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MS and GC-MS and biological activities of Chenopodium album subsp. album var. microphyllum
Industrial Crops & Products 141 (2019) 111755
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Chemical compositions by LC-MS/MS and GC-MS and biological activities of Chenopodium album subsp. album var. microphyllum
T
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Pelin Köseoğlu Yılmaza, , Abdulselam Ertaşb, Mehmet Akdenizc, Mine Koçyiğit Avcıd, Ufuk Kolaka a
Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul University, 34116 Istanbul, Turkey Department of Pharmacognosy, Faculty of Pharmacy, Dicle University, 21280 Diyarbakir, Turkey c The Council of Forensic Medicine, Ministry of Justice, 21100 Diyarbakir, Turkey d Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University, 34116 Istanbul, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: Anticholinesterase Antioxidant Chenopodium album Fatty acid Phenolic
Chenopodium species have been used in folk medicine and as vegetable for years. In the present study, the total phenolic and flavonoid contents, biological activities, phenolic constituents and fatty acid profile of Chenopodium album subsp. album var. microphyllum (Boen.) Aellen were determined for the first time. The antioxidant effects were investigated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and cupric ion reducing antioxidant capacity assays (CUPRAC). The Ellman method was applied for the determination of the cholinesterase inhibition activity. The phenolic constituents of the methanol extract and the fatty acid profile of the nhexane extract were evaluated by LC–MS/MS and GC–MS, respectively. Acetone and methanol extracts showed similar DPPH free radical scavenging activities (0.68 ± 0.07 and 0.68 ± 0.05 mmol Trolox/g extract, respectively), whereas the cupric ion reducing capacity of the acetone extract was the highest (0.41 ± 0.05 mmol Trolox/g extract). Acetone and methanol extracts had moderate butyrylcholinesterase inhibitory activities as 65.29 ± 1.56% and 52.64 ± 2.78%, respectively, whereas non of the extracts possessed anti-acetylcholinesterase effect. The methanol extract was found to contain significant amounts of hesperidin (9769.13 ± 158.26 μg/g extract) and rutin (2935.19 ± 39.92 μg/g extract). The major fatty acid constituents of the n-hexane extract were identified as myristic acid (18.26%) and cis-10-pentadecanoic acid (15.39%).
1. Introduction The Chenopodiaceae family (Goosefoot) includes approximately 100 genus and 1500 species (Glimn-Lacy and Kaufman, 2006). The genus Chenopodium L., belonging to the Chenopodiaceae family, comprises about 120 species all over the world and about 17 species in Turkey (Corio-Costet et al., 1998; Davis, 1988). Chenopodium species were found to contain several secondary metabolites such as flavonoids (Gohara and Elmazar, 1997), other phenolics (Cutillo et al., 2006), saponins (Dini et al., 2001), terpenes (Ahmed, 2000), sterols (Salt and Adler, 1985), alkaloids (Cutillo et al., 2004) and vitamins (Guill et al., 1997). Some of these species have antimicrobial, diuretic, laxative, cardiotonic, antiphlogistic, astringent, sedative, analgesic, hepatoprotective, antihelmintic, antiparasitic and antiseptic effects and have been used as folk medicine (Kokanova-Nedialkova et al., 2009). Chenopodium species have been also consumed as a leafy vegetable (C. album) and as a pseudocereal (C. album and C. quinoa) (Bhargava et al., 2006). Chenopodium album, one of the important species of the
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Chenopodiaceae family, is commonly known as lambsquarter, wild spinach, white goosefoot or as bathua in Hindi and bathuwa in English contexts (Jan et al., 2017). C. album has starch-rich seeds that can be used like cereals. Also, C. album was found to have a high nutritional content of protein, carbohaydrate, vitamin C, carotenoids, lipids and oxalic acid (Guerrero and Torija, 1997). Due to its content of phenolics, it exhibits high antioxidant activity by inhibition of free radicals and oxidative chain reactions within tissues and membranes (Jan et al., 2016). Khomarlou et al. reported the ethanolic extract and the fractions of C. album subsp. striatum as a potential antioxidant and antibacterial agent (Khomarlou et al., 2017). In another study, C. album’s ethanolic leaf extract was found to possess potent DNA protective capacity beside its antibacterial and antioxidant effects (Elif Elif Korcan et al., 2013). Laghari et al. (2011) investigated the antioxidant activity and the free phenolic acids of C. album and determined that the methanolic extract of the leaves had a DPPH scavenging activity higher than 70%. The fatty acid composition of C. album was also investigated since it is an edible plant and the omega fatty acids provides more nutritional value
Corresponding author. E-mail addresses: [email protected], [email protected] (P. Köseoğlu Yılmaz).
https://doi.org/10.1016/j.indcrop.2019.111755 Received 22 May 2019; Received in revised form 30 August 2019; Accepted 2 September 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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were expressed as quercetin equivalents (QE) (Park et al., 1997).
than the saturated ones (Ksouda et al., 2018) In a research the fatty acid profile of C. album leaves was determined and α-linolenic acid was found as the main component (Guerrero and Torija Isasa, 1997). The present study aimed to evaluate the total phenolic and flavonoid contents, the antioxidant potentials and cholinesterase inhibition effects of n-hexane, acetone and methanol extracts of C. album subsp. album var. microphyllum with phenolic constituents of the methanol extract by LC–MS/MS and fatty acid profile of n-hexane extract by GC–MS. DPPH free radical scavenging activity and cupric ion reducing antioxidant capacity (CUPRAC) assays were performed to investigate the antioxidant activity. The anticholinesterase effect was analyzed by the Ellman method. To the best of our knowledge, this work is the first report on biological activities and chemical composition of C. album subsp. album var. microphyllum.
