Chemical compositions by using LC-MS/MS and GC/MS and biological activities of Sedum sediforme (Jacq.) Pau

Subscriber access provided by ISTANBUL TEKNIK UNIVERSITESI

Journal of Agricultural and Food Chemistry is published by the American ChemicalSociety. 1155 Sixteenth Street N.W., Washington, DC 20036Published by American Chemical Society. Copyright © American Chemical Society.However, no copyright claim is made to original U.S. Government works, or worksproduced by employees of any Commonwealth realm Crown government in thecourse of their duties.

Article

Chemical compositions by using LC-MS/MS and GC/MSand biological activities of Sedum sediforme (Jacq.) Pau

Abdulselam Erta#, Mehmet Bo#a, Mustafa Abdullah Y#lmaz, Yeter Ye#il,Nesrin Ha#imi, Meryem #eyda Kaya, Hamdi Temel, and Ufuk Kolak

J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf500067q • Publication Date (Web): 28 Apr 2014

Downloaded from http://pubs.acs.org on May 4, 2014

Just Accepted

“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are postedonline prior to technical editing, formatting for publication and author proofing. The American ChemicalSociety provides “Just Accepted” as a free service to the research community to expedite thedissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscriptsappear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have beenfully peer reviewed, but should not be considered the official version of record. They are accessible to allreaders and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offeredto authors. Therefore, the “Just Accepted” Web site may not include all articles that will be publishedin the journal. After a manuscript is technically edited and formatted, it will be removed from the “JustAccepted” Web site and published as an ASAP article. Note that technical editing may introduce minorchanges to the manuscript text and/or graphics which could affect content, and all legal disclaimersand ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errorsor consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Table of Contents Graphic

Chemical compositions by using LC-MS/MS and GC/MS and biological

activities of Sedum sediforme (Jacq.) Pau

Page 1 of 33

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1



Short title: Investigations on Sedum sediforme (Jacq.) Pau

Abdulselam Ertaş*,†

, Mehmet Boğa‡, Mustafa Abdullah Yılmaz

§, Yeter Yeşil⊥, Nesrin

Haşimi∥, Meryem Şeyda Kaya∇, Hamdi Temel#,§

and Ufuk Kolak○

†Department of Pharmacognosy, Faculty of Pharmacy, Dicle University, 21280 Diyarbakır,

Turkey

‡Department of Pharmaceutical Technology, Faculty of Pharmacy, Dicle University, 21280

Diyarbakır, Turkey

§Dicle University Science and Technology Research and Application Center (DÜBTAM),

Dicle University, 21280 Diyarbakır, Turkey

⊥Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University, Istanbul

34116, Turkey

∥Department of Nutrition and Dietetics, School of Health, Batman University, 72060 Batman,

Turkey

∇Department of Pharmacology, Faculty of Pharmacy, Dicle University, 21280 Diyarbakır,

Turkey

#Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Dicle University, 21280

Diyarbakır, Turkey

○Department of General and Analytical Chemistry, Faculty of Pharmacy, Istanbul University,

Istanbul 34116, Turkey

Page 2 of 33



2

ABSTRACT: In this research, chemical composition and biological activities of various 1

extracts obtained from whole parts of Sedum sediforme (Jacq.) Pau were compared. The 2

amounts of total phenolic and flavonoid components in crude extracts were determined by 3

expressing as pyrocatechol and quercetin equivalents, respectively. All of the extracts 4

(petroleum ether, acetone, methanol and water) obtained from S. sediforme showed strong 5

antioxidant activity in four tested methods. Particularly, IC50 values of methanol extract that 6

was richest in terms of total phenolic and flavonoid contents, were found to be lower than α-7

tocopherol and BHT in β-carotene bleaching (9.78 ± 0.06 µg/mL), DPPH free (9.07 ± 0.07 8

µg/mL) and ABTS cation radical scavenging (5.87 ± 0.03 µg/mL) methods. Furthermore, 9

methanol extract of S. sediforme showed higher inhibition activity than galanthamine against 10

acetyl- and butyryl-cholinesterase enzymes. Also, acetone and methanol extracts exhibited 11

moderate antimicrobial activity against C. albicans. The main constituents of fatty acid and 12

essential oil were identified as palmitic acid (C16:0) (28.8%) and α-selinene (20.4%), 13

respectively, by GC/MS. In methanol extract of S. sediforme, quercetin, rutin, naringenin, 14

protocatechuic, p-coumaric, caffeic and chlorogenic acids were detected and quantified by 15

LC-MS/MS. Results of the current study showed that methanol extract of S. sediforme may 16

also be used as food supplement. 17

18

19

KEYWORDS: Sedum sediforme, phenolic content, essential oil, fatty acid, antioxidant, 20

anticholinesterase, antimicrobial, quercetin, LC-MS/MS, GC/MS. 21

22

23

Page 3 of 33



3

� INTRODUCTION 24

Belonging to the Crasulaceae family, genus Sedum L. comprises approximately 348 species in 25

the world and 33 species in Turkey. Additionally, it is named as Kayakoruğu and Damkoruğu 26

in Anatolia.1,2 Sedum species have been known as both vegetables and folk medicines. They 27

are used for the treatment of many diseases such as wounds, hemorrhoids, constipation, foot 28

fungi and used as laxative and diuretic.3-5 Being a mediterranean element, S. sediforme (Jacq.) 29

Pau is named as Altın otu (goldherb) due to its yellow flowers and used as food4-6 and 30

ornamental globally. 7 31

Previous phytochemical studies indicated that Sedum species contained different 32

natural compounds such as new isoflavone derivatives like sedacin A and B,8 arbutin, 33

hydroquinone,9 phenolic acids and flavonoids,10 flavonol glycosides, sarmenosides V–VII,11 34

and alkaloids.12 In addition, several researches exhibited that Sedum species had strong 35

antioxidant potential.5,8,10,13,14 36

Several reports can be found on S. sediforme in literature. In the study of Sakar et al., 37

it is reported that two new compounds, whose structures were elucidated by spectroscopic 38

means as (2R,3R)-7,4'-dihydroxy-5,3',5'-trimethoxyflavan 3-O-gallate and 1-β-D-39

glucopyranosyloxy-3-methoxy-5-hydroxybenzene that were isolated from S. sediforme 40

flowers.15 They were accompanied by limocitrin 3-glucoside, 1-β-D-glucopyranosyloxy-3,5-41

dihydroxybenzene, kaempferol 3-rhamnoside, quercetin 3-rhamnoside, (-)-epicatechin 3-42

gallate, (-)-epigallocatechin 3-gallate, myricetin 3-rhamnoside, and gallic acid.15 43

In a former study, an HPLC method was established to determine quercetin which is a 44

common hydrolyzate of the flavonoid glycosides in Sedum sarmentosum, S. lineare and S. 45

erythrostictum.16 Besides, Romojaro et al. reported that S. sediforme had high phenol content 46

and hydrophilic total antioxidant activity.5 These aforementioned studies triggered us to focus 47

on S. Sediforme that is an edible species. The high phenolic content of S. sediforme and the 48

Page 4 of 33



4

quercetin content of Sedum species show that chemical and biological activities of these 49

species are worth studying deeply. It is a known fact that phenolic compounds especially 50

quercetin shows high antioxidant and anticholinesterase activities.17-19 Thus, it is aimed to 51

investigate the relationship between the chemical composition and biological activities of S. 52

Sediforme. 53

To the best of our knowledge, there aren’t any reports on the essential oil, fatty acid 54

and phenolic profile, and antioxidant (β-carotene-linoleic acid test system, DPPH free radical 55

scavenging activity and cupric reducing antioxidant capacity (CUPRAC)), anticholinesterase 56

and antimicrobial activities of S. sediforme in the literature. At the beginning, the fatty acid 57

and essential oil compositions of S. sediforme were determined by using GC/MS in the 58

current study. In the next step, related antioxidant, anticholinesterase, and antimicrobial 59

activities; total phenolic and flavonoid contents were analyzed. Moreover, the phenolic and 60

flavonoid content of S. sediforme methanol extract was also determined using UHPLC-ESI-61

