Article pubs.acs.org/JAFC
Chemical Compositions by Using LC-MS/MS and GC-MS and Biological Activities of 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 (DÜ BTAM), 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 ‡
S Supporting Information *
ABSTRACT: In this research, the chemical composition and biological activities of various extracts obtained from whole parts of Sedum sediforme (Jacq.) Pau were compared. The amounts of total phenolic and flavonoid components in crude extracts were determined by expression as pyrocatechol and quercetin equivalents, respectively. All of the extracts (petroleum ether, acetone, methanol, and water) obtained from S. sediforme showed strong antioxidant activity in four tested methods. Particularly, the IC50 values of the methanol extract, which was the richest in terms of total phenolic and flavonoid contents, were found to be lower than those of α-tocopherol and BHT in β-carotene bleaching (9.78 ± 0.06 μg/mL), DPPH free radical scavenging (9.07 ± 0.07 μg/mL), and ABTS cation radical scavenging (5.87 ± 0.03 μg/mL) methods. Furthermore, the methanol extract of S. sediforme showed higher inhibition activity than galanthamine against acetyl- and butyryl-cholinesterase enzymes. Also, acetone and methanol extracts exhibited moderate antimicrobial activity against Candida albicans. The main constituents of fatty acid and essential oil were identified as palmitic acid (C16:0) (28.8%) and α-selinene (20.4%), respectively, by GC-MS. In the methanol extract of S. sediforme, quercetin, rutin, naringenin, and protocatechuic, p-coumaric, caffeic, and chlorogenic acids were detected and quantified by LC-MS/MS. Results of the current study showed that the methanol extract of S. sediforme may also be used as a food supplement. KEYWORDS: Sedum sediforme, phenolic content, essential oil, fatty acid, antioxidant, anticholinesterase, antimicrobial, quercetin, LC-MS/MS, GC-MS
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INTRODUCTION Belonging to the Crassulaceae family, the genus Sedum L. comprises approximately 348 species in the world and 33 species in Turkey. Additionally, it is named Kayakoruğu and Damkoruğu in Anatolia.1,2 Sedum species have been known as both vegetables and folk medicines. They are used for the treatment of many diseases such as wounds, hemorrhoids, constipation, and foot fungi and as a laxative and diuretic.3−5 Being a Mediterranean element, Sedum sediforme (Jacq.) Pau is named Altın otu (goldherb) due to its yellow flowers and is used as a food4−6 and ornamental globally.7 Previous phytochemical studies indicated that Sedum species contained different natural compounds such as new isoflavone derivatives sedacins A and B,8 arbutin and hydroquinone,9 phenolic acids and flavonoids,10 flavonol glycosides and sarmenosides V−VII,11 and alkaloids.12 In addition, several studies exhibited that Sedum species had strong antioxidant potential.5,8,10,13,14 Several papers can be found on S. sediforme in the literature. In the study of Sakar et al., it is reported that two new © 2014 American Chemical Society
compounds, the structures of which were elucidated by spectroscopic means as (2R,3R)-7,4′-dihydroxy-5,3′,5′-trimethoxyflavan 3-O-gallate and 1-β-D-glucopyranosyloxy-3-methoxy-5-hydroxybenzene, were isolated from S. sediforme flowers.15 They were accompanied by limocitrin 3-glucoside, 1-β-D-glucopyranosyloxy-3,5-dihydroxybenzene, kaempferol 3rhamnoside, quercetin 3-rhamnoside, (−)-epicatechin 3-gallate, (−)-epigallocatechin 3-gallate, myricetin 3-rhamnoside, and gallic acid.15 In a former study, an HPLC method was established to determine quercetin, which is a common hydrolysate of the flavonoid glycosides in Sedum sarmentosum, Sedum lineare, and Sedum erythrostictum.16 Besides, Romojaro et al. reported that S. sediforme had a high phenol content and hydrophilic total antioxidant activity.5 These aforementioned studies triggered us Received: Revised: Accepted: Published: 4601
January 5, 2014 April 11, 2014 April 28, 2014 April 28, 2014 dx.doi.org/10.1021/jf500067q | J. Agric. Food Chem. 2014, 62, 4601−4609
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Table 1. Analytical Parameters of UHPLC-ESI-MS/MS Method analyte
RTa
quercetin protocatechuic acid chrysin rutin hesperetin naringenin rosmarinic acid vanillin p-coumaric acid caffeic acid chlorogenic acid
5.369 1.380 10.086 4.373 6.445 6.112 4.609 4.207 4.407 2.580 1.661
equation f(x) f(x) f(x) f(x) f(x) f(x) f(x) f(x) f(x) f(x) f(x)
= = = = = = = = = = =
1829.35x 783.913x 520.665x 788.005x 570.363x 1298.14x 168.107x 52.7804x 69.0997x 2783.56x 1448.01x
+ 32208.90 + 9758.52 + 866.55 − 9096.62 + 2835.74 − 6598.05 − 428.04 + 1089.93 + 264.38 + 45880.00 + 2695.90
R2b
RSD%c
linearity range (mg/L)
LOD/LOQd (μg/L)
recovery (%)
Ue (%)
0.999 75 0.999 70 0.999 68 0.999 27 0.999 72 0.999 82 0.999 59 0.999 82 0.999 97 0.999 50 0.999 86
1.33 2.19 3.59 0.95 2.16 1.50 2.60 2.75 1.24 1.10 0.62
0.025−1.000 0.025−1.000 0.025−1.000 0.025−1.000 0.025−1.000 0.025−1.000 0.025−1.000 0.025−1.000 0.025−1.000 0.025−1.000 0.025−1.000
0.63/1.93 0.74/2.25 0.60/1.85 0.53/1.59 0.68/2.10 0.80/2.45 0.93/2.86 0.85/2.61 0.76/2.34 0.66/2.01 0.72/2.22
97.1 102.3 98.4 99.0 101.2 96.5 91.2 100.8 99.3 95.9 97.8
7.6 10.1 5.9 7.1 6.5 3.5 4.9 5.1 4.6 5.9 9.5
a Retention time. bCoefficient of determination. cRelative standard deviation. dLimit of deteection/limit of quantification. ePercent relative uncertainty at 95% confidence level (k = 2).
