlkay YILDIZ

YAYIN İDARE MERKEZİ ADRESİ : Ankara Üniversitesi, Eczacılık Fakültesi,

Dekanlığı, 06100 Tandoğan/Ankara

YAYIN İDARİ MERKEZİ ADRESİ TEL : 0 (312) 213 54 62

0 (312) 203 30 69

YAYIN TÜRÜ : Bilimsel Periyodik Elektronik Dergi, Yılda 3 Sayı

Özgün Makaleler / Original Articles Sayfa / Page

Ufuk ÖZGEN, Sıla Özlem ŞENER, Merve BADEM, Hatice SEÇİNTİ, Seda Damla HATİPOĞLU, Ahmet Ceyhan GÖREN, Cavit KAZAZ, Erhan PALASKA - Evaluation of HPLC, phytochemical, anticholinesterase and antioxidant profiles of the aerial parts of Asperula taurina subsp. caucasica - Asperula taurina subsp. caucasica'nın toprak üstü kısımlarının YBSK, fitokimyasal, antikolinesteraz ve antioksidan profillerinin değerlendirilmesi

1

Lyudmila KUCHERENKO, Igor BELENICHEV, Ivan MAZUR, Olga KHROMYLOVA, Natalia PARNIUK - Influence of the fixed

combination of glycine with thiotriazoline on energy metabolism parameters in brain in conditions of experimental cerebral ischemia - Glisin ile tiyotriazolin sabit kombinasyonunun

deneysel serebral iskemi şartlarında beyin enerji metabolizması göstergelerine etkisi

14

Arezoo VIEW, Aras RAFIEE - Upregulation of MIR-17 and MIR-221 by benomyl,

carbaryl, malathion and diazinon pesticides in mice blood - Fare kanında MIR-17 ve MIR-221'in benomil, karbaril, malatiyon ve diazinon pestisitleri ile upregülasyonu

22

Anastasiia DONCHENKO, Svitlana VASYUK - Spectrophotometric determination

of metoprolol tartrate in pure and dosage forms - Saf ve dozaj

formlarında metoprolol tartaratın spektrofotometrik tayini

33

Roman SHCHERBYNA, Volodymyr PARCHENKO, Volodymyr MARTYNYSHYN, Vasyl HUNCHAK - Evaluation of acute and subacute toxicity of oil liniment based on 4-((5-(decylthio)-4-methyl-4H-1,2,4-triazol-3-yl)methyl)morpholine - 4-((5-(Desiltiyo)-4-metil-4H-1,2,4-triazol-3-il)metil)morfolin esaslı yağ merhemi akut ve subakut toksisite parametreleri tayini

43

İÇİNDEKİLER / CONTENTS

ANKARA ÜNİVERSİTESİ ECZACILIK FAKÜLTESİ

DERGİSİ

Cilt / Vol : 42

Sayı / No : 1

Yıl / Year : 2018

eISSN : 2564-6524

JOURNAL OF FACULTY OF

PHARMACY

OF ANKARA UNIVERSITY

Documents

ANKARA ÜNİVERSİTESİ ECZACILIK FAKÜLTESİ …journal.pharmacy.ankara.edu.tr/wp-content/uploads/sites/...Ozgen et al. J. Fac. Pharm. Ankara, 42(1): 1-13, 2018 2 the plant did not