MS/MS for quantitative and qualitative purposes. 62

� MATERIALS AND METHODS 63

Chemicals and instruments. Phenolic content and fatty acid composition of S. sediforme 64

was determined by using LC-ESI-MS/MS (Shimadzu, Kyoto, Japan) and GC/MS (Thermo 65

Scientific Polaris Q) instruments, respectively. A Shimadzu UV spectrophotometer and 66

BioTek Power Wave XS microplate reader (USA) were used for the activity assays. 2,2′-67

azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (purity: 97.5%) 68

and butylated hydroxytoluene (BHT) (≥99%) were purchased from Merck (Germany); 69

quercetin (95%), protocatechuic acid (97%), chrysin (97%), rutin (94%), hesperetin (95%), 70

naringenin (95%), rosmarinic acid (96%), vanillin (99%), p-coumaric acid (98%), caffeic acid 71

(98%), chlorogenic acid (95%), formic acid (≤ 100%), 2,2-diphenyl-1-picrylhydrazyl (DPPH) 72

(≥ 95%), β-carotene (≥ 93%), linoleic acid (≥ 99%), Tween 40, pyrocathecol (≥ 99%), 5,5-73

Page 5 of 33



5

dithiobis-(2-nitro benzoic acid) (DTNB) (≥ 98%), copper (II) chloride dihydrate 74

(CuCl2.2H2O) (≥ 99%), neocuproine (2,9-dimethyl-1,10-phenanthroline) (≥ 98%), EDTA 75

(Ethylenediaminetetraacetic acid) (≥ 98%), acetylcholinesterase (AChE) (Type-VI-S, EC 76

3.1.1.7, 425.84 U/mg), and butyrylcholinesterase (BChE) (EC 3.1.1.8, 11.4 U/mg) were 77

obtained from Sigma (Germany); α-tocopherol (≥ 95.5%) and acetylthiocholine iodide (≥ 78

98%) were from Aldrich (Germany); galanthamine hydrobromide (≥ 94%) was from Sigma-79

Aldrich (Germany); Folin Ciocalteu Phenol reagent was from Applichem (Germany); 80

butyrylthiocholine iodide (≥ 99%) was from Fluka (Germany). 81

Plant material. Sedum sediforme (Jacq.) Pau, which was collected by Dr. A. Ertaş from 82

Western Turkey (Istanbul) in May 2012 and characterized by Dr. Y. Yeşil (Department of 83

Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University). Voucher specimens have 84

been strored in the Herbarium of Istanbul University, Faculty of Pharmacy (ISTE: 9805). 85

Identification and Quantitation of Phenolic Compounds. Instruments and 86

Chromatographic Conditions. LC-ESI-MS/MS analysis of the methanol extract was 87

performed by using a Shimadzu UHPLC instrument coupled to a tandem MS instrument. The 88

liquid chromatograph was equipped with LC-30AD binary pumps, DGU-20A3R degasser, 89

CTO-10ASvp column oven and SIL-30AC autosampler. 90

For the chromatographic seperation, a C18 reversed-phase Inertsil ODS-4 (100 91

mm×2,1 mm i.d., 2µm) analytical column was used. The column temperature was fixed at 92

40˚C. The elution gradient consisted of mobile phase (A) water (5mM ammonium acetate and 93

0.1% formic acid) and (B) acetonitrile (0.1% formic acid). The gradient was used as the 94

following: at t = 0.00 min, 20% B; at t = 3.00 min, 20% B; at t = 3.01 min 50% B; at t = 8.99 95

min 50% B; at t = 9.01 min, 90% B; at t = 11.99 min 90% B; at t = 12.00 min, 20% B; at t = 96

Page 6 of 33



6

14.99 min 20% B. The solvent flow rate was maintained at 0.5 mL/min and injection volume 97

was settled as 10 µL. 98

MS Instrumentation. MS detection was performed using Shimadzu LCMS 8040 model 99

triple quadrupole mass spectrometer equipped with an ESI source operating in negative ion 100

mode. LC-ESI-MS/MS data were collected and processed by LabSolutions software 101

(Shimadzu, Kyoto, Japan). The multiple reaction monitoring (MRM) mode was used to 102

quantify the analytes: the assay of phenolic compounds was performed following two or three 103

transitions per compound, the first one for quantitative purposes and the second and/or the 104

third one for confirmation. 105

Optimization of LC-ESI-MS/MS Method. Subsequent to several combinations of trials, 106

a gradient of acetonitrile (0.1% formic acid, 5mM ammonium acetate) and water (0.1% 107

formic acid) system was concluded to be the best mobile phase solution. For rich ionization 108

and the seperation of the molecules, the mentioned mobile phase proved to be the best of all. 109

ESI source was chosen instead of APCI (Atmospheric Pressure Chemical Ionization) and 110

APPI (Atmospheric Pressure Photoionization) sources as the phenolic compounds were small 111

and relatively polar molecules. Tandem mass spectrometry was decided to be used for the 112

current study since this system is commonly used for its fragmented ion stability.17 The 113

working conditions were determined as interface temperature; 350 °C, DL temperature; 250 114

°C, heat block temperature; 400 °C, nebulizing gas flow (Nitrogen); 3L/min and drying gas 115

flow (Nitrogen); 15L/min. 116

Method Validation Parameters. In the current study, eleven phenolic compounds 117

(quercetin, protocatechuic acid, chrysin, rutin, hesperetin, naringenin, rosmarinic acid, 118

vanillin, p-coumaric acid, caffeic acid and chlorogenic acid) were quantified in S. sediforme. 119

In the chromatographic analysis of phenolic compounds, gradient seperation was applied. 120

Linear regression equations of the phenolic compound standards were represented in Table 1. 121

Page 7 of 33



7

The linearity of the LC-MS/MS conditions for phenolic compounds was affirmed in the range 122

from 0.025 to 1 mg/L by the high coefficient of determination (R2 > 0.999) obtained. The 123

limit of detection (LOD) and limit of quantitation (LOQ) of the method reported in this study 124

were dependent on the calibration curve established from six measurements. LOD and LOQ 125

of the method were determined by using the equations 3S/N and 10S/N respectively (S/N 126

refers to the Signal to noise ratio) (Table 1). For different compounds, LOD ranged from 0.53 127

to 0.93 µg/L and LOQ ranged from 1.59 to 2.86 µg/L (Table 1). Furthermore, the recovery of 128

the phenolic compounds standards ranged from 91.2% to 102.3%. 129

Estimation of Uncertainty. Identification of uncertainty sources. The sources of the 130

uncertainty for the applied method were evaluated and calculated using EURACHEM Guide, 131

2004.17,20,21 132

The following parameters were used for the calculations of uncertainties: 133

1. Calibration curve (cal), 134

2. Purity of reference standards (pur) 135

3. Stock solutions (Css) 136

4. Weighing of samples (msample) 137

5. Repeatabilitity (rep) 138

6. Recovery (rec) 139

Standard combined uncertainty is a function of the individual uncertainties of each 140

parameter and calculated by using equation (1): 141

)()()()()()()( 222222recurepuwuCssupurucaluCu ++++++= (1) 142

Main uncertainty sources defined as purity of standards and calibration curve. 143

Standard combined uncertainties multiplied by two for the calculation of expanded 144

Page 8 of 33



8

uncertainties by accepting 95% confidence level. Calculated uncertainties are shown in 145