Plant Material. S. sediforme (Jacq.) Pau was collected by Dr. A. Ertaş from western Turkey (Istanbul) in May 2012 and characterized by Dr. Y. Yeşil (Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University). Voucher specimens have been strored in the Herbarium of Istanbul University, Faculty of Pharmacy (ISTE: 9805). Identification and Quantitation of Phenolic Compounds. Instruments and Chromatographic Conditions. LC-ESI-MS/MS analysis of the methanol extract was performed by using a Shimadzu UHPLC instrument coupled to a tandem MS instrument. The liquid chromatograph was equipped with LC-30AD binary pumps, a DGU20A3R degasser, a CTO-10ASvp column oven, and an SIL-30AC autosampler. For the chromatographic separation, a C18 reversed-phase Inertsil ODS-4 (100 mm × 2.1 mm i.d., 2 μm) analytical column was used. The column temperature was fixed at 40 °C. The elution gradient consisted of mobile phases (A) water (5 mM ammonium acetate and 0.1% formic acid) and (B) acetonitrile (0.1% formic acid). The following gradient was used: 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 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 = 14.99 min, 20% B. The solvent flow rate was maintained at 0.5 mL/min, and the injection volume was settled as 10 μL. MS Instrumentation. MS detection was performed using a Shimadzu LCMS 8040 model triple-quadrupole mass spectrometer equipped with an ESI source operating in negative ion mode. LC-ESIMS/MS data were collected and processed by LabSolutions software (Shimadzu). The multiple reaction monitoring (MRM) mode was used to quantify the analytes: the assay of phenolic compounds was performed following two or three transitions per compound, the first one for quantitative purposes and the second and/or third one for confirmation. Optimization of LC-ESI-MS/MS Method. Subsequent to several combinations of trials, a gradient acetonitrile (0.1% formic acid, 5 mM ammonium acetate) and water (0.1% formic acid) system was concluded to be the best mobile phase solution. For rich ionization and the separation of the molecules, the mentioned mobile phase proved to be the best of all. ESI source was chosen instead of atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) sources as the phenolic compounds were small and relatively polar molecules. Tandem mass spectrometry was chosen to be used for the current study because this system is commonly used for its fragmented ion stability.17 The working conditions were determined as interface temperature, 350 °C; DL temperature, 250 °C; heat block temperature, 400 °C; nebulizing gas flow (nitrogen), 3 L/min; and drying gas flow (nitrogen), 15 L/min. Method Validation Parameters. In the current study, 11 phenolic compounds (quercetin, protocatechuic acid, chrysin, rutin, hesperetin, naringenin, rosmarinic acid, vanillin, p-coumaric acid, caffeic acid, and chlorogenic acid) were quantified in S. sediforme. In the chromatographic analysis of phenolic compounds, gradient separation was
to focus on S. sediforme, which is an edible species. The high phenolic content of S. sediforme and the quercetin content of Sedum species show that chemical and biological activities of these species are worth studying deeply. It is a known fact that phenolic compounds, especially quercetin, show high antioxidant and anticholinesterase activities.17−19 Thus, we aimed to investigate the relationship between the chemical composition and biological activities of S. sediforme. To the best of our knowledge, there are no studies on the essential oil, fatty acid, and phenolic profiles and antioxidant (βcarotene−linoleic acid test system, DPPH free radical scavenging activity, and cupric reducing antioxidant capacity (CUPRAC)), anticholinesterase and antimicrobial activities of S. sediforme in the literature. At the beginning, the fatty acid and essential oil compositions of S. sediforme were determined by using GC-MS in the current study. In the next step, related antioxidant, anticholinesterase, and antimicrobial activities and total phenolic and flavonoid contents were analyzed. Moreover, the phenolic and flavonoid contents of S. sediforme methanol extract was also determined using UHPLC-ESI-MS/MS for quantitative and qualitative purposes.
