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International Journal of Food Properties

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Chemical profile and biological activities of two edible
plants: Chemical investigation and quantitative analysis
using LC-MS/MS and GC-MS
a b c d e
Abdulselam Ertas , Mustafa Abdullah Yilmaz , Mehmet Boga , Nesrin Hasimi , Yeter Yesil ,
f g h
Ahmet C. Goren , Hamdi Temel & Gulacti Topcu
a
Department of Pharmacognosy, Faculty of Pharmacy, Dicle University, 21280 Diyarbakir,
Turkey,
b
Dicle University Science and Technology Research and Application Center (DUBTAM), Dicle
University, 21280 Diyarbakir, Turkey,
c
Department of Pharmaceutical Technology, Faculty of Pharmacy, Dicle University, 21280
Diyarbakir, Turkey,
d
Department of Nutrition and Dietetics, School of Health, Batman University, 72060
Batman, Turkey,
e
Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University, 34116
Istanbul, Turkey,
f
TUBITAK UME, National Metrology Institute, Chemistry Group, Organic Chemistry
Laboratory, 41470 Kocaeli, Turkey,
g
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Dicle University, 21280
Diyarbakır, Turkey,
h
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Bezmialem Vakif
University, 34093 Istanbul, Turkey
Accepted author version posted online: 18 May 2015.

To cite this article: Abdulselam Ertas, Mustafa Abdullah Yilmaz, Mehmet Boga, Nesrin Hasimi, Yeter Yesil, Ahmet C.
Goren, Hamdi Temel & Gulacti Topcu (2015): Chemical profile and biological activities of two edible plants: Chemical
investigation and quantitative analysis using LC-MS/MS and GC-MS, International Journal of Food Properties, DOI:
10.1080/10942912.2015.1020437

To link to this article: http://dx.doi.org/10.1080/10942912.2015.1020437

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 Chemical profile and biological activities of two edible
plants: Chemical investigation and quantitative
analysis using LC-MS/MS and GC-MS
Running Title: A detailed study on two edible plants

t
ip
Abdulselam Ertas,1* Mustafa Abdullah Yilmaz,2 Mehmet Boga,3 Nesrin Hasimi,4 Yeter Yesil,5
Ahmet C. Goren,6 Hamdi Temel,7 and Gulacti Topcu8*

cr
1
Department of Pharmacognosy, Faculty of Pharmacy, Dicle University, 21280 Diyarbakir,
Turkey,

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2
Dicle University Science and Technology Research and Application Center (DUBTAM), Dicle
University, 21280 Diyarbakir, Turkey,
3

Diyarbakir, Turkey ,
4
an
Department of Pharmaceutical Technology, Faculty of Pharmacy, Dicle University, 21280

Department of Nutrition and Dietetics, School of Health, Batman University, 72060 Batman,
M
Turkey,
5
Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University, 34116
Istanbul, Turkey,
ed

6
TUBITAK UME, National Metrology Institute, Chemistry Group, Organic Chemistry
Laboratory, 41470 Kocaeli, Turkey,
pt

7
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Dicle University, 21280
Diyarbakır, Turkey,
ce

8
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Bezmialem Vakif
University, 34093 Istanbul, Turkey

*Correspondence to: Assistant Prof. Dr. A. Ertas, E-mail: [email protected];

Ac

[email protected], Phone: +90 412 248 8030/7536 / +90505 628 3447.

*Correspondence to: Prof. Dr. G. Topcu, E-mail: [email protected], Phone:

+902125232288 , Fax:+902125332326.

1
 Abstract

The objectives of this study were to define the phenolic and fatty acid profiles,

anticholinesterase, antioxidant, antimicrobial activities and total phenolic-flavonoid contents of

Lycopsis orientalis and Tragopogon latifolius var. angustifolius which have been used as food

t
ip
source and food supplement in Anatolia and have never been examined before. Rosmarinic and

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quinic acids (21.11 and 11.46 mg g-1 extract, respectively) were found to be the most abundant

constituents in L. orientalis and T. latifolius var. angustifolius among the studied twenty-seven

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compounds by LC-MS/MS. In the fatty acid compositions of L. orientalis and T. latifolius var.

angustifolius that were determined by GC-MS, oleic (29.1 %) and palmitic (28.7 %) acids were
an
identified as the major components, respectively. The high antioxidant activity of the methanol
M
extract of L. orientalis shows parallelism to its rosmarinic acid content. Besides, this extract

showed medium anticholinesterase activity. The results of the present study proves that the L.
ed

orientalis might also be used as a food source owing to its high phenolic acid content and strong

antioxidant property.
pt

Keywords: Lycopsis orientalis; Tragopogon latifolius var. angustifolius; anticholinesterase;

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antimicrobial; antioxidant; LC-MS/MS.

Ac

2
 INTRODUCTION

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ip
Genus Tragopogon L. which belongs to the Asteraceae family, consists of 84 species in the

world and 21 species in Turkey.[1] Tragopogon species are known as “Salsify” throughout the

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world. Tragopogon latifolius Boiss. is grown in south and east Transcaucasia; and its two

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varieties, T. latifolius Boiss. var. angustifolius Boiss. and T. latifolius Boiss. var. latifolius Boiss,

were reported in Turkey.[1,2] T. latifolius var. angustifolius is used to heal wounds and as food
an
source; and are known as Ispınk and Yemlik, in Anatolia.[1,2] T. latifolius var. angustifolius is

consumed either raw or cooked.[2] The previous studies on a Tragopogon species show that T.
M
porrifolius L. contains gallic acid, catechin, epigallocatechin and epigallocatechin gallate.[3] In

other reports it was mentioned that the ethanolic extract of T. pratensis L. which contains some
ed

polyphenolic compounds, displays antileukemic activity by inducing apoptosis on J-45.01 human

acute T leukaemia cell line.[4,5] In some studies, volatile constituents and oleanane triterpenoids
pt

of T. porrifolius L. and T. pratensis L. were also determined.[6-8] According to the literature

ce

survey, chemical properties and biological activities of T. latifolius var. angustifolius have not

been studied yet.