Table 1. 146

Preparation of plant extracts for LC-ESI-MS/MS. For sample preparation, initially, 147

the whole parts of the dried and powdered plants (10 g) were extracted by MeOH (3 × 50) mL 148

in 24 h at room temperature (Extraction yield: 8.3%). The extract was subsequently filtered 149

and evaporated under reduced pressure. Then, dry filtrate diluted until 250 mg/L and passed 150

through the microfiber filter 0.2 µm for LC-ESI-MS/MS. 151

Esterification of total fatty acids and GC/MS conditions. Esterification of 152

petroleum ether extract (100 mg) of S. sediforme was performed according to the report of 153

Kılıc et al.22 In this study, Thermo Scientific Polaris Q GC/MS was used. GC/MS study 154

conditions and comparison of identification and quantification of the compounds were done 155

exactly same manner according to Kılıc et al.22 156

Preparation of plant extracts for biological activities and GC/MS. In this analysis, 157

primarily, whole plant material was dried and powdered, 100 g of plant material was 158

sequentially macerated with petroleum ether (3 × 250 mL), acetone (3 × 250 mL), methanol (3 159

× 250 mL) and water (3 × 250 mL) for 24 h at room temperature. Subsequent to filtration, the 160

solvents were evaporated to handle the crude extracts. 161

Preparation of essential oil and GC/MS conditions. Essential oil was obtained using 162

a Clevenger apparatus from the whole parts of plant (100 g), which was crumbled into small 163

pieces and soaked in distilled water (500 ml) for 3 h. The obtained essential oil was dried over 164

anhydrous Na2SO4 and stored at +4 °C for a sufficient period of time. The essential oil was 165

diluted using CH2Cl2 (1:3 volume/volume) prior to GC/FID (Gas Chromatography/Flame 166

Ionization Detector) and GC/MS analysis. GC/FID analysis was performed using Thermo 167

Page 9 of 33



9

Electron Trace GC/FID detector and GC/MS analysis was performed using the same GC and 168

Thermo Electron DSQ MS. 169

The following GC conditions were applied for both GC/MS and GC/FID analyses. 170

The GC oven temperature was kept at 60 °C for 10 min and programmed to 280 °C at a rate 171

of 4 °C/min and then kept constant at 280 °C for 10 min. A nonpolar Phenomenex DB5 fused 172

silica column (30 m × 0.32 mm, 0.25 µm film thickness) was used with helium at 1 mL/min 173

(20 psi) as a carrier gas. The split ratio was adjusted to 1:50, the injection volume was 0.1 µL, 174

and EI/MS was recorded at 70 eV ionization energy. The mass range was m/z 35–500 amu. 175

Alkanes (C8-C24) were used as reference points in the calculation of Kovats Indices (KI) by 176

the same conditions.23,24 177

Identification of the compounds was based on comparing their retention times and 178

mass spectra with those obtained from authentic samples and/or the NIST and Wiley spectra 179

as well as data from the published literature. GC/FID and GC/MS analyses were replicated 180

three times (Mean RSD% < 0.1). 181

Determination of total phenolic and flavonoid contents. Total phenolic and 182

flavonoid amounts in the crude extracts expressing as pyrocatechol and quercetin equivalents, 183

respectively, were calculated according to the following equations:25,26 184

Absorbance = 0.0126 pyrocatechol (µg) + 0.0314 (R2 = 0.9936) 185

Absorbance = 0.1495 quercetin (µg) – 0.0958 (R2 = 0.9994) 186

Antioxidant activity of the extracts. In order to determine the antioxidant activity, 187

the following tests were applied: β-carotene-linoleic acid test system, DPPH free radical and 188

ABTS cation radical scavenging activity and cupric reducing antioxidant capacity (CUPRAC) 189

methods.27-30 190

191

Page 10 of 33



10

Anticholinesterase activity of the extracts. A spectrophotometric method developed 192

by Ellman et al. was used to indicate the acetyl- and butyryl-cholinesterase inhibitory 193

activities.31 194

Determination of Antimicrobial activity and Minimum Inhibition concentration 195

(MIC). Five different microorganisms including gram positive bacteria (Streptococcus 196

pyogenes ATCC19615 and Staphylococcus aureus ATCC 25923), gram negative bacteria 197

(Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922) and yeast (Candida 198

albicans ATCC10231) which were purchased from Refik Saydam Sanitation Center (Turkey) 199

were used for detecting the antimicrobial activity of the samples. The disc diffusion method 200

was employed for this purpose.32 The minimum inhibition concentration determined by the 201

broth macrodilution method according to NCCLS.33 Ampicillin and fluconazole were used as 202

positive controls for bacteria and yeast, respectively. 203

Statistical analysis. The results of the antioxidant and anticholinesterase activity assays 204

were represented as means ± SD. The results were evaluated using an unpaired t-test and 205

ANOVA variance analysis with the NCSS statistical computer package. The differences were 206

considered statistically significant at p < 0.05. 207

� RESULTS AND DISCUSSION 208

Quantitative analysis of phenolic and flavonoid compounds by UHPLC-ESI 209

(QqQ)/MS/MS. Having the same general structure with an aromatic hydroxyl nucleus, 210

phenolic compounds exist in nature with a number of almost 8000.17 Phenolic compounds that 211

exist in plants constitute one of the most important groups acting as free radical terminators 212

and primary antioxidants. Plant polyphenols are multifunctional in a way that they act as 213

reducing agents, hydrogen atom donors and singlet oxygen scavengers. Besides, being the 214

most diverse and prevalent natural compounds, flavonoids are the most important phenolics. 215

Page 11 of 33



11

Being a member of polyphenol family, flavonoids have more than 4000 species which exist in 216

the roots, flowers and leaves of the plants.17 217

In a literature survey, it can be found that there are several reports about the use of liquid 218

chromatography electrospray ionization tandem mass spectrometry to perform quantitative 219

analyses.17,34 Therefore, for quantitative purpose, the analyses of eleven phenolic and 220

flavonoid compounds in the methanol extract of S. sediforme were done by an accurate 221

method on a mass spectrometer equipped with a triple quadrupole analyzer. Due to the fact 222

that negative ionization mode was more sensitive and selective for phenolics and flavonoids, 223

it was preffered in the current study. 224

The specific fragmentation reactions were selected in order to monitor the aforesaid 225

phenolic and flavonoid compounds by MRM. Eleven compounds which were five flavonoids, 226

five phenolic acids and one phenolic aldehyde were monitored by the transition from the 227

specific deprotonated molecular ions to the corresponding fragment ions. Molecular ions, 228

fragments observed in MS/MS, related collision energies for these fragments and the 229

quantified result for S. sediforme was presented in Table 2. 230

Quercetin, rutin and naringenin were detected and quantified out of five flavonoids; 231

however, chrysin, hesperetin and vanillin were not found. Furthermore, four phenolic acids 232

(protocatechuic acid, p-coumaric acid, caffeic acid and chlorogenic acid) were characterized 233

in S. sediforme (Table 2, Figure 1, Figure S1 in the Supporting Information). Quercetin was 234

found to be the most abundant flavonoid compound (1813.51±137.82 µg/g extract) in the 235

methanol extract of S. sediforme (Table 2, Figure 1, 3). Besides, caffeic acid (151.25±8.93 236

µg/g extract) was found to be the most plentiful phenolic acid in S. sediforme (Table 2, Figure 237

1). 238

In literature, there are few studies on phenolic and flavonoid compounds of Sedum 239

species by HPLC and LC-MS techniques.16,35 Xu et al. reported a HPLC methodology that 240