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MATERIALS AND METHODS
Chemicals and Instruments. The phenolic content and fatty acid composition of S. sediforme were determined by using LC-ESI-MS/MS (Shimadzu, Kyoto, Japan) and GC-MS (Thermo Scientific Polaris Q) instruments, respectively. A Shimadzu UV spectrophotometer and a BioTek Power Wave XS microplate reader (USA) were used for the activity assays. 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (purity = 97.5%) and butylated hydroxytoluene (BHT) (≥99%) were purchased from Merck (Darmstadt, Germany); quercetin (95%), protocatechuic acid (97%), chrysin (97%), rutin (94%), hesperetin (95%), naringenin (95%), rosmarinic acid (96%), vanillin (99%), p-coumaric acid (98%), caffeic acid (98%), chlorogenic acid (95%), formic acid (≤100%), 2,2diphenyl-1-picrylhydrazyl (DPPH) (≥95%), β-carotene (≥93%), linoleic acid (≥99%), Tween 40, pyrocathecol (≥99%), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) (≥98%), copper(II) chloride dihydrate (CuCl2·2H2O) (≥99%), neocuproine (2,9-dimethyl-1,10-phenanthroline) (≥98%), ethylenediaminetetraacetic acid (EDTA) (≥98%), acetylcholinesterase (AChE) (type VI-S, EC 3.1.1.7, 425.84 U/mg), and butyrylcholinesterase (BChE) (EC 3.1.1.8, 11.4 U/mg) were obtained from Sigma (Germany); α-tocopherol (≥95.5%) and acetylthiocholine iodide (≥98%) were from Aldrich (Germany); galanthamine hydrobromide (≥94%) was from Sigma-Aldrich (Germany); Folin−Ciocalteu phenol reagent was from Applichem (Germany); butyrylthiocholine iodide (≥99%) was from Fluka (Germany). 4602
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applied. Linear regression equations of the phenolic compound standards are represented in Table 1. The linearity of the LC-MS/MS conditions for phenolic compounds was affirmed in the range from 0.025 to 1 mg/L by the high coefficient of determination (R2 > 0.999) obtained. The limit of detection (LOD) and limit of quantitation (LOQ) of the method reported in this study were dependent on the calibration curve established from six measurements. The LOD and LOQ of the method were determined by using the equations 3S/N and 10S/N, respectively (S/N refers to the signal to noise ratio) (Table 1). For different compounds, LOD ranged from 0.53 to 0.93 μg/L and LOQ ranged from 1.59 to 2.86 μg/L (Table 1). Furthermore, the recovery of the phenolic compound standards ranged from 91.2 to 102.3%. Estimation of Uncertainty. Identification of Uncertainty Sources. The sources of the uncertainty for the applied method were evaluated and calculated using EURACHEM Guide, 2004.17,20,21 The following parameters were used for the calculations of uncertainties: (1) calibration curve (cal); (2) purity of reference standards (pur); (3) stock solutions (Css); (4) weighing of samples (msample); (5) repeatabilitity (rep); (6) recovery (rec). Standard combined uncertainty is a function of the individual uncertainties of each parameter and calculated by using eq 1:
amu. Alkanes (C8−C24) were used as reference points in the calculation of Kovats indices (KI) by the same conditions.23,24 Identification of the compounds was based on comparing their retention times and mass spectra with those obtained from authentic samples and/or the NIST and Wiley spectra as well as data from the published literature. GC-FID and GC-MS analyses were replicated three times (mean RSD% < 0.1). Determination of Total Phenolic and Flavonoid Contents. Total phenolic and flavonoid amounts in the crude extracts expressed as pyrocatechol and quercetin equivalents, respectively, were calculated according to the following equations:25,26 absorbance = 0.0126 pyrocatechol (μg) + 0.0314 (R2 = 0.9936)
absorbance = 0.1495 quercetin (μg) − 0.0958 (R2 = 0.9994) Antioxidant Activity of the Extracts. To determine the antioxidant activity, the following tests were applied: β-carotene− linoleic acid test system, DPPH free radical and ABTS cation radical scavenging activity, and cupric reducing antioxidant capacity (CUPRAC) methods.27−30 Anticholinesterase Activity of the Extracts. A spectrophotometric method developed by Ellman et al. was used to indicate the acetyl- and butyryl-cholinesterase inhibitory activities.31 Determination of Antimicrobial Activity and Minimum Inhibitory Concentration (MIC). Five different microorganisms including Gram-positive bacteria (Streptococcus pyogenes ATCC19615 and Staphylococcus aureus ATCC 25923), Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922), and yeast (Candida albicans ATCC10231), which were purchased from Refik Saydam Sanitation Center (Turkey), were used for detecting the antimicrobial activity of the samples. The disc diffusion method was employed for this purpose.32 The minimum inhibitory concentration was determined by the broth macrodilution method according to NCCLS.33 Ampicillin and fluconazole were used as positive controls for bacteria and yeast, respectively. Statistical Analysis. The results of the antioxidant and anticholinesterase activity assays were represented as means ± SD. The results were evaluated using an unpaired t test and ANOVA variance analysis with the NCSS statistical computer package. The differences were considered statistically significant at p < 0.05.