2.4. Antioxidant capacity The antioxidant capacities of the extracts were calculated in terms of trolox-equivalent antioxidant capacity (TEAC). The calibration curve of trolox was prepared with different concentrations in the range of 10–80 μmol/L. The sample solutions were prepared with ethanol in DPPH free radical scavenging assay and with distilled water in cupric ion reducing antioxidant capacity (CUPRAC) assay at 1000 mg/L. 2.4.1. DPPH free radical scavenging assay Four milligrams of DPPH were dissolved in 100 mL of ethanol and the solution is mixed for half an hour in dark. The absorbance was measured after 30 min of DPPH˙ addition (10 μL of sample solution and 30 μL of distilled water were mixed with 160 μL of DPPH˙ solution) at ambient temperature and 517 nm (Blois, 1958).
2. Materials and methods 2.1. Chemicals and reagents
2.4.2. Cupric ion reducing antioxidant capacity assay (CUPRAC) Twelve and 0.5 μL of the sample solution and 54.5 mL of distilled water were added to a solution prepared by adding 61.0 μL of 10 mmol/ L CuCl2, 61.0 μl of 7.5 mmol/L neocuproine and 61.0 μL of 1.0 mmol/L NH4CH3COO buffer (pH 7), respectively. The absorbance of the solution was measured after an hour at ambient temperature and 450 nm (Apak et al., 2004).
Ethanol, methanol, n-hexane, acetone, potassium acetate, aluminum nitrate nonahydrate and HPLC grade methanol were supplied from Merck (Darmstadt, Germany); ammonium formate, pyrocathecol, quercetin, 1,1-diphenyl-2-picrylhydrazyl (DPPH), copper (II) chloride dihydrate, 5,5′-dithio-bis(2-nitrobenzoic) acid (DTNB), electric eel acetylcholinesterase (AChE, Type-VI-S, EC 3.1.1.7, 425.84 U/mg) and horse serum butyrylcholinesterase (BChE, EC 3.1.1.8, 11.4 U/mg) were from Sigma (Steinheim, Germany); acetylthiocholine iodide and FolinCiocalteu reagent were from Applichem (Darmstadt, Germany); neocuproine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and galantamine hydrobromide were from Sigma-Aldrich (Sternheim, Germany); butyrylthiocholine iodide was from Fluka Chemie (Fluka Chemie, Steinheim, Germany); ammonium acetate, sodium carbonate, sodium hydrogen phosphate and sodium dihydrogen phosphate were from Reidel de Haen (Seelze, Germany).
2.5. Anticholinesterase assay The solutions of the extracts in ethanol were prepared at 4000 mg/ L. The DTNB solution was prepared by adding 2.0 mL of pH 7.0 and 4.0 mL of pH 8.0 phosphate buffer to a mixture of 1.0 mL of 16 mg/mL DTNB and 7.5 mg/mL NaHCO3 in pH 7.0 phosphate buffer. Aliquots of 130 mL of 100 mmol/L phosphate buffer (pH 8.0), 10 μL of sample solution and 20 μL AChE (or BChE) solution were mixed and incubated at 25°C for 15 min. After that, 20 μL of DTNB solution was added and the reaction was initiated by 20 μL acetylthiocholine iodide (or butyrylthiocholine iodide). The hydrolysis of these substrates were monitored at 412 nm (Ellman et al., 1961).
2.2. Plant material and extraction The aerial parts and roots of C. album var. album subsp. microphyllum were collected from Yenifoça, İzmir (Turkey) in June 2012 and identified by Assoc. Prof. Dr. Mine Koçyiğit Avcı (Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University, Turkey). Voucher specimen has been deposited in the Herbarium of Faculty of Pharmacy, Istanbul University, Turkey (ISTE: 116005). The air-dried and grounded aerial parts and roots of C. album var. album subsp. microphyllum (2750 g) were macerated with 4 L of nhexane (24 h x 4), 4 L of acetone (24 h x 4) and 4 L of methanol (24 h x 4), respectively at ambient temperature. The solvent was evaporated under reduced pressure following filtration.