Page 12 of 33



12

was established to determine quercetin which is a common hydrolyzate of the flavonoid 241

glycosides in the S. sarmentosum, S. lineare and S. erythrostictum.16 242

Fatty acid and essential oil composition by GC/MS. GC/MS analysis was used to 243

determine the fatty acid composition of the petroleum ether extract. As represented in Table 3, 244

ten components were identified, constituting 100.0% of the petroleum ether extract of S. 245

sediforme, and the major constituents were characterized as palmitic acid (C16:0) (28.8%), 246

stearic acid (C18:0) (24.6%), and linolenic acid (C18:3 omega-3) (12.9%). This is the very 247

first report on the fatty acid composition of S. sediforme. The amount of saturated fatty acids 248

was found more than the amount of unsaturated fatty acid in the present study. There has been 249

no reports regarding direct fatty acid analysis of Sedum species by GC/MS, except the 250

lipophilic extract of obtained from Sedum hispanicum.36 But, in this study, 0.89% of the fatty 251

acid content was identified in the lipophilic extract of S. hispanicum. In that sense, it could be 252

said that current study is the first report on the fatty acid composition of Sedum species. 253

The essential oil composition was examined by GC/MS analysis. Twenty-four 254

components were determined, constituting 91.6% of the essential oil composition of S. 255

sediforme. The main components of the essential oil of S. sediforme were identified as α-256

selinene (20.4%), 2,5-di-tert octyl-p-benzoquinone (13.1%), valencene (6.3%) and carvone 257

oxide (4.3%) (Table 4). There have been few reports regarding GC/MS analysis of essential 258

oil composition of Sedum species.37,38 Yaylı et al. reported that thirty-eight and thirty-five 259

components were identified in the essential oils of S. pallidum var. bithynicum and S. spurium 260

respectively. Besides, in their study, the main components of these species were found to be 261

caryophyllene oxide for S. pallidum var. bithynicum and hexahydrofarnesyl acetone for S. 262

spurium in the ratios of 12.8% and 15.7%, respectively.38 263

Antioxidant activity and total phenolic and flavonoid content. The antioxidant 264

activity studies of the petroleum ether (SSP), acetone (SSA), methanol (SSM) and water 265

Page 13 of 33



13

(SSW) extracts prepared from the whole plant of S. sediforme were carried out by β-carotene 266

bleaching, DPPH free radical scavenging, ABTS cation radical decolorisation and cupric 267

reducing antioxidant capacity (CUPRAC) assays. The SSM extract showed the highest 268

extraction yield, but no significant differences in the extraction yields of other extracts were 269

observed. 270

In the crude extracts, total phenolic and flavonoid amounts were determined 271

expressing as pyrocatechol and quercetin equivalents, respectively (y = 0.0126 pyrocatechol 272

(µg) + 0.0314, R2 = 0.9936 and y = 0.1495 quercetin (µg) – 0.0958, R2 = 0.9994). The 273

phenolic and flavonoid amounts of the SSM extract were identified to be the richest. The 274

amounts of total phenolic and flavonoid from SSM were 335.71 ± 4.81 µg/mg extract and 275

26.66 ± 0.75 µg/mg extract, respectively. The amount of phenolic components were seen to be 276

higher than that of flavonoid components. The results were shown in Table 5. In the literature, 277

total phenolic and flavonoid content in the examined Sedum acre extracts were expressed in 278

terms of gallic acid and rutin equivalents respectively.14 Moreover, Stankovic et al. reported 279

that the total phenolic and flavonoid amounts in the examined acetone extract of S. acre were 280

181.75 mg/g and 173.42 mg/g respectively.14 Additionally, in the report of Romojaro et al. S. 281

sediforme showed high levels of total phenolic content, 191.53 mg /100 g fresh weight (gallic 282

acid equivalent).5 With this regard, the richness of Sedum species in terms of phenolic and 283

flavonoid compounds that are known for their important pharmacological properties, 284

increases the importance of these species. 285

As indicated in Table 6, the SSP and SSA extracts showed moderate lipid peroxidation 286

activity (IC50: 51.34 ± 0.92 and 54.61 ± 0.31 µg/mL, respectively) and the SSW extract 287

showed weak lipid peroxidation activity (153.05 ± 1.71 µg/mL) in β-carotene bleaching 288

method. However, the SSM extract showed very strong lipid peroxidation activity (9.78 ± 289

0.06 µg/mL) in β-carotene bleaching method. Furthermore, the SSM extract exhibited higher 290

Page 14 of 33



14

activity than α-tocopherol (15.54 ± 0.21 µg/mL) and BHT (10.35 ± 0.03 µg/mL), that were 291

used as standards in the β-carotene bleaching method. As seen in Table 6, the SSP and SSW 292

extracts showed weak and moderate activity (174.55 ± 0.91 and 104.45 ± 1.28 µg/mL) in 293

DPPH free radical scavenging activity, respectively. On the other hand, the SSA and SSM 294

extracts exhibited very strong activity in DPPH free radical scavenging activity. Besides, the 295

SSA (17.20 ± 0.33 µg/mL) and SSM (9.07 ± 0.07 µg/mL) extracts showed higher activity than 296

α-tocopherol (18.76 ± 0.31 µg/mL) and BHT (48.86 ± 0.09 µg/mL). In the previous studies, 297

Mavi et al. Reported that S. sempervivoides showed very strong activity (88.9 and 86.0% 298

inhibition, respectively) in DPPH free radical scavenging assay and lipid peroxidation-299

thiobarbituric acid method at 200 µg/mL concentration.13 In addition, Stankovic et al. reported 300

that the acetone extract of S. acre exhibited very strong activity in DPPH free radical 301

scavenging assay.14 The largest capacity to neutralize DPPH radicals was found for the 302

acetone extract, which neutralized 50% of free radicals at the concentration of 29.57 µg/mL.14 303

Morover, in the study of Thuong et al., the MeOH, EtOAc and BuOH-soluble fractions 304

exhibited significant scavenging activities against free radicals (DPPH and superoxide) as 305

well as remarkable inhibitory effects on lipid peroxidation.10 306

As shown in Table 6, the SSP extract indicated moderate activity (75.03 ± 0.45 µg/mL) 307

in ABTS cation radical scavenging assay. However, the SSA, SSM and SSW extracts 308

exhibited very strong effects in ABTS cation radical scavenging assay. In addition to that, the 309

SSA (8.76 ± 0.52 µg/mL), SSM (5.87 ± 0.03 µg/mL) and SSW (9.01 ± 0.29 µg/mL) extracts 310

showed higher activity than α-tocopherol (9.88 ± 0.08 µg/mL) and BHT (10.67 ± 0.11 311

µg/mL). According to Romojaro et al., S. sediforme showed very strong effects in hydrophilic 312

and lipophilic total antioxidant activities, with 588.87 ± 35.52 mg of Trolox equivalent 100 g-313

1 FW.5 Morover, S. sediforme showed good activity (81.60% inhibition) in peroxyl radical 314

(H2O2) scavenging potential assay.5 The SSM extract and α-tocopherol indicated 1.73 and 315

Page 15 of 33



15

1.62 absorbance in CUPRAC at 100 µg/mL, respectively (Figure 2). According to our 316

literature survey this is the first study about cupric reducing antioxidant capacity of Sedum 317

species. Therefore this study is important in this field. 318

When we look at the antioxidant results of the four tested extracts, we can see the 319

parallelism between antioxidant activities and total phenolic content. In particular, looking at 320

the quantitative phenolic analysis of the methanol extract by LC-MS/MS, it can be deduced 321

that this high activity may be related to the quercetin content. Quercetin is known to have 322

various pharmacological effects. 17-19 Particularly, flavonoids are present in plant sources as 323

flavonoid glycosides. Therefore, this high activity also might be said to arise from quercetin 324

gycosides. If we were to express in a different way, this high activity can be attributed to the 325

synergic effect between quercetin, quercetin glycosides and other phenolic compounds.39 In 326

further studies, our group plan to purify, structurally determine and quantify the secondary 327

metabolites, especially flavonoid glycosides of Sedum species. Furthermore, we also aim to 328

study in vivo pharmacological effects of the purified compounds. 329

Anticholinesterase activity. As demonstrated in Table 5, the SSA extract exhibited 330

good inhibitory activity (65.69% inhibition) against butyryl-cholinesterase enzyme. The SSW 331

extract showed moderate inhibitory activity (40.61% inhibition) against acetyl-cholinesterase 332

enzyme, at 200 µg/mL. On the other hand, the SSM extract showed 85.09 and 89.57% 333

inhibition activity which is higher than galanthamine inhibitory activity against acetyl- and 334

butyryl-cholinesterase enzymes at 200 µg/mL, respectively. 335

The anticholinesterase activity of the SSM extract shows parallelism to its high total 336

phenolic ve flavonoid contents. This high activity of the SSM extract might be related not 337

only to its high total phenolic ve flavonoid contents but also to its quercetin content directly or 338

to the synergic effect of quercetin with other phenolic compounds.39 Because, when previous 339

studies are examined, it can be seen that quercetin has strong antioxidant properties as well as 340