Ac

Genus Lycopsis L. which belong to the Boraginaceae family, is represented by 1 species

(Lycopsis orientalis L.) in Turkey and 2 species in the world.[9] Being common in Balkan

Peninsula, Anchusa arvensis L. Bieb. subsp. orientalis L. Nordh. is the synonym of L. orientalis

in Norsk Flora.[10] It is reported that A. arvensis subsp. orientalis, known as Frez, Kara dinding

3
 and Mıjık, is used as diuretic and food supplement.[2] In Anatolia, same as T. latifolius var.

angustifolius, L. orientalis is also consumed extensively as raw and cooked.[2] In a prior study,

the effects of the aqueous extract of L. orientalis on algaesthesis and skin ulcer in mice were

investigated.[11] Additionally, pyrrolizidine alkaloids were isolated from the different parts of A.

t
arvensis (Syn. Lycopsis arvensis).[12]

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Recently, several scientific studies have been focused on the phenolic compounds of the edible

plants having a number of pharmacological effects and the biological activities.[13] From this

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point of view, in the current study, it was aimed to investigate and compare the results of L.

orientalis and T. latifolius var. angustifolius as their cooking and consumption patterns are

similar. [2]
an
To the best of our knowledge, there are no reports about the chemical properties and
M
biological activities of L. orientalis and T. latifolius var. angustifolius in literature. In the present

study, total phenolic-flavonoid contents, anticholinesterase, antioxidant and antimicrobial

ed

activities of L. orientalis and T. latifolius var. angustifolius were studied. Besides, the fatty acid

compositions of L. orientalis and T. latifolius var. angustifolius petroleum ether extracts were
pt

examined by GC-MS technique. Finally, the chemical composition of their methanol extracts

were determined qualitatively and quantitatively using LC-MS/MS.

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MATERIAL AND METHODS

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Chemicals and Instruments

4
 Phenolic contents and fatty acid compositions of L. orientalis and T. latifolius var. angustifolius

were determined by LC-MS/MS (Shimadzu, Kyoto, Japan) and GC-MS (Thermo Scientific

Polaris Q) instruments, respectively. A Shimadzu UV spectrophotometer and BioTek Power

Wave XS microplate reader (USA) were used for the activity assays. 2,2′-Azinobis (3-

t
ip
ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (purity: 97.5 %) and butylated

hydroxytoluene (BHT) (≥99 %) were purchased from Merck (Germany); (L)-malic acid (95-100

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%), quercetin (95 %), protocatechuic acid (97 %), chrysin (97 %), rutin (94 %), hesperetin (95

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%), naringenin (95 %), rosmarinic acid (96 %), vanillin (99 %), p-coumaric acid (98 %), caffeic

acid (98 %), chlorogenic acid (95 %), hyperoside (≥9 7 %), myricetin (≥96 %), coumarin (≥99

an
%), kaempferol (≥97 %), formic acid (≤100 %), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (≥95 %),

β-carotene (≥93 %), linoleic acid (≥99 %), Tween 40, pyrocathecol (≥99 %), 5,5-dithiobis-(2-
M
nitro benzoic acid) (DTNB) (≥98 %), copper (II) chloride dihydrate (CuCl 2 .2H 2 O) (≥99 %),

neocuproine (2,9-dimethyl-1,10-phenanthroline) (≥98 %), EDTA (Ethylenediaminetetraacetic

ed

acid) (≥98 %), acetylcholinesterase (AChE: from electric eel) (Type-VI-S, EC 3.1.1.7, 425.84

U/mg), and butyrylcholinesterase (BChE: from horse serum) (EC 3.1.1.8, 11.4 U/mg) were
pt

obtained from Sigma (Germany); quinic acid (98 %), tr-aconitic acid (98 %), 4-hydroxybenzoic
ce

acid (≥99 %), fisetin (≥98 %), α-tocopherol (≥95.5 %) and acetylthiocholine iodide (≥ 98 %)

were from Aldrich (Germany); gallic acid (≥99 %), tannic acid (puris), salicylic acid (≥99 %),
Ac

galanthamine hydrobromide (≥94 %) were from Sigma-Aldrich (Germany); Folin Ciocalteu

Phenol reagent was from Applichem (Germany); hesperidin (≥97 %), luteolin (≥97 %), apigenin

(≥99 %), rhamnetin (≥99 %), (butyrylthiocholine iodide (≥99%) were from Fluka (Germany).

5
 Plant material

The whole plants of Lycopsis orientalis L. and Tragopogon latifolius Boiss. var. angustifolius

Boiss. were collected from south eastern part of Turkey (Malatya) in June 2012 and identified by

t
Dr. Yeter Yeşil. These specimens have been stored at the Herbarium of Istanbul University

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(ISTE 9803 and ISTE 10012, respectively).

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Preparation of plant extracts for LC-MS/MS

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10 g of the powdered plants were extracted three times with MeOH (50 mL each) at room

an
temperature for 24 h. Afterwards, the extracts obtained were combined, filtered and evaporated

under low pressure. Dry filtrates were reconstituted in methanol at a concentration of 250 mg L-1
M
and filtered through the 0.2 µm PTFE filter prior to LC-MS/MS analysis.
ed

Preparation of plant extracts for biological activities and GC-MS

Powdered form of the whole plant material was weighed (100 g) and sequentially macerated
pt

three times with petroleum ether (250 mL each), acetone (250 mL each), methanol (250 mL
ce

each) and water (250 mL each) at 25 ºC for 24 hours. After filtration, the solvent was evaporated

to get the crude extracts. The yields of the petroleum ether extracts were calculated as T latifolius
Ac

var. angustifolius petroleum ether extract (TLAP) 1.50 %, L. orientalis petroleum ether extract

(LOP) 0.80 %, the acetone extracts as TLAA 1.65 %, LOA 0.87 %, the methanol extracts as

TLAM 2.89 %, LOM 2.60 %, and the water extracts as TLAW 2.01 %, LOW 1.29 % (w/w).