2.6. LC–MS/MS analysis The quantitative analysis of the phenolic compounds were performed by an LC–MS/MS system composed of Shimadzu Nexera UHPLC instrument coupled with Shimadzu LCMS 8040 triple quadrupole mass spectrometer equipped with an LC-30AD model gradient pump, DGU20A3R degasser, CTO-10ASvp column owen and SIL-30AC autosampler. The chromatographic separation was achieved using an Agilent Poroshell 120 column (EC-C18 2.7 μm, 4.6 mm × 150 mm). The column temperature was set to 40°C. A gradient program with a mobile phase system consisting of two parts as eluate A (5 mM ammonium formate and 0.15% formic acid in ultrapure water) and B (5 mM ammonium formate and 0.15% formic acid in methanol) was established for the elution of the analytes. After the stabilization of the system with 20% eluent B flow, a linear gradient from 0.0 to 25.0 min from 20% to 100% of eluent B flow, from 25.0 to 35.0 min an isocratic hold at 100% of eluent B flow, from 35.0 to 45.0 min from 100% to 20% of eluent B flow was applied. The flow rate was adjusted to 0.5 mL/min and the injection volume was 2 μL. The triple quadrapole mass spectrometer had an electrospray ionization (ESI) source that could work both in positive and negative ionization modes. LC–MS/MS data were collected and processed by LabSolutions software (Shimadzu, Kyoto, Japan). The analytes were quantified by the multiple reaction monitoring (MRM) mode. The analysis was performed following one or two transitions per compound, the first one for quantitative purposes and the second one for confirmation. The optimum ESI conditions were determined as interface
2.3. Total phenolic and flavonoid contents The solutions of the extracts were prepared with ethanol at 1000 mg/L. For the determination of the total phenolic content, 180 μL of distilled water, 4.0 μL Folin-Ciocalteu reagent and 12.0 μL of 2% Na2CO3 solution were added to 4.0 μL of sample solution and the solution was kept at ambient temperature for 2 h. Following that, the absorbance of the sample was measured at 760 nm. The calibration curve of pyrocatechol was prepared with the standard solutions in the concentration range of 0.5–4.0 μg/mL and the total phenolic content results were calculated in terms of pyrocatechol equivalents (PE) (Slinkard and Singleton, 1977). For the determination of total flavonoid content, 172 μL of 80% ethanol and 4 μL of 1.0 mol/L KCH3COO were added to 20 μL of sample solution. After 1 min, 4 μL of 10% Al(NO3)3 solution was added to the mixture and the absorbance was measured at 415 nm after 40 min. The calibration curve of quercetin was prepared in the concentration range of 5–40 μg/mL and the total flavonoid contents 2
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The fatty acid analysis of the species was achived by a HeadspaceGC–MS/FID (Agilent) GC–MS system with a nonpolar Phenomenex DB5 column (30 m - 0.32 mm, 0.25 μm film thickness). The flow rate of helium was 1 L/min (20 psi). A temperature program was applied. The column temperature was hold at 40°C for 5 min, then increased to 280°C with a rate of 5°C/min and hold for 10 min. The split ratio was adjusted to 1:20 and the injection volume was 0.1 μL. The ionization energy was 70 eV. The scanning range was set as m/z to 35–450 atomic mass unit (amu). The GC–MS libraries of NIST and Wiley were used to identify the components. The relative percentages of the separated components were calculated by computerized integrator using total ion chromatography.
0.02 0.03 0.01 0.02 0.01 0.01 0.06 0.02 0.02 23.74/31.66 17.05/21.16 24.33/33.73 12.17/16.64 13.09/19.51 24.39/34.35 13.52/21.07 13.29/20.07 13.55/20.74
0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01
0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01
1.00 0.99 1.01 1.00 1.01 1.00 1.00 1.00 1.00
2.7. GC–MS analysis
100–3200 75–2400 100–3200 50–1600 50–1600 100–3200 50–1600 50–1600 50–1600 0.991 0.991 0.990 0.994 0.990 0.990 0.996 0.990 0.991
In the present study, n-hexane (CAH), acetone (CAA) and methanol (CAM) extracts of C. album subsp. album var. micropyhllum were obtained with yields of 1.00%, 0.84% and 0.98%, respectively. 3.1. Total phenolic and flavonoid contents Total phenolic (TPC) and flavonoid contents (TFC) of CAH, CAA and CAM were determined (Table 2). The highest TPC was found for CAA as 64.37 μg PEs/mg extract. The richest sample in flavonoids was also the acetone extract (CAA, 126.67 μg QEs/mg extract). Since acetone is a good solvent for phenolics and flavonoids, the results obtained were reasonable. Adedapo et al. (2011) also determined that the TPC and TFC of the acetone extracts of C. album leaves were higher than the ones of ethanol and water extracts (Adedapo et al., 2011). 3.2. Antioxidative effect The antioxidant abilities of CAH, CAA and CAM were investigated in terms of free radical scavenging activity and cupric ion reducing antioxidant capacity (Table 2). CAA and CAM possessed similar moderate DPPH free radical scavenging activities as 0.68 mmol TR/g extract. CAA had the highest cupric ion reducing antioxidant capacity (0.41 mmol TR/g extract) among all the extracts analyzed. Balpetek Külcü et al. (2019) investigated the antioxidant activities of methanol, chloroform and hexane extracts of C. album by DPPH free radical activity scavenging and CUPRAC assays and determined that the acetone extract had a higher DPPH scavenging activity, whereas the methanol extract exhibited a higher cupric ion reducing capacity (Balpetek Külcü et al., 2019). The antioxidant activity of CAA might be caused by its higher phenolic and flavonoid contents. It is usually complicated to compare the results of various antioxidant activity studies because of different conditions and expressions. However, the results given in terms of trolox equivalents in the present work could indicate the importance of this species among other antioxidants and plants. 3.3. Anticholinesterase activity β-amyloid, the abnormal protein, might be one of the key factors in the progress of Alzheimer’s disease and the cholinesterases promote the aggregation of β-peptides and amyloid formation (Köseoğlu Yilmaz et al., 2014). The anticholinesterase activity of the C. album subsp. album var. micropyhllum extracts were determined by the Ellman method (Table 2). The method is based on the measurement of the
d
c
tR – Retention time. RSD – Relative standard deviation. RME – Relative mean error. U – Relative standard uncertainity at 95% confidence level (k = 2). b
a
153.40 353.30 609.10 611.10 431.00 447.00 301.20 285.20 269.20 7.00 8.03 13.67 13.68 14.54 15.13 17.09 17.78 19.20 Protocatechuic acid Chlorogenic acid Rutin Hesperidin Apigetrin Astragalin Quercetin Luteolin Apigenin
109.10-108.00 191.20-85.00 300.10-301.10 303.00-449.30 268.10-269.10 284.10-227.10 151.10-179.10 133.10-151.00 117.00-151.10
Neg Neg Neg Pos Neg Neg Neg Neg Neg
y = 120,226 + 590.460x y = 87,418.5 + 697.935x y = 30,144.8 + 469.333x y = 123,981 + 2539.52x y = 146,897 + 1052.01x y = 44,598.6 + 329.506x y=-146948 + 1826.89x y = 495,252 + 3166.03x y = 483,037 + 3115.89x
Intraday
Linear range (μg/L) r2 Ion mode Molecular ions (m/z) Parent ion (m/z) tRa Analytes
Table 1 Analytical performance of the LC–MS/MS method.