u(C) =
u 2(cal) + u 2(pur) + u 2(Css) + u 2(w) + u 2(rep) + u 2(rec) (1)
Main uncertainty sources are defined as purity of standards and calibration curve. Standard combined uncertainties are multiplied by 2 for the calculation of expanded uncertainties by accepting a 95% confidence level. Calculated uncertainties are shown in Table 1. Preparation of Plant Extracts for LC-ESI-MS/MS. For sample preparation, initially, the whole parts of the dried and powdered plants (10 g) were extracted by MeOH (3 × 50 mL) in 24 h at room temperature (extraction yield, 8.3%). The extract was subsequently filtered and evaporated under reduced pressure. Then, dry filtrate was diluted until 250 mg/L and passed through the 0.2 μm microfiber filter for LC-ESI-MS/MS. Esterification of Total Fatty Acids and GC-MS Conditions. Esterification of petroleum ether extract (100 mg) of S. sediforme was performed according to the report of Kılıc et al.22 In this study, Thermo Scientific Polaris Q GC-MS was used. GC-MS study conditions and comparison of identification and quantification of the compounds were done in exactly same manner according to Kılıc et al.22 Preparation of Plant Extracts for Biological Activities and GC-MS. In this analysis, primarily, whole plant material was dried and powdered, and 100 g of plant material was sequentially macerated with petroleum ether (3 × 250 mL), acetone (3 × 250 mL), methanol (3 × 250 mL), and water (3 × 250 mL) for 24 h at room temperature. Subsequent to filtration, the solvents were evaporated to handle the crude extracts. Preparation of Essential Oil and GC-MS Conditions. Essential oil was obtained using a Clevenger apparatus from the whole parts of plant (100 g), which was crumbled into small pieces and soaked in distilled water (500 mL) for 3 h. The obtained essential oil was dried over anhydrous Na2SO4 and stored at 4 °C for a sufficient period of time. The essential oil was diluted using CH2Cl2 (1:3 v/v) prior to gas chromatography−flame ionization detector (GC-FID) and GC-MS analysis. GC-FID analysis was performed using a Thermo Electron Trace GC-FID detector, and GC-MS analysis was performed using the same GC and Thermo Electron DSQ MS. The following GC conditions were applied for both GC-MS and GC-FID analyses. The GC oven temperature was kept at 60 °C for 10 min and programmed to 280 °C at a rate of 4 °C/min and then kept constant at 280 °C for 10 min. A nonpolar Phenomenex DB5 fused silica column (30 m × 0.32 mm, 0.25 μm film thickness) was used with helium at 1 mL/min (20 psi) as a carrier gas. The split ratio was adjusted to 1:50, the injection volume was 0.1 μL, and EI/MS was recorded at 70 eV ionization energy. The mass range was m/z 35−500
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RESULTS AND DISCUSSION Quantitative Analysis of Phenolic and Flavonoid Compounds by UHPLC-ESI (QqQ)/MS/MS. Having the same general structure with an aromatic hydroxyl nucleus, almost 8000 phenolic compounds exist in nature.17 Phenolic compounds that exist in plants constitute one of the most important groups acting as free radical terminators and primary antioxidants. Plant polyphenols are multifunctional in that they act as reducing agents, hydrogen atom donors, and singlet oxygen scavengers. Besides being the most diverse and prevalent natural compounds, flavonoids are the most important phenolics. Being members of the polyphenol family, flavonoids have more than 4000 species that exist in the roots, flowers, and leaves of plants.17 A literature survey reveals several studies on the use of liquid chromatography electrospray ionization tandem mass spectrometry to perform quantitative analyses.17,34 Therefore, for quantitative purpose, the analyses of 11 phenolic and flavonoid compounds in the methanol extract of S. sediforme were done by an accurate method on a mass spectrometer equipped with a triple-quadrupole analyzer. Due to the fact that negative ionization mode was more sensitive and selective for phenolics and flavonoids, it was preferred in the current study. The specific fragmentation reactions were selected to monitor the aforesaid phenolic and flavonoid compounds by 4603
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MRM. Eleven compounds, which were five flavonoids, five phenolic acids, and one phenolic aldehyde, were monitored by the transition from the specific deprotonated molecular ions to the corresponding fragment ions. Molecular ions, fragments observed in MS/MS, related collision energies for these fragments, and the quantified result for S. sediforme are presented in Table 2. Table 2. Identification and Quantification of Phenolic Compounds of Methanol Extract of S. sediforme by UHPLCESI-MS/MS compound
parent ion (m/z)a
quercetin
300.90
protocatechuic acid chrysin
152.90
rutin
609.00
hesperetin
300.90
naringenin
270.90
rosmarinic acid
358.90
vanillin
150.90
p-coumaric acid
162.90
caffeic acid
178.90
chlorogenic acid
353.00
252.90
MS2(CE)b 151.0 (22), 121.0 (26), 107.0 (29) 108.9 (15), 90.9 (25) 62.9 (33),143.0 (28) ,107.0 (26) 300.1 (39), 271.0 (53) 164.0 (24), 136.0 (30), 108.0 (37) 151.0 (18), 119.0 (25), 107.0 (26) 132.9 (41), 161.0 (17) 136.0 (17), 92.0 (21), 107.8 (26) 119.1 (15), 92.9 (28) 134.9 (14), 134.0 (25) 191.1 (15), 84.8 (45)
quantificationc (μg analyte/g extract) 1813.51 ± 137.82 71.23 ± 7.22 NDd 138.62 ± 8.17 ND 39.67 ± 1.38 ND ND
Figure 1. UHPLC-ESI-MS/MS chromatograms of (A) 250 μg/mL standard mix and (B) S. sediforme methanol extract.