6
 GC/MS conditions and esterification of the fatty acids

Esterification of the petroleum ether extract and GC/MS procedure described by Ertas et al.[14]

were applied. Thermo Scientific Polaris Q GC-MS/MS was used.

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Determination of total phenolic and flavonoid contents

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Total phenolic and flavonoid contents expressed as pyrocatechol and quercetin equivalents

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respectively, were determined as reported in the literature.[15,16] The following equations were

used to calculate total phenolic and flavonoid contents of the extracts:

an
Absorbance = 0.0164 pyrocatechol (μg) + 0.0266 (R2 = 0.9969)
M
Absorbance = 0.1519 quercetin (μg) – 0.1294 (R2 = 0.9986)

Antioxidant activity assays

ed

β-Carotene-linoleic acid test system, DPPH free radical scavenging activity, ABTS cation radical
pt

decolorisation and cupric reducing antioxidant capacity (CUPRAC) assays were carried out to
ce

determine the antioxidant activity. [17-20]

β-Carotene bleaching method: 0.5 mg of β-carotene in 1 mL of chloroform was added into

Ac

linoleic acid (25 µL) and Tween 40 emulsifier (200 mg) mixture. After evaporating chloroform,

100 mL of distilled water saturated with oxygen was added followed by shaking, 160 μL of this

mixture was transferred into different test tubes containing 40 μL of the sample solutions at

different concentrations. The emulsion was added to each tube, the zero time absorbances of the

7
 values were read at 470 nm. The mixture was incubated for 2 h at 50 °C. [17] A blank, devoid of

β-carotene, was prepared for back ground subtraction. α-Tocopherol was used as a standard. The

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

t
a
ln

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R= b
t

cr
Where: ln=natural log, a=absorbance at time zero, b=absorbance at time t (120 min).

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

control, using following equation:

AA (Inhibition%) =
an
RControl − RSample
RControl
x 100
M
DPPH Free radical scavenging activity method: 0.1 mM, 160 µL of DPPH solution in methanol
ed

was added to 40 µL of sample solutions in methanol at different concentrations. After 30 min.,

the absorbance values were read at 517 nm. The DPPH free radical scavenging potential was
pt

calculated using the following equation:

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Acontrol − Asample
DPPH free radical scavenging effect (Inhibition %) = × 100
Acontrol
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A Control is the initial concentration of the DPPH•

A Sample is the absorbance of the remaining concentration of DPPH• in the presence of the

extracts or positive controls. [18]

8
 ABTS cation radical decolorization assay: Seven milimolar ABTS in H 2 O was added to 2.45

mM potassium persulfate to produce ABTS•+ and solution was stored in the dark at 25 °C for 12-

16 h. The prepared solution was diluted with ethanol to get an absorbance of 0.700 ± 0.025 at

734 nm. ABTS•+ solution (160 µL) was added to each sample solution (40 μL) at different

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concentrations. After 30 min., the percentage inhibition at 734 nm was read for each

concentration relative to a blank absorbance (methanol). The following equation was used to

cr
calculate the scavenging capability of ABTS•+ [19]:

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Acontrol − Asample
ABTS•+ scavenging effect (Inhibition %) = × 100
Acontrol
an
Cupric reducing antioxidant capacity (CUPRAC) method: Aliquots of 61 μL of 1.0 × 10−2 M
M
copper (II) chloride, 61 μL of NH 4 OAc buffer (1 M, pH 7.0), and 61 μL of 7.5 × 10−3 M

neocuproine solution were stirred, x μL sample solution (2.5, 6.25, 12.5, and 25 μL) and (67 − x)
ed

μL distilled water were added to reach the final volume 250 μL. The tubes were left to stand for

one hour. Afterwards, the absorbance at 450 nm was measured against a reagent blank. [20]
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Anticholinesterase activity
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A spectrophotometric method developed by Ellman et al.[21] was established to indicate the

Ac

acetyl- and butyryl-cholinesterase inhibitory effects. Aliquots of 150 µL of 100 mM sodium

phosphate buffer (pH 8.0), 10 μL of sample solution and 20 μL BChE (or AChE) solution were

stirred and incubated for 15 min at 25 ºC, then DTNB (10 μL) is added to mixture. In the next

step, by the addition of butyrylthiocholine iodide (or acetylthiocholine iodide) (10 μL) the

9
 reaction was started. At the end, final concentration of the tested solutions was 200 μg/mL.

BioTek Power Wave XS at 412 nm was used to monitor the hydrolysis of these substrates. The

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

percentages of inhibition was calculated by using the following equation:

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AControl − ASample
(Inhibition %) = x 100
AControl

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Determination of antimicrobial activity and Minimum Inhibition

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concentration (MIC)
an
The antimicrobial activities of extracts against different microorganisms were assessed according
M
to inhibition zone diameter and MIC value.[22,23] Five different microorganisms including gram

positive bacteria (Streptococcus pyogenes ATCC 19615 and Staphylococcus aureus ATCC
ed

25923), gram negative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC

25922) and yeast (Candida albicans ATCC 10231) which were purchased from Refik Saydam
pt

Sanitation Center (Turkey) were used for detecting the antimicrobial activity of the samples. The
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disc diffusion method was employed for this purpose. The minimum inhibitory concentration

determined by the broth macrodilution method according to NCCLS. Ampicillin and fluconazole
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were used as positive controls for bacteria and yeast, respectively.