temperature; 350°C, desolvation line temperature; 250°C, heat block temperature; 400°C, nebulizing gas flow (nitrogen); 3 L/min and drying gas flow (nitrogen); 15 L/min. The analytical performance of the LC–MS/MS method was given in Table 1.
3. Results and discussion
Calibration equation
LOD/LOQ (μg/L)
RSD (%)b
Interday
Ud RME (%)c
P. Köseoğlu Yılmaz, et al.
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Table 2 Total phenolic and flavonoid contents, and biological activities of C. album subsp. album var. microphyllum extractsa. Extract
CAH CAA CAM
Total phenolic content (μg PEs/mg)d
44.46 ± 1.26 62.32 ± 1.30 40.89 ± 1.36
Total flavonoid content (μg QEs/mg)e
DPPHb (mmol TrEs/g)f
52.99 ± 3.26 126.67 ± 3.08 30.22 ± 2.77
0.03 ± 0.00 0.68 ± 0.07 0.68 ± 0.05
CUPRACc (mmol TrEs/g)
0.16 ± 0.06 0.41 ± 0.05 0.30 ± 0.01
Cholinesterase inhibition (%) AchEg
BchEh
NAi NA NA
44.31 ± 2.13 65.29 ± 1.56 52.64 ± 2.78
Calibration curve equation of total phenolic content analysis – y = 0.0336 + 0.0280x, r2 = 0.9957. Calibration curve equation of total flavonoid content analysis – y = 0.0404 + 0.0366x, r2 = 0.9946. Calibration curve equation of DPPH analysis – y = 0.0089 + 0.0232x, r2 = 0.9950. Calibration curve equation of CUPRAC analysis – y = 0.0047 + 0.0184x, r2 = 0.9975. a The results are average of three measurements ± standard deviation. b DPPH – 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay. c CUPRAC – cupric ion reducing antioxidant capacity assay. d PEs – pyrocatechol equivalents. e QEs – quercetin equivalents. f TrEs – trolox equivalents. g AChE – Acetylcholinesterase. h BChE – Butyrylcholinesterase. i NA – not active.
of whole plant methanolic extract was found as hesperidin. In another report, the polyphenolic profiles of six quinoa (C. quinoa) flours were determined by UHPLC-ES-MS/MS and phenolics such as rutin, hesperidin, quercetin and chlorogenic acid, which were also detected in this study, were quantified in all of the samples (Pellegrini et al., 2018). Ozer et al. (2016) determined the phenolic phytochemicals of ethanol and water extracts of C. botrys including chlorogenic acid, luteolin and apigenin. Apigenin was not detected and chlorogenic acid was quantified in the water extract (118.83 μg/g extract) similar to the present report, whereas luteolin was also determined in the methanol extract (11.28 μg/g extract) in this study, differently. In another report, astragalin (below quantification limit), apigetrin (below quantification limit) and rutin (2.80 μg/g extract in leaves, 1.95 μg/g extract in stems) were detected in the methanol extract of C. hybridum (Podolak et al., 2016). In the present study, apigetrin and astragalin were also quantified at concentrations of 7.94 μg/g extract and 40.58 μg/g extract, respectively. Repo-Carrasco-Valencia et al. (2010) investigated flavonoids and other phenolic compounds in cultivated C. quinoa samples and among the analyzed phenolics quercetin was determined in all of the samples with the highest concentration of 55.5 mg/100 g. Protocatechuic acid was not detected in any of the quinoa. On the other hand Laghari et al. (2011) determined protocatechuic acid in the methanolic extract of the fruits of C. album. In the present study quercetin and protocatechuic acid were quantified in the methanol extract at 112.21 μg/g extract and 13.79 μg/g extract, respectively. Laghari et al. also determined gallic acid, protocatechuic aldehyde, vanillic acid, caffeic acid, syringic acid, syringe aldehyde, p-coumaric acid, ferulic acid, m-coumaric acid, and o-coumaric acid, differently (Laghari et al., 2011).