94.08 ± 4.33 151.25 ± 8.93
Table 3. GC-MS Analysis of S. sediforme Petroleum Ether Extract
23.30 ± 2.18
a
Molecular ions of the standard compounds (mass to charge ratio). 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. dNot detected.
b
Quercetin, rutin, and naringenin were detected and quantified of five flavonoids; however, chrysin, hesperetin, and vanillin were not found. Furthermore, four phenolic acids (protocatechuic acid, p-coumaric acid, caffeic acid, and chlorogenic acid) were characterized in S. sediforme (Table 2; Figure 1; Figure S1 in the Supporting Information). Quercetin was found to be the most abundant flavonoid compound (1813.51 ± 137.82 μg/g extract) in the methanol extract of S. sediforme (Table 2; Figure 1). Besides, caffeic acid (151.25 ± 8.93 μg/g extract) was found to be the most plentiful phenolic acid in S. sediforme (Table 2; Figure 1). In the literature, there are few studies on phenolic and flavonoid compounds of Sedum species by HPLC and LC-MS techniques.16,35 Xu et al. reported a HPLC methodology that was established to determine quercetin, which is a common hydrolysate of the flavonoid glycosides in S. sarmentosum, S. lineare, and S. erythrostictum.16 Fatty Acid and Essential Oil Composition by GC-MS. GC-MS analysis was used to determine the fatty acid composition of the petroleum ether extract. As represented in Table 3, 10 components were identified, constituting 100.0% of the petroleum ether extract of S. sediforme, and the major
tRa (min)
constituentb
9.69 14.39 18.60 25.27 30.64 30.77 30.86 31.54 37.38 43.82
octanedioic acid 10-undecenoic acid myristic acid palmitic acid linoleic acid oleic acid linolenic acid stearic acid arachidic acid behenic acid total
a
compositionc (%) 1.2 1.2 2.5 28.8 9.7 12.6 12.9 24.6 3.3 3.2
± 0.05 ± 0.06 ± 0.04 ± 0.30 ± 0.18 ± 0.12 ± 0.15 ± 0.29 ± 0.06 ± 0.10
100.0 b
Retention time (in minutes). Nonpolar Phenomenex DB-5 fused silica colum. cPercentage of relative weight.
constituents were characterized as palmitic acid (C16:0) (28.8%), stearic acid (C18:0) (24.6%), and linolenic acid (C18:3 omega-3) (12.9%). This is the first report on the fatty acid composition of S. sediforme. The amount of saturated fatty acids was found to be greater than the amount of unsaturated fatty acid in the present study. There have been no reports regarding direct fatty acid analysis of Sedum species by GC-MS, except the lipophilic extract obtained from Sedum hispanicum.36 However, in this study, 0.89% of the fatty acid content was identified in the lipophilic extract of S. hispanicum. In that sense, 4604
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it could be said that the current study is the first report on the fatty acid composition of Sedum species. The essential oil composition was examined by GC-MS analysis. Twenty-four components were determined, constituting 91.6% of the essential oil composition of S. sediforme. The main components of the essential oil of S. sediforme were identified as α-selinene (20.4%), 2,5-di-tert-octyl-p-benzoquinone (13.1%), valencene (6.3%), and carvone oxide (4.3%) (Table 4). There have been few papers regarding GC-MS
pallidum var. bithynicum and hexahydrofarnesyl acetone for S. spurium in the ratios of 12.8 and 15.7%, respectively.38 Antioxidant Activity and Total Phenolic and Flavonoid Content. The antioxidant activity studies of the petroleum ether (SSP), acetone (SSA), methanol (SSM), and water (SSW) extracts prepared from the whole plant of S. sediforme were carried out by β-carotene bleaching, DPPH free radical scavenging, ABTS cation radical decolorisation, and CUPRAC assays. The SSM extract showed the highest extraction yield, but no significant differences in the extraction yields of other extracts were observed. In the crude extracts, total phenolic and flavonoid amounts were determined and expressed as pyrocatechol and quercetin equivalents, respectively (y = 0.0126 pyrocatechol (μg) + 0.0314, R2 = 0.9936 and y = 0.1495 quercetin (μg) − 0.0958, R2 = 0.9994). The phenolic and flavonoid amounts of the SSM extract were identified to be the richest. The amounts of total phenolic and flavonoid from SSM were 335.71 ± 4.81 and 26.66 ± 0.75 μg/mg extract, respectively. The amount of phenolic components was seen to be higher than that of flavonoid components. The results are shown in Table 5. In the literature, total phenolic and flavonoid contents in the examined Sedum acre extracts were expressed in terms of gallic acid and rutin equivalents, respectively.14 Moreover, Stankovic et al. reported that the total phenolic and flavonoid amounts in the examined acetone extract of S. acre were 181.75 and 173.42 mg/g, respectively.14 Additionally, in the study of Romojaro et al. S. sediforme showed high levels of total phenolic content, 191.53 mg/100 g fresh weight (gallic acid equivalent).5 In this regard, the richness of Sedum species in terms of phenolic and flavonoid compounds, which are known for their important pharmacological properties, increases the importance of these species. As indicated in Table 6, the SSP and SSA extracts showed moderate lipid peroxidation activity (IC50 = 51.34 ± 0.92 and
Table 4. Chemical Composition of the Essential Oil from S. sediforme RIa
constituentb
compositionc (%)
865 954 1193 1197 1249 1276 1299 1376 1409 1442 1481 1484 1498 1677 1712 1746 1890 2109 2156 2259 2366 2407 2896 2900
isononane camphene mrytenal mrytenol 1,3-di-tert-butylbenzene carvone oxide carvacrol α-copaene longifolene aromadendrene α-curcemene valencene α-selinene cadalene curcumen-15-al 2-methylheptadecane 2-methyl-1-hexadecanol heneicosane 1-nonadecanol 2,5-di-tert-octyl-p-benzoquinone arachidic acid tetracosane choleic acid nonacosane
2.1 1.2 1.0 1.1 3.5 4.3 1.3 1.8 3.2 3.5 1.3 6.3 20.4 2.1 1.5 4.0 2.3 4.2 2.1 13.1 2.6 4.2 2.1 2.4
total
91.6
± 0.02 ± 0.03 ± 0.01 ± 0.03 ± 0.04 ± 0.04 ± 0.01 ± 0.02 ± 0.03 ± 0.03 ± 0.01 ± 0.02 ± 0.09 ± 0.03 ± 0.03 ± 0.02 ± 0.02 ± 0.04 ± 0.03 ± 0.07 ± 0.05 ± 0.03 ± 0.04 ± 0.01
Table 6. Antioxidant Activity of the Extracts and Standardsa IC50 (μg/mL) sample
a
Retention indices (DB-5 column). bNonpolar Phenomenex DB-5 fused silica column. cPercentage of relative weight.
SSP SSA SSM SSW α-TOC BHT
analysis of essential oil composition of Sedum species.37,38 Yaylı et al. reported that 38 and 35 components were identified in the essential oils of S. pallidum var. bithynicum and S. spurium, respectively. Besides, in their study, the main components of these species were found to be caryophyllene oxide for S.
lipid peroxidation 51.34 54.61 9.78 153.05 15.54 10.35
± ± ± ± ± ±
0.92a 0.31b 0.06c 1.71d 0.21e 0.03f
DPPH free radical 174.55 17.20 9.07 104.45 18.76 48.86
± ± ± ± ± ±
0.91a 0.33b 0.07c 1.28d 0.31e 0.09f
ABTS cation radical 75.03 8.76 5.87 9.01 9.88 10.67
± ± ± ± ± ±
0.45a 0.52b 0.03c 0.29b 0.08d 0.11e
Values are means ± SD, n = 3; values with different letters in the same column are significantly different (p < 0.05).
a
Table 5. Total Phenolic and Flavonoid Contents, Extraction Yield, and Anticholinesterase Activity of Extracts and Galanthamine at 200 μg/mLa sample SSP SSA SSM SSW galanthaminee
inhibition % against AChE NAd 28.31 85.09 40.61 79.91
± 1.12a ± 0.21b ± 0.60c ± 0.42d
inhibition % against BChE 11.53 65.69 89.57 12.32 81.21
± 0.20a ± 2.91b ± 0.86c ± 1.01a ± 0.59d
phenolic content (μg PEsb/mg extract) 137.30 254.37 335.71 184.92
± 0.85a ± 2.30b ± 4.81c ± 5.91d
flavonoid content (μg QEsc/mg extract) 10.91 21.23 26.66 15.93
± 0.14a ± 0.71b ± 0.75c ± 0.81d
extraction yield % (w/w) 3.02 3.11 7.20 2.31
a Values expressed are means ± SD of three parallel measurements, and values were calculated according to negative control. Values with different letters in the same column are significantly different (p < 0.05). bPyrocatechol equivalents (y = 0.0126x + 0.0314, R2 = 0.9936). cQuercetin equivalents (y = 0.1495x − 0.0958, R2 = 0.9994). dNot active. eStandard drug.