Page 16 of 33



16

anticholinesterase effect.17-19 Additionally, Min et al. reported that quercetin had showed 341

potential inhibitory activity against AChE.18 Furthermore, Choi et al. reported that 342

quercetin might improve cognitive ability against TMT-induced neuronal deficit and also had 343

an inhibitory action against AChE.19 To our knowledge, there is not any reports about the 344

anticholinesterase activity of Sedum species. Taking into account that our results were higher 345

than galanthamine and there has been no such study about Sedum species, the 346

anticholinesterase activity results of S. sediforme will be important data in this field. 347

Antimicrobial activity. The antimicrobial activities of S. sediforme extracts against 348

different microorganisms were measured by disc diffusion method and the results were 349

assessed according to inhibition zone diameter. Results are presented in Table 7. No 350

antimicrobial activity of the water extract against the five tested microorganisms was detected 351

(Data not shown). However the petroleum ether, acetone and methanol extracts were active on 352

tested microorganisms and the sensitivity of active extracts was found not to differ 353

significantly among tested microorganisms. While the petroleum ether extract exhibited weak 354

antimicrobial activity (inhibition zone < 12) against all tested microorganisms, the acetone 355

and methanol extracts showed moderate antimicrobial activity against C. albicans (inhibition 356

zone < 20-12) and weak antimicrobial activity against Gram positive/negative bacteria. The 357

highest activities were exhibited by acetone and methanol extracts against C. albicans with 18 358

± 0 mm and 15 ± 0.1 mm inhibition zone diameter, respectively. 359

For a more reliable assessment of antimicrobial activity, a broth dilution assay was 360

carried out. The sensitivity of the test microorganisms against active extracts was evaluated 361

and results were shown as MIC (Table 7). Values ranged from 8–19 mg/mL for the petroleum 362

ether extract, 1–17 mg/mL for the acetone extracts and 1–9 mg/mL for the methanol extracts. 363

The MIC results indicate that the methanol extract was found to be the most active extract. 364

The lowest MIC value was recorded by the acetone and methanol extracts against C. albicans 365

Page 17 of 33



17

(1 mg/mL). This is the first study dealing with antimicrobial activity of S. sediforme. On the 366

other hand, in a previous report, the methanol, acetone and ethyl acetate extracts of S. acre 367

revealed high antibacterial activity against gram positive bacteria and low to moderate 368

antifungal activity. 14 Besides, the essential oil of S. pallidum Bieb. var. bithynicum and S. 369

spurium showed low antimicrobial activity against Gram-negative/positive bacteria and yeast-370

like fungi38 and S. sormentosum Bunge showed weak inhibitory activity against B. subtilis 371

and S. aureus.40 372

The present study concluded that the methanol extract of S. sediforme showed very 373

strong antioxidant and anticholinesterase activities. These properties of the methanol extract 374

of S. sediforme were parallel to the total phenolic content. Based on our results, quercetin was 375

found to be the most abundant phenolic compound in S. sediforme. Many studies in literature 376

showed that quercetin and its glycosides had potent biological properties, in particular 377

antioxidant and anticholinesterase activities. Thus, these high activities of S. sediforme may 378

be related to either high total phenolic or quercetin contents.17-19,41 From a broader 379

perspective, these high activities of S. sediforme might be related not only to its high total 380

phenolic and flavonoid contents but also directly to its quercetin content or to the synergic 381

effect of quercetin with other phenolic compounds.39 382

Furthermore, protocatechuic, p-coumaric and chlorogenic acids were found for the 383

first time in Sedum species. Although the total phenolic content was found as very rich in S. 384

sediforme, phenolic constituents were detected in low amounts. Therefore, these results could 385

be the effect of some other phenolic constituents such as flavonoid glycosides that we have 386

not studied yet. 387

In conclusion, it is found that S. sediforme had very high antioxidant and 388

anticholinesterase activities. Therefore, the results of the current study showed that the 389

Page 18 of 33



18

methanol extract of S. sediforme can also be used as a food source because of its high 390

quercetin amount, total phenolic content, strong antioxidant and anticholinesterase properties. 391

All in all, rich total phenolic content and high antioxidant and anticholinesterase 392

capacity of the methanol extract of S. sediforme indicated that more future studies should be 393

done in this field. 394

� AUTHOR INFORMATION 395

*Corresponding Author 396

A. Ertaş, Dicle University, Faculty of Pharmacy, Department of Pharmacognosy, 21280 397

Diyarbakir, Turkey. Phone: +90 412 2488030/7512. Email: [email protected]; 398

[email protected]. 399

Funding 400

We acknowledge the Dicle University for financial support (Research University Grant 401

DUBAP: 13-ASMYO-61). 402

Notes 403

The authors declare no competing financial interest. 404

� ABBREVIATIONS USED 405

LC-MS/MS, Liquid Chromatography-Tandem Mass Spectrometry; GC-MS, Gas 406

Chromatography-Mass Spectrometry; GC/FID, Gas Chromatography/Flame Ionization 407

Detector; UHPLC-ESI-MS/MS, Ultra High Performance Liquid Chromatography-408

Electrospray Ionisation-Tandem Mass Spectrometry; ABTS, 2,2′-azinobis(3-409

ethylbenzothiazoline-6-sulfonic acid) diammonium salt; BHT, butylated hydroxytoluene; 410

DPPH, 2,2-diphenyl-1-picrylhydrazyl; DTNB, 5,5-dithiobis-(2-nitro benzoic acid); 411

CuCl2.2H2O, copper (II) chloride dihydrate; neocuproine, 2,9-dimethyl-1,10-phenanthroline; 412

EDTA, ethylenediaminetetraacetic acid; APCI, Atmospheric Pressure Chemical Ionization; 413

APPI, Atmospheric Pressure Photoionization; LOD, limit of detection; LOQ, limit of 414

Page 19 of 33



19

quantitation; CUPRAC, cupric reducing antioxidant capacity; BChE, butyrylcholinesterase 415

enzyme; AChE, acetylcholinesterase enzyme; MIC, minimum inhibition concentration; 416

NCCLS, National Committee for Clinical Laboratory Standards; SD,Standard Deviation; 417

MRM, Multiple Reaction Monitoring; SSP, the petroleum ether extract of S. sediforme; SSA, 418

acetone extract of S. sediforme; SSM, methanol extract of S. sediforme, SSW, water extract of 419

S. sediforme. 420

ACKNOWLEDGMENTS 421

Thanks to Dicle University Science and Technology Research and Application Center 422

(DÜBTAM) for the partial support in this study. 423

� ASSOCIATED CONTENT 424

*S Supporting Information 425

Figure S1. Identified compounds from S. sediforme. Figure S2. LC-MS/MS TIC 426

chromatogram for 250 µg/L standard mix. Figure S3. LC-MS/MS TIC chromatogram for 427

methanol extract of S. sediforme. Figure S4 and S5. Total ion current chromatogram (TIC) of 428

fatty acid and essential oil of S. sediforme, respectively. Equations S1-7. equations related to 429

the calculations of uncertainty parameters. The antioxidant and anticholinesterase activities 430

methods were given. Table S1-S14. The quantification and statistical results of total phenolic 431

and total flavonoid contents, antioxidant and anticholinesterase activities. This material is 432

available free of charge via the Internet at http://pubs.acs.org. 433

434

� REFERENCES 435 1) Alpınar, K. Sedum L. In Türkiye Bitkileri Listesi/Damarlı Bitkiler, Güner, A.; Aslan, 436