absorbance of yellow-colored solution occured as a result of the thio anion produced by the enzymatic hydrolysis of the substrate (AcI or BuI) reacting with DTNB (Ellman et al., 1961). None of the extracts exhibited acetylcholinesterase inhibition. On the other hand, CAA and CAM had moderate anti-butyrylcholinesterase activity as 65.29% and 52.64% inhibitions, respectively. To the best of our knowledge, this study is the first report on the in vitro anticholinesterase activity of a Chenopodium species. 3.4. Quantification of phenolics by LC–MS/MS The phenolic compounds from natural sources may have antioxidant properties which can be counteracting for the reactive oxygen species associated diseases. Considering this, the phenolic content of CAM was investigated by LC–MS/MS. The analyzed phenolics as protocatechuic acid, chlorogenic acid, rutin, hesperidin, apigetrin, astragalin, quercetin, luteolin and apigenin were selected considering the reports on phytochemical studies on other Chenopodium species in the literature. It was found that CAM had significant amounts of hesperidin (9769.13 μg/g extract) and rutin (2935.19 μg/g extract). Hesperidin (Wilmsen et al., 2005) and rutin (Yang et al., 2008) have high antioxidant properties similar to synthetics, which might be the reason of the activity of CAM. Also protocatechuic acid, chlorogenic acid, apigetrin, astragalin, and quercetin were quantified (Table 3). Similar to the present work, Paśko et al. (2008) quantified hesperidin (1.86 mg/kg extract) and rutin (360 mg/kg extract) in methanolhydrochloric acid-water extract (8:1:1, v/v) of C. quinoa seeds in which rutin was the main flavonoid. In the present study, the major flavonoid Table 3 Phenolic content of C. album subsp. album var. microphyllum methanolic extracta. Analyte
Phenolic content (μg/g)
Protocatechuic acid Chlorogenic acid Rutin Hesperidin Apigetrin Astragalin Quercetin Luteolin Apigenin
13.79 ± 0.30 20.34 ± 0.61 2935.19 ± 39.92 9769.13 ± 158.26 7.94 ± 0.10 40.58 ± 0.62 112.31 ± 6.44 ND ND
3.5. Fatty acid analysis by GC–MS The fatty acid composition of CAH was analyzed by GC–MS (Table 4). Nine components were identified including 100% of CAH. The main constituents of the fatty acid composition were identified as myristic acid (C14:0) (18.26%) and cis-10-pentadecanoic acid (C15:1) (15.39%). Differently, the main component of the fatty acid composition of C. album was determined as C18:3w3 (α-lineolenic acid) in another study (Guerrero and Torija Isasa, 1997). 4. Conclusions Plants have been used as folk medicine and with nutritional purposes for centruies. Today biologically active compounds isolated from
a The results are average of three measurements ± standard deviation.
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Table 4 Fatty acid composition of C. album subsp. album var. microphyllum n-hexane extracta. tR (min)b
Components
Fatty acid composition (%)
18.95 22.95 27.72 28.73 29.42 30.48 31.38 32.19 33.35
Myristic acid (C14:0) cis-10-Pentadecanoic (C15:1) Elaidic acid (C18:1n9t) Linoleaidic acid (C18:2n6t) Linoleic acid (C18:2n6c) Arachidic acid (C20:0) cis-11-Eicosenoic acid (C20:1) Heneicosanoic acid (C21:0) cis-11,14-Eicosadienoic acid (C20:2) Total:
18.26 ± 0.96 15.39 ± 0.54 11.86 ± 0.71 3.97 ± 0.09 14.78 ± 0.18 9.85 ± 0.59 8.99 ± 0.48 11.27 ± 0.12 5.63 ± 0.06 100.0
a b
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Phenolic composition, antioxidant and enzyme inhibitory activities of ethanol and water extracts of Chenopodium botrys. RSC Adv. 6, 64986–64992. https://doi.org/10.1039/C6RA13229D. Park, Y.K., Koo, M.H., Ikegaki, M., Contado, J.L., 1997. Comparison of the flavonoid aglycone contents of Apis mellifera propolis from various regions of Brazil. Arq. Biol. Tecnol. 40, 97–106. Paśko, P., Sajewicz, M., Gorinstein, S., Zachwieja, Z., 2008. Analysis of selected phenolic acids and flavonoids in Amaranthus cruentus and Chenopodium quinoa seeds and sprouts by HPLC. Acta Chromatogr. 20, 661–672. https://doi.org/10.1556/AChrom. 20.2008.4.11. Pellegrini, M., Lucas-Gonzales, R., Ricci, A., Fontecha, J., Fernández-López, J., PérezÁlvarez, J.A., Viuda-Martos, M., 2018. Chemical, fatty acid, polyphenolic profile, techno-functional and antioxidant properties of flours obtained from quinoa (Chenopodium quinoa Willd) seeds. Ind. Crops Prod. 111, 38–46. https://doi.org/10. 1016/j.indcrop.2017.10.006. Podolak, I., Olech, M., Galanty, A., Załuski, D., Grabowska, K., Sobolewska, D., Michalik, M., Nowak, R., 2016. Flavonoid and phenolic acid profile by LC-MS/MS and biological activity of crude extracts from Chenopodium hybridum aerial parts. Nat. Prod. Res. 30, 1766–1770. https://doi.org/10.1080/14786419.2015.1136908. Repo-Carrasco-Valencia, R., Hellström, J.K., Pihlava, J.-M., Mattila, P.H., 2010. Flavonoids and other phenolic compounds in Andean indigenous grains: quinoa (Chenopodium quinoa), kañiwa (Chenopodium pallidicaule) and kiwicha (Amaranthus caudatus). Food Chem. 120, 128–133. https://doi.org/10.1016/j.foodchem.2009.09. 087. Salt, T.A., Adler, J.H., 1985. Diversity of sterol composition in the family Chenopodiaceae. Lipids 20, 594–601. https://doi.org/10.1007/BF02534285. Slinkard, K., Singleton, V.L., 1977. Total phenol analysis automation and comparison with manual methods. Am. J. Enol. Viticult. 28, 49–55. Wilmsen, P.K., Spada, D.S., Salvador, M., 2005. Antioxidant activity of the flavonoid hesperidin in chemical and biological systems. J. Agric. Food Chem. 53, 4757–4761. https://doi.org/10.1021/jf0502000. Yang, J., Guo, J., Yuan, J., 2008. In vitro antioxidant properties of rutin. LWT - Food Sci. Technol. 41, 1060–1066. https://doi.org/10.1016/j.lwt.2007.06.010.