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54.61 ± 0.31 μg/mL, respectively) and the SSW extract showed weak lipid peroxidation activity (153.05 ± 1.71 μg/mL) in the β-carotene bleaching method. However, the SSM extract showed very strong lipid peroxidation activity (9.78 ± 0.06 μg/mL) in the β-carotene bleaching method. Furthermore, the SSM extract exhibited higher activity than α-tocopherol (15.54 ± 0.21 μg/mL) and BHT (10.35 ± 0.03 μg/mL), which were used as standards in the β-carotene bleaching method. As seen in Table 6, the SSP and SSW extracts showed weak and moderate activity (174.55 ± 0.91 and 104.45 ± 1.28 μg/mL) in DPPH free radical scavenging activity, respectively. On the other hand, the SSA and SSM extracts exhibited very strong DPPH free radical scavenging activity. Besides, the SSA (17.20 ± 0.33 μg/mL) and SSM (9.07 ± 0.07 μg/mL) extracts showed higher activity than α-tocopherol (18.76 ± 0.31 μg/mL) and BHT (48.86 ± 0.09 μg/mL). In previous studies, Mavi et al. reported that S. sempervivoides showed very strong activity (88.9 and 86.0% inhibition, respectively) in the DPPH free radical scavenging assay and lipid peroxidation−thiobarbituric acid method at 200 μg/mL concentration.13 In addition, Stankovic et al. reported that the acetone extract of S. acre exhibited very strong activity in the DPPH free radical scavenging assay.14 The largest capacity to neutralize DPPH radicals was found for the acetone extract, which neutralized 50% of free radicals at the concentration of 29.57 μg/mL.14 Morover, in the study of Thuong et al., the MeOH-, EtOAc-, and BuOH-soluble fractions exhibited significant scavenging activities against free radicals (DPPH and superoxide) as well as remarkable inhibitory effects on lipid peroxidation.10 As shown in Table 6, the SSP extract indicated moderate activity (75.03 ± 0.45 μg/mL) in the ABTS cation radical scavenging assay. However, the SSA, SSM, and SSW extracts exhibited very strong effects in the ABTS cation radical scavenging assay. In addition to that, the SSA (8.76 ± 0.52 μg/ mL), SSM (5.87 ± 0.03 μg/mL), and SSW (9.01 ± 0.29 μg/ mL) extracts showed higher activity than α-tocopherol (9.88 ± 0.08 μg/mL) and BHT (10.67 ± 0.11 μg/mL). According to Romojaro et al., S. sediforme showed very strong effects in hydrophilic and lipophilic total antioxidant activities, with 588.87 ± 35.52 mg of Trolox equivalent 100 g−1 FW.5 Morover, S. sediforme showed good activity (81.60% inhibition) in peroxyl radical (H2O2) scavenging potential assay.5 The SSM extract and α-tocopherol indicated 1.73 and 1.62 absorbance in CUPRAC at 100 μg/mL, respectively (Figure 2). According to our literature survey this is the first study about the cupric reducing antioxidant capacity of Sedum species. Therefore, this study is important in this field. When we look at the antioxidant results of the four tested extracts, we can see the parallelism between antioxidant activities and total phenolic content. In particular, looking at the quantitative phenolic analysis of the methanol extract by LC-MS/MS, it can be deduced that this high activity may be related to the quercetin content. Quercetin is known to have various pharmacological effects.17−19 Particularly, flavonoids are present in plant sources as flavonoid glycosides. Therefore, this high activity also might be said to arise from quercetin gycosides. If we were to express this in a different way, this high activity can be attributed to the synergic effect between quercetin, quercetin glycosides, and other phenolic compounds.39 In further studies, our group plans to purify, structurally determine, and quantify the secondary metabolites, especially flavonoid glycosides of Sedum species. Furthermore,
Figure 2. Cupric reducing antioxidant capacity of S. sediforme, αtocopherol, and BHT.
we also aim to study in vivo pharmacological effects of the purified compounds. Anticholinesterase Activity. As demonstrated in Table 5, the SSA extract exhibited good inhibitory activity (65.69% inhibition) against butyrylcholinesterase enzyme. The SSW extract showed moderate inhibitory activity (40.61% inhibition) against acetylcholinesterase enzyme, at 200 μg/mL. On the other hand, the SSM extract showed 85.09 and 89.57% inhibition activities, which are higher than galanthamine inhibitory activity against acetyl- and butyryl-cholinesterase enzymes at 200 μg/mL, respectively. The anticholinesterase activity of the SSM extract shows parallelism to its high total phenolic and flavonoid contents. This high activity of the SSM extract might be related not only to its high total phenolic and flavonoid contents but also to its quercetin content directly or to the synergic effect of quercetin with other phenolic compounds.39 When previous studies are examined, it can be seen that quercetin has strong antioxidant properties as well as an anticholinesterase effect.17−19 Additionally, Min et al. reported that quercetin had shown potential inhibitory activity against AChE.18 Furthermore, Choi et al. reported that quercetin might improve cognitive ability against TMT-induced neuronal deficit and also had an inhibitory action against AChE.19 To our knowledge, there are no reports about the anticholinesterase activity of Sedum species. Because our results were higher than those for galanthamine and there has been no such study about Sedum species, the anticholinesterase activity results of S. sediforme will be important data in this field. Antimicrobial Activity. The antimicrobial activities of S. sediforme extracts against different microorganisms were measured by using the disc diffusion method, and the results were assessed according to inhibition zone diameter. Results are presented in Table 7. No antimicrobial activity of the water extract against the five tested microorganisms was detected (data not shown). However, the petroleum ether, acetone, and methanol extracts were active on tested microorganisms, and the sensitivity of active extracts was found not to differ significantly among tested microorganisms. Whereas the petroleum ether extract exhibited weak antimicrobial activity (inhibition zone < 12) against all tested microorganisms, the acetone and methanol extracts showed moderate antimicrobial activity against C. albicans (inhibition zone < 20−12) and weak antimicrobial activity against Gram-positive and -negative bacteria. The highest activities were exhibited by acetone and methanol extracts against C. albicans, with 18 ± 0 and 15 ± 0.1 mm inhibition zone diameters, respectively. 4606
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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 Gram-positive S. aureus S. pyogenes Gram-negative E. coli P. aeruginosa yeast C. albicans
acetone extract
methanol extract
positive controls
DDa
MIC
DDa
MIC
DDa
MIC
10 ± 0.4 9 ± 0.4
18 ± 0.2 19 ± 0.7
7 ± 0.2 9 ± 0.1
8 ± 0.2 7 ± 0.1
9 ± 0.3 9 ± 0.3
5 ± 0.6 8 ± 0.5
35 ± 0.2 19 ± 0.2
1.95 ± 0.3 7.815 ± 0.1
9 ± 0.3 7 ± 0.2
18 ± 0.5 8 ± 0.5
7 ± 0.1 10 ± 0
17 ± 0.3 5 ± 0.5
10 ± 0.1 10 ± 0.2
9 ± 0.4 6 ± 0.2
20 ± 0.1 NAc
7.815 ± 0.4 NA
8 ± 0.3
8 ± 0.4
1 ± 0.2
15 ± 0.1
1 ± 0.3
30 ± 0.3
3.125 ± 0.2
18 ± 0
a
DDb
MIC
b
Inhibition zone in diameter (mm) around the disks (6 mm) impregnated with 30 mg/mL of plant extracts. 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 as μg/mL for antibiotics. cNot active.