S.; Ekim, T.; Vural, M.; Babaç, M.T., Eds.; Nezahat Gökyiğit Botanik Bahçesi ve 437

Flora Araştırmaları Derneği Yayını: Istanbul, Turkey, 2012; pp. 384-386. 438

Page 20 of 33



20

2) Chamberlain, D. F. Sedum L. In Flora of Turkey and the East Aegean Islands, Davis 439

P.H., Ed.; Edinburgh University Press: Edinburgh, Scotland, 1972; 4, pp. 224-244. 440

3) Baser, K. H. C. The Medicinal Plants of Korean; Kyo-Hak Publishing Co.: Seoul, 441

Korea, 1999; pp. 198-203. 442

4) Baytop, T. Tükçe Bitki Adları Sözlüğü; Türk Tarih Kurumu Basımevi: Ankara, 443

Turkey, 1994; p.163. 444

5) Romojaro, A.; Botella, M. A.; Obon, C.; Pretel, M. T. Nutritional and antioxidant 445

properties of wild edible plants and their use as potential ingredients in the modern 446

diet. Int. J. Food Sci. Nutr. 2013, 64(8), 944-952. 447

6) Elmadfa, I. In Local Mediterranean Food Plants and Nutraceuticals, Heinrich, M.; 448

Müller, W. E.; Galli, C., Eds.; Karger Medical and Scientific Publishers: Basel, 449

Sweden, 2006; 59, p. 49. 450

7) Andry, H.; Yamamoto, T.; Inoue, M. Effectiveness of hydrated lime and artificial 451

zeolite amendments and Sedum (Sedum sediforme) plant cover in controlling soil 452

erosion from an acid soil. Aust. Soil Res. 2007, 45(4), 266-279 453

8) Li, W. L.; Luo, Q. Y.; Wu, L. Q. Two new prenylated isoflavones from Sedum aizoon 454

L. Fitoterapia 2011, 82(3), 405-407. 455

9) Stanislaw, G.; Wanda, D. M.; Joanna, K. Occurrence of arbutin and hydroquinone in 456

the genus Sedum L. Farm. Pol. 1984, 40, 211-213. 457

10) Thoung, P. T.; Kang, J. H.; Na, M.; Jin, W.; Youn, U. J.; Seeong, Y. H.; Song, K. S.; 458

Min B. K.; Bae, K. Anti-oxidant constituents from Sedum takesimense. 459

Phytochemistry 2007, 68(19), 2432-2438. 460

11) Morikawa, T.; Ninomiya, K; Zhang, Y.; Yamada, T.; Nakamura, S.; Matsuda, H.; 461

Muraoka, O.; Hayakawa, T.; Yoshikawa, M. Flavonol glycosides with lipid 462

Page 21 of 33



https://www.researchgate.net/publication/255953866_Nutritional_and_antioxidant_properties_of_wild_edible_plants_and_their_use_as_potential_ingredients_in_the_modern_diet?el=1_x_8&enrichId=rgreq-99c90c11-f9c8-4198-a405-4caa677692e7&enrichSource=Y292ZXJQYWdlOzI2MTk1MjQ4NTtBUzoxMDQ4NzgyMjMzOTY4NzVAMTQwMjAxNjMyNTg4MA==





https://www.researchgate.net/publication/49640226_Two_new_prenylated_isoflavones_from_Sedum_aizoon_L?el=1_x_8&enrichId=rgreq-99c90c11-f9c8-4198-a405-4caa677692e7&enrichSource=Y292ZXJQYWdlOzI2MTk1MjQ4NTtBUzoxMDQ4NzgyMjMzOTY4NzVAMTQwMjAxNjMyNTg4MA==



https://www.researchgate.net/publication/290890873_Occurrence_of_arbutin_and_hydroquinone_in_the_genus_Sedum_L?el=1_x_8&enrichId=rgreq-99c90c11-f9c8-4198-a405-4caa677692e7&enrichSource=Y292ZXJQYWdlOzI2MTk1MjQ4NTtBUzoxMDQ4NzgyMjMzOTY4NzVAMTQwMjAxNjMyNTg4MA==



21

accumulation inhibitory activity from Sedum sarmentosum. Phytochem. Lett. 2012, 463

5(1), 53-58. 464

12) Gill, S.; Raszeja W.; Szynkiewicz, G. Occurrence of nicotine in some species of the 465

genus Sedum. Farm. Pol. 1979, 35, 151-153. 466

13) Mavi, A.; Terzi, Z.; Ozgen, U.; Yıldırım, A.; Coskun, M. Antioxidant properties of 467

some medicinal plants: Prangos ferulacea (Apiaceae), Sedum sempervivoides 468

(Crassulaceae), Malva neglecta (Malvaceae), Cruciata taurica (Rubiaceae), Rosa 469

pimpinellifolia (Rosaceae), Galium verum subsp. verum (Rubiaceae), Urtica dioica 470

(Urticaceae). Biol. Pharm. Bull. 2004, 27(5), 702-705. 471

14) Stankovıć, M.; Radojevıć, I.; Ćurčıć, M.; Vasıć, S.; Topuzovıć, M.; Čomıć, L.; 472

Markovıć, S. Evaluation of biological activities of goldmoss stonecrop (Sedum acre 473

L.). Turk. J. Biol. 2012, 36(5), 580-588. 474

15) Sakar, M. K.; Petereit, F.; Nahrstedt, A. Two phloroglucinol glucosides, flavan 475

gallates and flavonol glycosides from Sedum Sediforme flowers. Phytochemistry 1993, 476

33(1), 171-174. 477

16) Xu, R.; Chen, Y. J.; Wan D. R.; Wang, J. HPLC determination of quercetin in three 478

plant drugs from genus Sedum, In Proceedings of 2009 International Conference of 479

Natural Product and Traditional Medicine, Liu, J., Vittori, S., Yang, C., Eds.; Xian, 480

China, 2009; 1&2, 643-646. 481

17) Gulcin, I.; Bursal, E,; Sehitoglu, H. M.; Goren, A. C. Polyphenol contents and 482

antioxidant activity of lyophilized aqueous extract of propolis from Erzurum, Turkey. 483

Food Chem. Toxicol. 2010, 48(8-9), 2227-2238. 484

18) Min, B. S.; Cuong, T. D.; Lee, J. S.; Shin, B. S.; Woo, M. H.; Hung, T. M. 485

Cholinesterase inhibitors from Cleistocalyx operculatus Buds. Arch. Pharm. Res. 486

2010, 33(10), 1665-1670. 487

Page 22 of 33



22

19) Choi, G. N; Kim, J. H.; Kwak, J. H.; Jeong, C. H.; Jeong, J. H.; Jeong, H. R.; Lee, U.; 488

Heo, H. J. Effect of quercetin on learning and memory performance in ICR mice 489

under neurotoxic trimethyltin exposure. Food Chem. 2012, 132(2), 1019-1024. 490

20) Binici, B.; Bilsel, M.; Karakas, M.; Koyuncu, I.; Goren, A. C. An efficient GC-IDMS 491

method for determination of PBDEs and PBB in plastic materials. Talanta 2013, 116, 492

417-426. 493

21) EURACHEM CITAC Guide CG4. Quantifiying Uncertainty in Analytical 494

Measurement, Third edition. Editors: Ellison, S. L. R. (LGC, UK); Williams, A. (UK) 495