The results are average of three measurements ± standard deviation. tR – Retention time.
plants are still important sources for modern drug formulations and nutraceuticals. The present study provided information on chemical composition and biological activities of C. album subsp. album var. micropyhllum for the first time. For years, the seeds and the leaves of C. album have been used for nutriton and as folk medicine around the world. This report could be the first step for this species to be utilized similar to C. album. On the other hand, further nutritional, toxicological and pharmacological studies must be performed for nutritional and pharmaceutical purposes. References Adedapo, A., Jimoh, F., Afolayan, A., 2011. Comparison of the nutritive value and biological activities of the acetone, methanol and water extracts of the leaves of Bidens pilosa and Chenopodium album. Acta Pol. Pharm. 68, 83–92. Ahmed, A.A., 2000. Highly oxygenated monoterpenes from Chenopodium ambrosioides. J. Nat. Prod. 63, 989–991. https://doi.org/10.1021/np990376u. Apak, R., Guclu, K., Ozyurek, M., Karademir, S.E., 2004. Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. J. Agric. Food Chem. 52, 7970–7981. 7970-7981. https://doi.org/10.1021/jf048741x. Balpetek Külcü, D., Demir Gökışık, C., Aydın, S., 2019. An investigation of antibacterial and antioxidant activity of nettle (Urtica dioicaL.), mint (Mentha piperita), thyme (Thyme serpyllum) and Chenopodium album L. plants from Yaylacık plateau, Giresun. Turkey J. Agric. Food Sci. Technol. 7, 73–80. https://doi.org/10.24925/turjaf.v7i1. 73-80.2123. Bhargava, A., Shukla, S., Ohri, D., 2006. Chenopodium quinoa—an Indian perspective. Ind. Crops Prod. 23, 73–87. https://doi.org/10.1016/j.indcrop.2017.10.006. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. https://doi.org/10.1038/1811199a0. Corio-Costet, M.F., Chapuis, L., Delbecque, J.P., 1998. Chenopodium album L. (fat hen): in vitro cell culture, and production of secondary metabolites (phytosterols and ecdysteroids). In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry 41: Medicinal and Aromatic Plants X. Springer-Verlag, Berlin, Heidelberg, New York, pp. 97–112. Cutillo, F., D’Abrosca, B., DellaGreca, M., Zarrelli, A., 2004. Chenoalbicin, a novel cinnamic acid amide alkaloid from Chenopodium album. Chem. Biodivers. 1, 1579–1583. https://doi.org/10.1002/cbdv.200490118. Cutillo, F., DellaGreca, M., Gionti, M., Previtera, L., Zarrelli, A., 2006. Phenols and lignans from Chenopodium album. Phytochem. Anal. 17, 344–349. https://doi.org/10.1002/ pca.924. Davis, P.H., 1988. Flora of Turkey and the East Aegean Island 2 University Press, Edinburgh. Dini, I., Schettino, O., Simioli, T., Dini, A., 2001. Studies on the constituents of Chenopodium quinoa seeds: isolation and characterization of new triterpene saponins. J. Agric. Food Chem. 49, 741–746. https://doi.org/10.1021/jf000971y. Elif Korcan, S., Aksoy, O., Erdoğmuş, S.F., Ciğerci, İ.H., Konuk, M., 2013. Evaluation of antibacterial, antioxidant and DNA protective capacity of Chenopodium album’s ethanolic leaf extract. Chemosphere 90, 374–379. https://doi.org/10.1016/j.