Method validation parameters

10
 In this study, twenty-four phenolic compounds (flavonoids, flavonoid glycosides, phenolic acids,

a phenolic aldehyde, coumarin) and three non-phenolic organic acids that are widespread in

edible plant materials were qualified and quantified in L. orientalis and T. latifolius var.

angustifolius. Rectilinear regression equations and the linearity ranges of the studied standard

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compounds were given in Table 1. Correlation coefficients were found to be higher than 0.99.

The limit of detection (LOD) and the limit of quantitation (LOQ) of the reported analytical

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method were shown in Table 1. For the studied compounds, LOD ranged from 0.05 to 25.8 µg/L

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and LOQ ranged from 0.17 to 85.9 µg/L (Table 1).[24] Moreover, the recoveries of the phenolic

compounds ranged from 96.9% to 106.2%.

Estimation of uncertainty
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M
Ertas et al. report was used to evaluate and quantify the uncertainty sources of the applied
[25,26]
method. Purity of reference standards (pur), sample weights (w), calibration curves (cal),
ed

repeatability (rep), stock solutions (Css) and recovery (rec) were the sources used to calculate

the uncertainties. To calculate the standard combined uncertainty the following equation (1) was
pt

applied:
ce

u (C ) = u 2 (cal ) + u 2 ( pur ) + u 2 (Css ) + u 2 ( w) + u 2 (rep) + +u 2 (rec) (1)

Ac

Calibration curve and the purity of standards were defined as the main sources of uncertainty. In

order to calculate expanded uncertainties at 95% confidence level (k:2), standard combined

uncertainties were multiplied by two. Calculated uncertainties were given in Table 1.

11
 Statistical analysis

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The total phenolic-flavonoid contents, antioxidant,anticholinesterase and antimicrobial activities

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results were shown as means ± standard deviation. The results were evaluated using an unpaired

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t-test and one way analysis of variance ANOVA. The differences were regarded as statistically

significant at p < 0.05.

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RESULT AND DISCUSSION

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Through the consumption of edible plants, various phenolic acids take part in the protection of
M
human body from oxidative damage diseases like coronary heart disease, cancers, and stroke,

inflammation and cardiovascular diseases. There has been an increasing interest on phenolic
ed

compounds due to such properties of them. Not only the researchers but also the food

manufacturers give importance to phenolic compounds owing to their strong antioxidant

pt

properties, ampleness in the human diet, and their probable role in the prevention of various
ce

diseases associated with oxidative stress.[27] Therefore, the interest to the natural antioxidants

sourced from fruits, vegetables, spices and other plants has been increased.
Ac

Quantitative analysis of phenolic and flavonoid compounds by LC-

MS/MS

12
 In literature, the LC-MS/MS technique is extensively used in quantitative analysis of phenolic

compounds.[28,29] Thus, an accurate quantitative method was developed on a mass spectrometer

equipped with a triple quadrupole analyzer for the analyses of twenty-seven compounds (Table

1). The methanol extracts of L. orientalis and T. latifolius var. angustifolius were analysed to

t
ip
quantify these compounds by the mentioned method. For the detection of the studied compounds

by MRM (Multiple reaction monitoring), the specific fragmentation reactions were selected.

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These compounds were monitored by the transition from the specific deprotonated and

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protonated pseudomolecular ions to the corresponding fragment ions. Molecular ions and

fragments observed in MS/MS, collision energies of these fragments and the quantified results

an
for L. orientalis and T. latifolius var. angustifolius were presented in Table 2.
M
In the methanol extracts of L. orientalis and T. latifolius var. angustifolius, rosmarinic and quinic

acids were found to be the most abundant compounds, (21.11 and 11.46 mg g-1 extract,
ed

respectively) (Table 2 and Figure 1A-C). Furthermore, rutin, hesperedin, hyperoside and

apigenin were also detected in the methanol extract of the studied plants (Table 2 and Figure 1B-
pt

C). Additionally, while quercetin, luteolin and kaempferol were specified in the methanol

extracts of T. latifolius var. angustifolius, they were not identified in the methanol extract of L.
ce

orientalis (Table 2 and Figure 1B-C). Malic, tr-aconitic, chlorogenic, protocatechuic, trans-

caffeic, p-coumaric, 4-OH benzoic and salicylic acids were determined in both studied methanol
Ac

extracts. Rosmarinic acid was found in high amounts in the methanol extract of L. orientalis.

Nevertheless, in the methanol extract of T. latifolius var. angustifolius, rosmarinic acid was not

detected (Table 2 and Figure 1). It is the first time that the examined compounds were identified

in Lycopsis species. Besides, the existance of most of these compounds were not reported in

13
 Tragopogon species before. In literature, there are few studies about chemical profile of

Tragopogon species with HPLC. In a previous study, gallic acid (1.35 mg g-1), ferulic acid (0.20

mg g-1), rutin (0.09 mg g-1), resveratrol (0.014 mg g-1), sinapic acid (0.11 mg g-1) and caffeic acid

(0.28 mg g-1) were detected in T. pratensis.[4] In the current study, rutin (0.28 mg g-1) and caffeic

t
acid (0.24 mg g-1) were identified in T. latifolius var. angustifolius. Moreover, while in the study

ip
of Kucekova et al. [4] p-coumaric acid and quercetin were not detected in T. pratensis, they were

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detected in our study. Another report indicated the existence of gallic acid, catechin,

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epigallocatechin and epigallocatechin gallate in T. porrifolius.[3] In this respect, this report is

significant in determining the chemical profile of Lycopsis and Tragopogon species.

Fatty acid composition by GC-MS

an
M
The fatty acid composition of the petroleum ether extracts were determined by GC-MS analyses.