2004. 496

22) Kılıc, T.; Dirmenci, T.; Goren, A. C. Chemotaxonomic evaluation of species of 497

Turkish Salvia: fatty acid composition of seed oils. II. Rec. Nat. Prod. 2007, 1(1), 17-498

23. 499

23) Altun, M.; Goren, A. C. Essential oil composition of Satureja cuneifolia by 500

simultaneous distillation-extraction and thermal desorption GC-MS techniques. J. 501

Essent. Oil Bear. Pl. 2007, 10(2), 139-144. 502

24) Polatoglu, K.; Demirci, B.; Demirci, F.; Goren, N.; Baser, K. H. C. The essential oil 503

composition of Tanacetum densum (Labill.) Heywood ssp eginense Heywood from 504

Turkey. Rec. Nat. Prod. 2012, 6(4), 402-406. 505

25) Slinkard, K.; Singleton, V. L. Total phenol analyses: Automation and comparison with 506

manual methods. Am J. Enol. Viticult. 1977, 28(49), 49-55. 507

26) Moreno, M. I. N.; Isla, M. I.; Sampietro, A. R.; Vattuone, M. A. Comparison of the 508

free radical-scavenging activity of propolis from several regions of Argentina. J. 509

Ethnopharmacol. 2000, 71(1-2), 109-114. 510

27) Miller, H. E. A simplified method for the evaluation of antioxidants. J Am Oil Chem 511

Soc. 1971, 48, 91. 512

Page 23 of 33



23

28) Blois, M. S. Antioxidant determinations by the use of a stable free radical. Nature 513

1958, 181, 1199-1200. 514

29) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C.; 515

Antioxidant activity applying an improved ABTS radical cation decolorization assay. 516

Free Radical Bio. Med. 1999, 26(9-10), 1231-1237. 517

30) Apak, R.; Guclu, K.; Ozyurek, M.; Karademir, S. E. Novel total antioxidant capacity 518

index for dietary polyphenols and vitamins C and E, using their cupric ion reducing 519

capability in the presence of neocuproine: CUPRAC Method. J. Agric. Food. Chem. 520

2004, 52(26), 7970-7981. 521

31) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. A new and rapid 522

colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 523

1961, 7, 88-95. 524

32) NCCLS (National Committee for Clinical Laboratory Standards). Performance 525

Standards for Antimicrobial Disk Susceptibility Test. 6th ed. Approved Standard, 526

Wayne Pa. M2-A6, 1997. 527

33) NCCLS (National Committee for Clinical Laboratory Standards). Methods for 528

Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; 529

Approved Standard 8th ed. Wayne Pa. M08-A8 2009. 530

34) Onder, F. C.; Ay, M.; Sarker, S. D. Comparative study of antioxidant properties and 531

total phenolic content of the extracts of Humulus lupulus L. and quantification of 532

bioactive components by LC−MS/MS and GC−MS. J. Agric. Food Chem. 2013, 533

61(44), 10498-10506. 534

35) Sturm, S.; Mulinacci, N.; Vincieri, E. E.; Stuppner, H. Analysis of flavonols of Sedum 535

telephium L. leaves by capillary electrophoresis and HPLC-Mass spectrometry. 536

Chromatographia 1999, 50(7/8), 433-438. 537

Page 24 of 33



24

36) Orhan, I.; Deliorman-Orhan, D.; Ozcelik, B. Antiviral activity and cytotoxicity of the 538

lipophilic extracts of various edible plants and their fatty acids. Food Chem. 2009, 539

115(2), 701-705. 540

37) Mesicek, N.; Perpar, M. Essential oils from the orpine, Sedum maximum. Farm. Vestn. 541

1973, 24, 123-124. 542

38) Yaylı, N.; Yasar, A.; Yılmaz Iskender, N.; Yaylı, N.; Cansu, T. B.; Coskuncelebi, K.; 543

Karaoglu, S. Chemical constituents and antimicrobial activities of the essential oils 544

from Sedum allidum var. bithynicum and S. spurium grown in Turkey. Pharm. Biol. 545

2010, 48(2), 191-194. 546

39) Ginsburg, H.; Deharo, E. A call for using natural compounds in the development of 547

new antimalarial treatments-an introduction. Malaria J. 2011, 10(Suppl 1), S1. 548

40) Kim, S. J.; Cho, A. R.; Han, J. Antioxidant and antimicrobial activities of leafy green 549

vegetable extracts and their applications to meat product preservation. Food Control 550

2013, 29(1),112-120. 551

41) Rice-Evans, C. A.; Miller, N. J.; Paganga, G.; Structure antioxidant activity 552

relationships of favonoids and phenolic acids. Free Radical Bio. Med. 1996, 20(3), 553

933-956.and HPLC-Mass Spectrometry 554

Figure Captions 555

Figure 1. UHPLC-ESI-MS/MS chromatograms of A: 250 µg/mL standard mix, B: S. 556

sediforme methanol extract. 557

Figure 2. Cupric reducing antioxidant capacity of S. sediforme, α-tocopherol and BHT. 558

Table Captions 559

Table 1. Analytical parameters of UHPLC-ESI-MS/MS method. 560

Page 25 of 33



25

Table 2. Identification and quantification of phenolic compounds of methanol extract of 561

S. sediforme by UHPLC-ESI-MS/MS. 562

Table 3. GC/MS analysis of S. sediforme petroleum ether extract. 563

Table 4. Chemical composition of the essential oil from S. sediforme. 564

Table 5. Total phenolic and flavonoid contents, extraction yield and anticholinesterase 565

activity of S. sediforme extracts and galanthamine at 200 µg/mL. 566

Table 6. Antioxidant activity of S. sediforme extracts and standards. 567

Table 7. Zones of growth inhibition (mm) and MIC values showing the antimicrobial 568

activity of S. sediforme extracts compared to positive controls.569

Page 26 of 33



26

Table 1. Analytical parameters of UHPLC-ESI-MS/MS method

aRT: Retention time bR2: coefficient of determination cRSD: relative standard deviation dLOD/LOQ(µg/L): Limit of deteection/Limit of quantification eU (%): Percent relative uncertainty at 95% confidence level (k=2)

Table 2. Identification and quantification of phenolic compounds of methanol extract of S. sediforme

by UHPLC-ESI-MS/MS

Compound Parent ion (m/z)

a MS

2(CE)

b Quantification

(µg analyte/g extract)c

Quercetin 300,90 151.0 (22), 121.0 (26), 107.0 (29) 1813.51±137.82

Protocatechuic acid 152,90 108.9 (15), 90.9 (25) 71.23±7.22

Chrysin 252,90 62.9 (33),143.0 (28),107.0 (26) N.Dd

Rutin 609,00 300.1 (39), 271.0 (53) 138.62±8.17

Hesperetin 300,90 164.0 (24), 136.0 (30), 108.0 (37) N.D

Naringenin 270,90 151.0 (18), 119.0 (25), 107.0 (26) 39.67±1.38

Rosmarinic acid 358,90 132.9 (41), 161.0 (17) N.D

Vanillin 150,90 136.0 (17), 92.0 (21), 107.8 (26) N.D

p-Coumaric acid 162,90 119.1 (15), 92.9 (28) 94.08±4.33

Caffeic acid 178,90 134.9 (14), 134.0 (25) 151.25±8.93

Chlorogenic acid 353,00 191.1 (15), 84.8 (45) 23.30±2.18 aParent ion (m/z): Molecular ions of the standard compounds (mass to charge ratio)

bMS2(CE): MRM fragments for the related molecular ions (CE refers to related collision energies of the fragment ions) cValues in µg/g (w/w) of plant methanol extract dN.D: not detected

Analytes RTa Equation R2b RSD%c Linearity

Range

(mg/L)

LOD/LOQ(µg/L)d Recovery (%) U (%)e

Quercetin 5.369 f(x)=1829.35x+32208.90 0.99975 1.33 0.025-1.000 0.63/1.93 97.1 7.6