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Chemical compositions by LC-MS/MS and GC-MS and biological activities of Chenopodium album subsp. album var. microphyllum
T
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Pelin Köseoğlu Yılmaza, , Abdulselam Ertaşb, Mehmet Akdenizc, Mine Koçyiğit Avcıd, Ufuk Kolaka a
Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul University, 34116 Istanbul, Turkey Department of Pharmacognosy, Faculty of Pharmacy, Dicle University, 21280 Diyarbakir, Turkey c The Council of Forensic Medicine, Ministry of Justice, 21100 Diyarbakir, Turkey d Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University, 34116 Istanbul, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: Anticholinesterase Antioxidant Chenopodium album Fatty acid Phenolic
Chenopodium species have been used in folk medicine and as vegetable for years. In the present study, the total phenolic and flavonoid contents, biological activities, phenolic constituents and fatty acid profile of Chenopodium album subsp. album var. microphyllum (Boen.) Aellen were determined for the first time. The antioxidant effects were investigated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and cupric ion reducing antioxidant capacity assays (CUPRAC). The Ellman method was applied for the determination of the cholinesterase inhibition activity. The phenolic constituents of the methanol extract and the fatty acid profile of the nhexane extract were evaluated by LC–MS/MS and GC–MS, respectively. Acetone and methanol extracts showed similar DPPH free radical scavenging activities (0.68 ± 0.07 and 0.68 ± 0.05 mmol Trolox/g extract, respectively), whereas the cupric ion reducing capacity of the acetone extract was the highest (0.41 ± 0.05 mmol Trolox/g extract). Acetone and methanol extracts had moderate butyrylcholinesterase inhibitory activities as 65.29 ± 1.56% and 52.64 ± 2.78%, respectively, whereas non of the extracts possessed anti-acetylcholinesterase effect. The methanol extract was found to contain significant amounts of hesperidin (9769.13 ± 158.26 μg/g extract) and rutin (2935.19 ± 39.92 μg/g extract). The major fatty acid constituents of the n-hexane extract were identified as myristic acid (18.26%) and cis-10-pentadecanoic acid (15.39%).
1. Introduction The Chenopodiaceae family (Goosefoot) includes approximately 100 genus and 1500 species (Glimn-Lacy and Kaufman, 2006). The genus Chenopodium L., belonging to the Chenopodiaceae family, comprises about 120 species all over the world and about 17 species in Turkey (Corio-Costet et al., 1998; Davis, 1988). Chenopodium species were found to contain several secondary metabolites such as flavonoids (Gohara and Elmazar, 1997), other phenolics (Cutillo et al., 2006), saponins (Dini et al., 2001), terpenes (Ahmed, 2000), sterols (Salt and Adler, 1985), alkaloids (Cutillo et al., 2004) and vitamins (Guill et al., 1997). Some of these species have antimicrobial, diuretic, laxative, cardiotonic, antiphlogistic, astringent, sedative, analgesic, hepatoprotective, antihelmintic, antiparasitic and antiseptic effects and have been used as folk medicine (Kokanova-Nedialkova et al., 2009). Chenopodium species have been also consumed as a leafy vegetable (C. album) and as a pseudocereal (C. album and C. quinoa) (Bhargava et al., 2006). Chenopodium album, one of the important species of the
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Chenopodiaceae family, is commonly known as lambsquarter, wild spinach, white goosefoot or as bathua in Hindi and bathuwa in English contexts (Jan et al., 2017). C. album has starch-rich seeds that can be used like cereals. Also, C. album was found to have a high nutritional content of protein, carbohaydrate, vitamin C, carotenoids, lipids and oxalic acid (Guerrero and Torija, 1997). Due to its content of phenolics, it exhibits high antioxidant activity by inhibition of free radicals and oxidative chain reactions within tissues and membranes (Jan et al., 2016). Khomarlou et al. reported the ethanolic extract and the fractions of C. album subsp. striatum as a potential antioxidant and antibacterial agent (Khomarlou et al., 2017). In another study, C. album’s ethanolic leaf extract was found to possess potent DNA protective capacity beside its antibacterial and antioxidant effects (Elif Elif Korcan et al., 2013). Laghari et al. (2011) investigated the antioxidant activity and the free phenolic acids of C. album and determined that the methanolic extract of the leaves had a DPPH scavenging activity higher than 70%. The fatty acid composition of C. album was also investigated since it is an edible plant and the omega fatty acids provides more nutritional value
Corresponding author. E-mail addresses: [email protected], [email protected] (P. Köseoğlu Yılmaz).
https://doi.org/10.1016/j.indcrop.2019.111755 Received 22 May 2019; Received in revised form 30 August 2019; Accepted 2 September 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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were expressed as quercetin equivalents (QE) (Park et al., 1997).
than the saturated ones (Ksouda et al., 2018) In a research the fatty acid profile of C. album leaves was determined and α-linolenic acid was found as the main component (Guerrero and Torija Isasa, 1997). The present study aimed to evaluate the total phenolic and flavonoid contents, the antioxidant potentials and cholinesterase inhibition effects of n-hexane, acetone and methanol extracts of C. album subsp. album var. microphyllum with phenolic constituents of the methanol extract by LC–MS/MS and fatty acid profile of n-hexane extract by GC–MS. DPPH free radical scavenging activity and cupric ion reducing antioxidant capacity (CUPRAC) assays were performed to investigate the antioxidant activity. The anticholinesterase effect was analyzed by the Ellman method. To the best of our knowledge, this work is the first report on biological activities and chemical composition of C. album subsp. album var. microphyllum.