quercetin amount, total phenolic content, strong antioxidant, and anticholinesterase properties. All in all, the rich total phenolic content and high antioxidant and anticholinesterase capacities of the methanol extract of S. sediforme indicated that more future studies should be done in this field.
For a more reliable assessment of antimicrobial activity, a broth dilution assay was carried out. The sensitivity of the test microorganisms against active extracts was evaluated, and results are shown as MIC (Table 7). Values ranged from 8 to 19 mg/mL for the petroleum ether extract, from 1 to 17 mg/ mL for the acetone extracts, and from 1 to 9 mg/mL for the methanol extracts. The MIC results indicate that the methanol extract was found to be the most active extract. The lowest MIC value was recorded by the acetone and methanol extracts against C. albicans (1 mg/mL). This is the first study dealing with the antimicrobial activity of S. sediforme. On the other hand, in a previous paper, the methanol, acetone, and ethyl acetate extracts of S. acre revealed high antibacterial activity against Gram-positive bacteria and low to moderate antifungal activity.14 Besides, the essential oils of S. pallidum Bieb. var. bithynicum and S. spurium showed low antimicrobial activity against Gram-negative and -positive bacteria and yeast-like fungi,38 and S. sormentosum Bunge showed weak inhibitory activity against B. subtilis and S. aureus.40 The present study concluded that the methanol extract of S. sediforme showed very strong antioxidant and anticholinesterase activities. These properties of the methanol extract of S. sediforme were parallel to the total phenolic content. On the basis of our results, quercetin was found to be the most abundant phenolic compound in S. sediforme. Many studies in the literature showed that quercetin and its glycosides had potent biological properties, in particular antioxidant and anticholinesterase activities. Thus, these high activities of S. sediforme may be related to either high total phenolic or quercetin contents.17−19,41 From a broader perspective, these high activities of S. sediforme might be related not only to its high total phenolic and flavonoid contents but also directly to its quercetin content or to the synergic effect of quercetin with other phenolic compounds.39 Furthermore, protocatechuic, p-coumaric, and chlorogenic acids were found for the first time in Sedum species. Although the total phenolic content was found as very rich in S. sediforme, phenolic constituents were detected in low amounts. Therefore, these results could be the effect of some other phenolic constituents such as flavonoid glycosides that we have not studied yet. In conclusion, it is found that S. sediforme had very high antioxidant and anticholinesterase activities. Therefore, the results of the current study showed that the methanol extract of S. sediforme can also be used as a food source because of its high
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ASSOCIATED CONTENT
* Supporting Information S
Figures S1−S5. Equations S1−S7. Antioxidant and anticholinesterase activities methods. Tables S1−S14. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(A.E.) Phone: +90 412 2488030/7512. E-mail:
[email protected] or
[email protected]. tr. Funding
We acknowledge the Dicle University for financial support (Research University Grant DUBAP: 13-ASMYO-61). We thank Dicle University Science and Technology Research and Application Center (DÜ BTAM) for the partial support of this study. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED LC-MS/MS, liquid chromatography−tandem mass spectrometry; GC-MS, gas chromatography−mass spectrometry; GCFID, gas chromatography−flame ionization detector; UHPLCESI-MS/MS, ultrahigh-performance liquid chromatography− electrospray ionization−tandem mass spectrometry; ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; BHT, butylated hydroxytoluene; DPPH, 2,2diphenyl-1-picrylhydrazyl; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); CuCl2·2H2O, copper(II) chloride dihydrate; neocuproine, 2,9-dimethyl-1,10-phenanthroline; EDTA, ethylenediaminetetraacetic acid; APCI, atmospheric pressure chemical ionization; APPI, atmospheric pressure photoionization; LOD, limit of detection; LOQ, limit of quantitation; CUPRAC, cupric reducing antioxidant capacity; BChE, butyrylcholinesterase enzyme; AChE, acetylcholinesterase enzyme; MIC, minimum inhibitory concentration; NCCLS, National Committee for Clinical Laboratory Standards; SD, standard deviation; MRM, 4607
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multiple reaction monitoring; SSP, petroleum ether extract of S. sediforme; SSA, acetone extract of S. sediforme; SSM, methanol extract of S. sediforme; SSW, water extract of S. sediforme
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