As it can be seen in Table 3, fourteen components were identified; constituting 99.8 % of the
ed

petroleum ether extract of T. latifolius var. angustifolius. The major constituents of the fatty

acids obtained from the petroleum ether extract were identified as palmitic (C16:0) (28.7 %),
pt

oleic (C 18:1 omega-9) (21.0 %), linoleic (C18:2 omega-6) (20.6 %) and linolenic acids (C18:3
ce

omega-3) (12.1 %). Additionally, fifteen components were identified, constituting 99.9 % of the

petroleum ether extract of L. orientalis. The major constituents of the fatty acid composition
Ac

obtained from the petroleum ether extract were characterized as oleic (29.1 %), palmitic (17.6

%), linoleic acids (17.2 %) and 1-octadecanol (8.2 %) (Table 3). When the fatty acid

compositions of T. latifolius var. angustifolius and L. orientalis were compared, they contain

palmitic acid (28.7 and 17.6 %, respectively), oleic acid (21.0 and 29.1 % ), 1-octadecanol (- and

14
 8.2 %) and arachidic acid (1.9 and 6.3 %). This is the first report on the fatty acid compositions

of L. orientalis and T. latifolius var. angustifolius. Also, according to our knowledge there is no

report on fatty acid composition of Tragopogon and Lycopsis species in literature.

There are several studies on fatty acid composition of Asteraceae and Boraginaceae families in

t
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literature. The major components of the fatty acid composition were identified as palmitic and

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linoleic acids for Asteraceae family[30,31]; and oleic, linoleic and linolenic acids for Boraginaceae

family.[32,33] Our results are in consistent with literature. In the current study, palmitic and oleic

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acids were found to be the main components of the fatty acid profile of T.

latifolius var. angustifolius, a member of Asteraceae family, and L. orientalis, a member

of Boraginaceae family, respectively.

an
M
Antioxidant activity and total phenolic-flavonoid content
ed

The antioxidant activity of the petroleum ether (TLAP and LOP, respectively), acetone (TLAA

and LOA), methanol (TLAM and LOM) and water (TLAW and LOW) extracts prepared from
pt

the whole plants of T. latifolius var. angustifolius and L. orientalis were studied using β-carotene

bleaching, DPPH free radical scavenging, cupric reducing antioxidant capacity and ABTS cation
ce

radical decolorisation assays. To our knowledge, there is no study about L. orientalis and T.
Ac

latifolius var. angustifolius regarding these antioxidant activities and total phenolic-flavonoid

contents in literature. In the crude methanol extracts prepared from the whole plants of L.

orientalis and T. latifolius var. angustifolius, total phenolic and flavonoid amounts were

determined expressing as pyrocatechol and quercetin equivalents, respectively (y = 0.0164

pyrocatechol (μg) + 0.0266, R2 = 0.9969 and y = 0.1519 quercetin (μg) – 0.1294, R2 = 0.9986).

15
 The total phenolic and flavonoid contents of the LOM and TLAM extracts were determined as

124.70 ± 1.74 mg PEs per g dry extract and 49.04 ± 0.09 mg QEs per g dry extract, respectively.

The total phenolic contents were detected to be higher than total flavonoid contents (Table 4).

As indicated in Table 4, while the LOM extract showed good lipid peroxidation activity (IC 50 :

t
ip
38.46±1.30 µg mL-1) in β-carotene bleaching method, the TLAA, TLAM, LOP and LOA

cr
extracts exhibited moderate lipid peroxidation activity (IC 50 : 105.32±0.98, 112.61±0.67, 109.

39±0.31 and 89.49±1.38 µg mL-1, respectively) in the same method. The other tested extracts

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indicate weak lipid peroxidation activity in β-carotene bleaching method. As it can be observed

in Table 4, the LOM and LOW extracts showed good activity (IC 50 : 50.82±0.33 and 59.31±1.18
an
µg mL-1, respectively) in DPPH free radical scavenging method. Besides, the TLAM, TLAW
M
and LOA extracts showed moderate activity (IC 50 : 117.72±0.91, 102.60±1.26 and 89.27±1.36 µg

mL-1, respectively) in DPPH free radical scavenging method. Other studied three extracts had
ed

weak activities in DPPH free radical scavenging activity test. As shown in Table 4, the TLAA,

TLAM, TLAW, LOA, LOM and LOW extracts showed the following IC 50 values in ABTS
pt

cation radical scavenging assay; 92.61±0.37, 55.51±0.62, 16.61±0.69, 27.81±0.17, 7.62±0.09,

and 8.51±0.12 µg mL-1, respectively. Particularly, the LOM and LOW extracts indicate very
ce

strong activity in ABTS cation radical scavenging assay. Also, these extracts exhibited higher

activity than α-tocopherol (IC 50 : 9.54±0.09 µg mL-1) and BHT (IC 50 : 10.31±0.14 µg mL-1),
Ac

which were used as standards in the ABTS cation radical scavenging assay. The other tested two

extracts had weak activity in ABTS cation radical scavenging assay. The LOM extract and α-

tocopherol exhibited 1.53 and 1.65 absorbance in CUPRAC at 100 µg mL-1, respectively (Figure

2). The other tested extracts showed weak or no activity in CUPRAC (data not shown). After

16
 examining the antioxidant properties of the eight extracts, the LOM extract showed highest

activity among the studied methods. This high antioxidant activity of LOM extract might be

stemmed from high total phenolic content or high rosmarinic acid amount that is known with its

high antioxidant capacity.[34]

t
ip
Anticholinesterase activity

cr
All of the extracts showed no or weak inhibitory activity against acetyl- and butyryl-

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cholinesterase enzymes, except from LOM extract which had moderate effect, at 200 µg mL-1

(Table 4). According to the literature research, this is the first study on the anticholinesterase
an
activity of Tragopogon and Lycopsis species.
M
Determination of antimicrobial activity and Minimum Inhibition

concentration (MIC)
ed

The antimicrobial activities of extracts against different microorganisms were assessed according
pt

to inhibition zone diameter and MIC value. Results were presented in Table 5. The petroleum
ce

ether and water extracts showed no activity against the five tested microorganisms (Data was not

shown). The acetone and methanol extracts were active on all microorganisms with different
Ac

zone diameters indicating weak antimicrobial activity (inhibition zone <12). Only the TLAM

extract possesses moderate activity (inhibition zone <20-12) against C. albicans (14 mm

inhibition zone diameter and 110 µg mL-1 MIC value). The lowest MIC value was recorded by

the LOM extract against C. albicans (25 ± 0.4 µg mL-1).