Protocatechuic acid 1.380 f(x)=783.913x+9758.52 0.99970 2.19 0.025-1.000 0.74/2.25 102.3 10.1

Chrysin 10.086 f(x)=520.665x+866.55 0.99968 3.59 0.025-1.000 0.60/1.85 98.4 5.9

Rutin 4.373 f(x)=788.005x-9096.62 0.99927 0.95 0.025-1.000 0.53/1.59 99.0 7.1

Hesperetin 6.445 f(x)=570.363x+2835.74 0.99972 2.16 0.025-1.000 0.68/2.10 101.2 6.5 Naringenin 6.112 f(x)=1298.14x-6598.05 0.99982 1.50 0.025-1.000 0.80/2.45 96.5 3.5

Rosmarinic acid 4.609 f(x)=168.107x-428.04 0.99959 2.60 0.025-1.000 0.93/2.86 91.2 4.9 Vanillin 4.207 f(x)=52.7804x+1089.93 0.99982 2.75 0.025-1.000 0.85/2.61 100.8 5.1

p-Coumaric acid 4.407 f(x)=69.0997x+264.38 0.99997 1.24 0.025-1.000 0.76/2.34 99.3 4.6

Caffeic acid 2.580 f(x)=2783.56x+45880.00 0.99950 1.10 0.025-1.000 0.66/2.01 95.9 5.9

Chlorogenic acid 1.661 f(x)=1448.01x+2695.90 0.99986 0.62 0.025-1.000 0.72/2.22 97.8 9.5

Page 27 of 33



27

Table 3. GC-MS analysis of S. sediforme petroleum ether extract

Rt (min)a Constituentsb Composition (%)c

9.69 Octanedioic acid 1.2±0.05

14.39 10-Undecenoic acid 1.2±0.06

18.60 Myristic acid 2.5±0.04

25.27 Palmitic acid 28.8±0.30

30.64 Linoleic acid 9.7±0.18

30.77 Oleic acid 12.6±0.12

30.86 Linolenic acid 12.9±0.15

31.54 Stearic acid 24.6±0.29

37.38 Arachidic acid 3.3±0.06

43.82 Behenic acid 3.2±0.10 Total 100.0

aRetention time (as minutes) bA nonpolar Phenomenex DB-5 fused silica colum cPercentage of relative weight Table 4. Chemical composition of the essential oil from S. sediforme

RIa Constituentsb

Composition (%)c

865 Isononane 2.1±0.02

954 Camphene 1.2±0.03

1193 Mrytenal 1.0±0.01

1197 Mrytenol 1.1±0.03

1249 1,3-Ditert butyl benzene 3.5±0.04

1276 Carvone oxide 4.3±0.04

1299 Carvacrol 1.3±0.01

1376 α-Copaene 1.8±0.02

1409 Longifolene 3.2±0.03

1442 Aromadendrene 3.5±0.03

1481 α-Curcemene 1.3±0.01

1484 Valencene 6.3±0.02

1498 α-Selinene 20.4±0.09

1677 Cadalene 2.1±0.03

1712 Curcumen-15-al 1.5±0.03

1746 2-Methyl heptadecane 4.0±0.02

1890 2-Methyl-1-hexadecanol 2.3±0.02

2109 Heneicosane 4.2±0.04

2156 1-Nonadecanol 2.1±0.03

2259 2,5-Di-tert octyl-p-benzoquinone 13.1±0.07

2366 Arachidic acid 2.6±0.05

2407 Tetracosane 4.2±0.03

2896 Choleic acid 2.1±0.04

2900 Nonacosane 2.4±0.01

Total 91.6 aRI Retention indices (DB-5 column) bA nonpolar Phenomenex DB-5 fused silica column cPercentage of relative weight

Page 28 of 33



28

Table 5. Total phenolic and flavonoid contents, extraction yield and anticholinesterase activity of extracts and galanthamine at 200 µg/mLx

Samples Inhibition %

against AChE Inhibition %

against BChE Phenolic content

(µg PEs/mg extract)z

Flavonoid content

(µg QEs/mg extract)t

Extraction Yield

% (W/W)

SSP NA 11.53± 0.20a 137.30± 0.85a 10.91± 0.14a 3.02

SSA 28.31± 1.12a 65.69± 2.91b 254.37± 2.30b 21.23± 0.71b 3.11

SSM 85.09± 0.21b

89.57± 0.86c 335.71± 4.81

c 26.66± 0.75c 7.20

SSW 40.61± 0.60c 12.32± 1.01a 184.92± 5.91d 15.93± 0.81d 2.31

Galanthaminey 79.91 ± 0.42d 81.21±0.59d

xValues expressed are means ± S.D. of three parallel measurements and values were calculated according to negative control. Values with different letters in the same column were significantly different (p < 0.05) yStandard drug zPes: pyrocatechol equivalents (y=0.0126x + 0.0314 R2=0.9936) tQes: quercetin equivalents (y=0.1495x – 0.0958 R2=0.9994) NA: Not active, W: Wight Table 6. Antioxidant activity of the extracts and standarts

IC50 (µg/mL)

Samples Lipid

Peroxidation

DPPH Free

Radical

ABTS Cation

Radical

SSP 51.34±0.92a 174.55±0.91a 75.03±0.45a

SSA 54.61±0.31b 17.20±0.33b 8.76±0.52b

SSM 9.78±0.06c

9.07±0.07c

5.87±0.03c

SSW 153.05±1.71d 104.45±1.28d 9.01±0.29b

α-TOC 15.54±0.21e 18.76±0.31e 9.88±0.08d

BHT 10.35±0.03f 48.86±0.09f 10.67±0.11e

Values are means ± S.D., n = 3, values with different letters in the same column were significantly different (p < 0.05)

Page 29 of 33



29

Table 7. Zones of growth inhibition (mm) and MIC values showing the antimicrobial activity of S. sediforme

extracts compared to positive controls

Petroleum ether extract Acetone extract Methanol extract Positive controls aDD MIC aDD MIC aDD MIC bDD MIC

Gram-positive

S.aureus 10±0.4 18±0.2 7±0.2 8±0.2 9±0.3 5±0.6 35±0.2 1.95±0.3

S.pyogenes 9±0.4 19±0.7 9±0.1 7±0.1 9±0.3 8±0.5 19±0.2 7.815±0.1

Gram-negative

E.coli 9±0.3 18±0.5 7±0.1 17±0.3 10±0.1 9±0.4 20±0.1 7.815±0.4

P.aeruginosa 7±0.2 8±0.5 10±0 5±0.5 10±0.2 6±0.2 NAc NA

Yeast C. albicans 8±0.3 8±0.4 18±0 1±0.2 15±0.1 1±0.3 30±0.3 3.125±0.2 aDD: Inhibition zone in diameter (mm) around the discs (6 mm) impregnated with 30 mg/mL of plant extracts bDD: Inhibition zone in diameter (mm) of positive controls that are ampicillin for bacteria and fluconazole for yeast. Minimum inhibitory concentration (MIC) values are given as mg/mL for plant extracts and µg/mL for antibiotics cNA: Not active

Page 30 of 33



30

Figure 1. UHPLC-ESI-MS/MS chromatograms of A: 250 µg/mL standard mix, B: S. sediforme methanol extract

A

B

Page 31 of 33



31

Figure 2. Cupric reducing antioxidant capacity of the S. sediforme, α-tocopherol and BHT

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0 10µg/mL 25µg/mL 50µg/mL 100µg/mL

Ab

sorb

an

ce

Concentration

SSP

SSA

SSM

SSW

BHT

α-TOC

Page 32 of 33



32

Table of Contents Graphic



Page 33 of 33



Documents

Chemical compositions by using LC-MS/MS and GC/MS and biological activities of Sedum sediforme (Jacq.) Pau