17
 CONCLUSION

t
ip
This report represents the first study on chemical compositions and biological activities of L.

orientalis and T. latifolius var. angustifolius. The antioxidant capacity of LOM extract was

cr
higher than the other seven extracts. The reason why LOM was the most active of all eight

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extracts tested for four antioxidants methods used, could be related to its high total phenolic

content or high amount of rosmarinic acid that have strong antioxidant activity.[34] According to
an
our study, quinic and rosmarinic acids (11.46 and 21.11 mg g-1 extract, respectively) were found

to be the most abundant phenolic compounds in TLAM and LOM extracts. Although the total
M
flavonoid content of TLAM extract was the richest, rutin (0.28 mg g-1 extract), hesperedin (0.29

mg g-1 extract), hyperoside (0.96 mg g-1 extract), apigenin (0.036 mg g-1 extract), quercetin
ed

(0.032 mg g-1 extract), luteolin (0.52 mg g-1 extract) and kaempferol (0.53 mg g-1 extract) were

in low amount and the other flavonoids were not detected in this extract. Thus, the high total
pt

flavonoid content of this extract could be related to some other flavonoids that we have not
ce

studied yet. As a result, rich total phenolic content and high antioxidant capacity of the LOM

necessitates further studies in this field. The results of the present study showed that LOM
Ac

extract can also be used as a food source due to its high phenolic acid content and strong

antioxidant properties.

ACKNOWLEDGEMENT

18
 The authors are thankful to Dicle University Science and Technology Research and Application

Center (DUBTAM) for its support in this study.

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23
 Figure Captions

FIGURE 1. LC-MS/MS chromatogram of A: 250 ppb standard mix, B: T. latifolius var.

angustifolius methanol extract, C: L. orientalis methnol extract. 1: Quinic acid, 2: Malic acid, 3:
tr-Aconitic acid, 4: Gallic acid, 5: Chlorogenic acid, 6: Protocatechuic acid, 7: Tannic acid, 8: tr-
caffeic acid, 9: Vanillin, 10: p-Coumaric acid, 11: Rosmarinic acid, 12: Rutin, 13: Hesperidin,
14: Hyperoside, 15: 4-OH Benzoic acid, 16: Salicylic acid, 17: Myricetin, 18: Fisetin,19:

t
Coumarin, 20: Quercetin, 21: Naringenin, 22: Hesperetin, 23: Luteolin, 24: Kaempferol, 25:

ip
Apigenin, 26: Rhamnetin, 27: Chrysin.

cr
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an
M
ed
pt
ce
Ac

24
 FIGURE 2. Cupric reducing antioxidant capacity of the T. latifolius var. angustifolius, L.
orientalis, α-tocopherol and BHT. Values are means ± S.D., n=3, p<0.05, significantly different
with Student’s t-test.

t
ip
cr
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TABLE 1. Analytical parameters of LC- MS/MS method.

Analyt

e no
RT Analytes
a
R2b an
Equation RSD%
c
Linearity

Range
LOD/L

OQ
Recover

y (%)
U
e
M
(mg/L) (µg/L)d
ed

1 Quinic acid 0.992 f(x)=33.6626*x+2513 0.038 250-10000 22.3 / 74.5 103.3 4.8

3.36 7 2.9 8
pt
ce

2 Malic acid 0.997 f(x)=93.6102*x- 0.121 250-10000 19.2 / 64.1 101.4 5.3

3.60 5 5673.77 4
Ac

3 tr-Aconitic acid 0.993 f(x)=79.2908*x- 0.390 250-10000 15.6 / 51.9 102.8 4.9

4.13 3 28416.2 8

25
 4 Gallic acid 0.990 f(x)=358.069*x+2641 0.473 25-1000 4.8 / 15.9 102.3 5.1

4.25 1 7.5 4

5 Chlorogenic acid 0.993 f(x)=48.9828*x+2677 0.188 250-10000 7.3 / 24.3 99.7 4.9

t
ip
5.29 2 9.7 2

cr
6 Protocatechuic 0.999 f(x)=36.8568*x+6197 0.595 100-4000 25.8 / 85.9 100.2 5.1

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5.51 acid 1 .38 8

6.30
Tannic acid 0.995

5
an
f(x)=90.2704*x+3023

3.2
0.907

5
100-4000 10.2 / 34.2 97.8 5.1
M
8 tr- caffeic acid 0.994 f(x)=1585.16*x+8395 1.008 25-1000 4.4 / 14.7 98.6 5.2
ed

7.11 2 7.5 0
pt

9 Vanillin 0.999 f(x)=44.5478*x- 0.409 250-10000 10.1 / 33.7 99.2 4.9

ce

8.57 5 574.867 4
Ac

10 p-Coumaric acid 0.990 f(x)=73.5303*x+2706 1.135 100-4000 15.2 / 50.8 98.4 5.1

9.17 9 4.3 8

11 9.19 Rosmarinic acid 0.999 f(x)=18.0298*x- 0.522 250-10000 10.4 / 34.8 101.7 4.9

26
 2 1149.86 0

12 Rutin 0.997 f(x)=51.8835*x+3841 0.814 250-10000 17.0 / 56.6 102.2 5.0

9.67 1 .66 6

t
ip
13 Hesperidin 0.997 f(x)=195.773*x+1056 0.136 250-10000 21.6 / 71.9 100.2 4.9

cr
9.69 3 41 3

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14 Hyperoside 0.954 f(x)=0.978146*x+827 0.213 100-4000 12.4 / 41.4 98.5 4.9

9.96 9
an .221 5
M
15 11.3 4-OH Benzoic 0.992 f(x)=635.003*x+5428 1.401 25-1000 3.0 / 10.0 106.2 5.2

8 acid 5 4.6 3
ed

16 11.3 Salicylic acid 0.990 f(x)=915.178*x+7257 0.661 25-1000 4 / 13.3 106.2 5.0
pt

9 4 1.4 9
ce

17 11.4 Myricetin 0.999 f(x)=54.2823*x+5414 2.824 100-4000 9.9 / 32.9 106.0 5.9

2 1 .67 7
Ac

18 12.1 Fisetin 0.998 f(x)=331.870*x+3440 2.426 100-4000 10.7 / 35.6 96.9 5.5

0 8 9.0 2

27
 19 12.1 Coumarin 0.992 f(x)=236.639*x+3437 0.420 100-4000 9.1 / 30.4 104.4 4.9

8 4 0.3 3

20 13.9 Quercetin 0.999 f(x)=206.102*x+1693 4.314 25-1000 2.0 / 6.8 98.9 7.1

t
ip
3 5 .14 9

cr
21 14.1 Naringenin 0.995 f(x)=1100.55*x+3905 2.020 25-1000 2.6 / 8.8 97.0 5.5

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5 6 5.7 0

22 14.8

0
Hesperetin 0.996

1
an
f(x)=160.323*x+6545

.07
1.016

4
25-1000 3.3/ 11.0 102.4 5.3
M
23 14.8 Luteolin 0.999 f(x)=111.474*x+3057 3.948 25-1000 5.8 / 19.4 105.4 6.9
ed

4 2 .10 7
pt

24 14.8 Kaempferol 0.991 f(x)=20.9677*x+571. 0.588 25-1000 2.0 / 6.6 99.1 5.2
ce

5 7 241 5
Ac

25 16.7 Apigenin 0.995 f(x)=543.793*x+1852 0.678 25-1000 0.1 / 0.3 98.9 5.3

3 4 5.6 2

26 Rhamnetin 0.999 f(x)=110.091*x+632. 2.567 25-1000 0.2 / 0.7 100.8 6.1

18.4

28
 1 4 444 8

27 20.6 Chrysin 0.996 f(x)=698.787*x+2353 1.553 25-1000 0.05 / 0.17 102.2 5.3

0 5 1.7 0

t
ip
cr
a
RT: Retention time

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b
R2: Coefficient of determination

c
RSD: Relative standard deviation
an
M
d
LOD/LOQ(µg/L): Limit of detection/Limit of quantification

e
U (%): Percent relative uncertainty at 95% confidence level (k=2).
ed

TABLE 2. Identification and quantification of compounds methanol extracts of T. latifolius var.

angustifolius (TLAM) and L. orientalis (LOM) by LC-MS/MS.
pt

Quantification
ce

Parent MS2(Collision
Compound (mg analyte/g extract)c
a b
ion (m/z) Energy)
Ac

TLAM LOM

Quinic acid 190.95 85 (22),93 (22) 11.46±0.55 2.98± 0.14

29
 Malic acid 133.05 115 (14),71 (17) 0.470±0.025 6.69±0.35

tr-Aconitic acid 172.85 85 (12).129 (9) 0.305±0.015 0.47±0.02

t
ip
Gallic acid 169.05 125 (14),79 (25) D.d D.

cr
Chlorogenic 353 191 (17) 2.12±0.10 0.26±0.13

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acid

Protocatechuic

acid
152.95 an
109 (16),108 (26) 0.16±0.008. 0.28±0.014
M
Tannic acid 182.95 124 (22),78 (34) N.D.e 0.08±0.004
ed

tr- Caffeic acid 178.95 135 (15),134 (24),89 (31) 0.24±0.012 0.295±0.015
pt
ce

Vanillin 151.05 136 (17),92 (21) N.D. N.D.

Ac

p-Coumaric acid 162.95 119 (15),93 (31) 0.39±0.02 0.21±0.01

Rosmarinic acid 358.9 161 (17),133 (42) N.D. 21.11±1.03

30
 Rutin 609.1 300 (37),271 (51),301 (38) 0.28±0.01 4.26±0.21

Hesperidin 611.1 303,465 0.29±0.014 2.39±0.12

t
ip
Hyperoside 463.1 300,301 0.96±0.05 332.37±15.85

cr
4-OH Benzoic 136.95 93,65 0.295±0.015 0.39±0.002

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acid

Salicylic acid 136.95 an

93,65,75 0.26±0.013 0.35±0.017
M
Myricetin 317 179,151,137 N.D. N.D.
ed

Fisetin 284.95 135,121 N.D. N.D.

pt

Coumarin 146.95 103,91,77 N.D. D.

ce

Quercetin 300.9 179,151,121 0.32±0.002 N.D.

Ac

Naringenin 270.95 151,119,107 N.D. N.D.

Hesperetin 300.95 164,136,108 N.D. N.D.

31
 Luteolin 284.95 175,151,133 0.52±0.035 D.

Kaempferol 284.95 217,133,151 0.53±0.027 N.D.

t
ip
Apigenin 268.95 151,117 0.037±0.002 0.005±0.0003

cr
Rhamnetin 314.95 165,121,300 N.D. N.D.

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Chrysin 253 143,119,107 N.D. N.D.

a
an
Parent ion (m/z): Molecular ions of the standard compounds (mass to charge ratio)
M
b
MS2(CE): MRM fragments for the related molecular ions (CE refers to related collision energies
ed

of the fragment ions)

c
Values in mg/g (w/w) of plant methanol extract
pt

d
D: peak observed, concentration is lower than the LOQ but higher than the LOD
ce

e
N.D: not detected.
Ac

TABLE 3. GC-MS Analysis of the petroleum ether of T. latifolius var. angustifolius (TLAP) and
L. orientalis (LOP).

32
 Composition Composition

Rt (min)a Constituentsb (%)c (%)

TLAP LOP

t
ip
14.39 10-Undecenoic 0.8 0.6

cr
acid

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18.60 Myristic acid 1.6 2.8

24.63 9-Hexadecenoic
an1.8 -
M
acid
ed

24.94 Palmitoleic acid - 0.2

pt

25.27 Palmitic acid 28.7 17.6

ce

29.75 Phytol 1.3 -

Ac

30.23 1-Octadecanol - 8.2

30.64 Linoleic acid 20.6 17.2

33
 30.77 Oleic acid 21.0 29.1

30.86 Linolenic acid 12.1 7.9

t
ip
31.54 Stearic acid 6.3 5.1

cr
36.23 Nonacosanol - 0.5

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36.55 Eicosane 0.9 -

36.69 11-Eicosenoic
an
- 2.5
M
acid
ed

37.38 Arachidic acid 1.9 6.3

pt

38.19 6-Hexadecenoic 0.9 0.6

acid
ce

39.36 Docosane 0.6 -

Ac

42.65 Pentacosane - 0.3

34
 43.82 Behenic acid 1.3 1.0

Total 99.8 99.9

t
ip
a
Retention time (as minutes)

cr
b
A nonpolar Phenomenex DB-5 fused silica colum

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c
Percentage of relative weight.

an
M
ed
pt
ce
Ac

35
 TABLE 4. Antioxidant activity*, total phenolic-flavonoid contents* and anticholinesterase
activity* of T. latifolius var. angustifolius and L. orientalis extracts, BHT, α-TOC and
galantamine.

Inhibition Inhibition IC 50
Flavonoid content

t
% % Phenolic content

ip
Lipid (µg/mL)
Samples ABTS
(mg QEs/g
(mg PEs/g extract)α

cr
against against
Peroxidati DPPH Free Cation
extract) β
AChE BChE
on Radical Radical

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TLAP NA NA
an
- - >200a >200a >200a
M
TLAA NA 19.45± - - 105.3± >200a 92.61±0.37b

0.30a 0.98b
ed

TLAM NA 35.61± 84.38± 2.98a 49.04± 0.09a 112.61±0.6 117.72±0.91b 55.51±0.62c

pt

0.76b 7c
ce

TLAW NA NA - - >200a 102.60±1.26c 16.61±0.69d

Ac

LOP NA 11.59± - - 109. >200a >200a

0.29c 39±0.31c

36
 LOA 17.41± 22.27± - - 89.49±1.38 89.27±1.36d 27.81±0.17e

0.84a 0.49a d

t
LOM 46.59± 47.82± 124.70± 1.74b 15.74± 0.32b 38.46±1.30e 50.82±0.33e 7.62±0.09f

ip
1.45b 0.52d

cr
us
172.33±2.1 59.31±1.18f 8.51±0.12g
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LOW NA 12.50± - -

0.39c 2f

Galantamin 85.07 ± 84.83±0.17e

an
- - >200a >200a >200a
M
e† 0.07c
ed

α-TOC† - - - - 15.21±0.09 17.41±0.39g 9.54±0.09h

g
pt
ce

BHT† - - - - 9.48±0.12h 49.91±0.17e 10.31±0.74h

Ac

*
Values expressed are means ± SEM of three parallel measurements (p<0.05)

†
Standard drug

α
PEs, pyrocatechol equivalents (y=0.0164x + 0.0266 R2=0.9969)

37
 β
QEs, quercetin equivalents (y=0.1519x – 0.1294 R2=0.9986)

NA: Not active.

TABLE 5. Zones of growth inhibition (mm) and MIC values showing the antimicrobial activities
of the extracts compared to positive controls.

t
ip
Microorganisms

cr
Gram positive Gram negative Yeast

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S. aureus
anS.pyogenes E.coli P. aeruginosa C. albicans

a
DD 10±0.5 11±0.3 11±0.1 12±0.3 14±0.5
M
MIC >1000 40±0.4 80±0.2 200±0.2 110±0.5
ed
TLAA

a
DD 9±0.4 10±0.2 10±0.6 10±0.4 11±0.5
pt
ce

MIC 90±0.5 >1000 30±0.1 50±0.7 35±0.1

TLAM
Ac

a
DD 12±0.2 12±0.1 12±0.2 11±0.1 10±0.1

MIC 30±0.2 >1000 45±0.2 >1000 >1000

LOA

38
 a
DD 11±0.3 10±0.1 11±0.2 9±0.3 12±0.4

MIC 42±0.3 35±0.2 20±0.7 50±0.3 25±0.4

LOM

t
ip
b
Positive DD 35±0.2 19±0.2 20±0.1 - 30±0.3

controls

cr
MIC 1.95±0.3 7.815±0.1 7.815±0.4 - 3.125±0.2

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-: Not active

a
an
DD: Inhibition zone in diameter (mm) around the discs (6 mm) impregnated with 30 mg mL-1 of
M
plant extracts.

b
DD: Inhibition zone in diameter (mm) of positive controls that are ampicillin for bacteria and
ed

fluconazole for yeast. Minimum inhibitory concentration (MIC) values are given as μg mL-1.
pt
ce
Ac

39