2.4. Antioxidant capacity The antioxidant capacities of the extracts were calculated in terms of trolox-equivalent antioxidant capacity (TEAC). The calibration curve of trolox was prepared with different concentrations in the range of 10–80 μmol/L. The sample solutions were prepared with ethanol in DPPH free radical scavenging assay and with distilled water in cupric ion reducing antioxidant capacity (CUPRAC) assay at 1000 mg/L. 2.4.1. DPPH free radical scavenging assay Four milligrams of DPPH were dissolved in 100 mL of ethanol and the solution is mixed for half an hour in dark. The absorbance was measured after 30 min of DPPH˙ addition (10 μL of sample solution and 30 μL of distilled water were mixed with 160 μL of DPPH˙ solution) at ambient temperature and 517 nm (Blois, 1958).
2. Materials and methods 2.1. Chemicals and reagents
2.4.2. Cupric ion reducing antioxidant capacity assay (CUPRAC) Twelve and 0.5 μL of the sample solution and 54.5 mL of distilled water were added to a solution prepared by adding 61.0 μL of 10 mmol/ L CuCl2, 61.0 μl of 7.5 mmol/L neocuproine and 61.0 μL of 1.0 mmol/L NH4CH3COO buffer (pH 7), respectively. The absorbance of the solution was measured after an hour at ambient temperature and 450 nm (Apak et al., 2004).
Ethanol, methanol, n-hexane, acetone, potassium acetate, aluminum nitrate nonahydrate and HPLC grade methanol were supplied from Merck (Darmstadt, Germany); ammonium formate, pyrocathecol, quercetin, 1,1-diphenyl-2-picrylhydrazyl (DPPH), copper (II) chloride dihydrate, 5,5′-dithio-bis(2-nitrobenzoic) acid (DTNB), electric eel acetylcholinesterase (AChE, Type-VI-S, EC 3.1.1.7, 425.84 U/mg) and horse serum butyrylcholinesterase (BChE, EC 3.1.1.8, 11.4 U/mg) were from Sigma (Steinheim, Germany); acetylthiocholine iodide and FolinCiocalteu reagent were from Applichem (Darmstadt, Germany); neocuproine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and galantamine hydrobromide were from Sigma-Aldrich (Sternheim, Germany); butyrylthiocholine iodide was from Fluka Chemie (Fluka Chemie, Steinheim, Germany); ammonium acetate, sodium carbonate, sodium hydrogen phosphate and sodium dihydrogen phosphate were from Reidel de Haen (Seelze, Germany).
2.5. Anticholinesterase assay The solutions of the extracts in ethanol were prepared at 4000 mg/ L. The DTNB solution was prepared by adding 2.0 mL of pH 7.0 and 4.0 mL of pH 8.0 phosphate buffer to a mixture of 1.0 mL of 16 mg/mL DTNB and 7.5 mg/mL NaHCO3 in pH 7.0 phosphate buffer. Aliquots of 130 mL of 100 mmol/L phosphate buffer (pH 8.0), 10 μL of sample solution and 20 μL AChE (or BChE) solution were mixed and incubated at 25°C for 15 min. After that, 20 μL of DTNB solution was added and the reaction was initiated by 20 μL acetylthiocholine iodide (or butyrylthiocholine iodide). The hydrolysis of these substrates were monitored at 412 nm (Ellman et al., 1961).
2.2. Plant material and extraction The aerial parts and roots of C. album var. album subsp. microphyllum were collected from Yenifoça, İzmir (Turkey) in June 2012 and identified by Assoc. Prof. Dr. Mine Koçyiğit Avcı (Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University, Turkey). Voucher specimen has been deposited in the Herbarium of Faculty of Pharmacy, Istanbul University, Turkey (ISTE: 116005). The air-dried and grounded aerial parts and roots of C. album var. album subsp. microphyllum (2750 g) were macerated with 4 L of nhexane (24 h x 4), 4 L of acetone (24 h x 4) and 4 L of methanol (24 h x 4), respectively at ambient temperature. The solvent was evaporated under reduced pressure following filtration.
2.6. LC–MS/MS analysis The quantitative analysis of the phenolic compounds were performed by an LC–MS/MS system composed of Shimadzu Nexera UHPLC instrument coupled with Shimadzu LCMS 8040 triple quadrupole mass spectrometer equipped with an LC-30AD model gradient pump, DGU20A3R degasser, CTO-10ASvp column owen and SIL-30AC autosampler. The chromatographic separation was achieved using an Agilent Poroshell 120 column (EC-C18 2.7 μm, 4.6 mm × 150 mm). The column temperature was set to 40°C. A gradient program with a mobile phase system consisting of two parts as eluate A (5 mM ammonium formate and 0.15% formic acid in ultrapure water) and B (5 mM ammonium formate and 0.15% formic acid in methanol) was established for the elution of the analytes. After the stabilization of the system with 20% eluent B flow, a linear gradient from 0.0 to 25.0 min from 20% to 100% of eluent B flow, from 25.0 to 35.0 min an isocratic hold at 100% of eluent B flow, from 35.0 to 45.0 min from 100% to 20% of eluent B flow was applied. The flow rate was adjusted to 0.5 mL/min and the injection volume was 2 μL. The triple quadrapole mass spectrometer had an electrospray ionization (ESI) source that could work both in positive and negative ionization modes. LC–MS/MS data were collected and processed by LabSolutions software (Shimadzu, Kyoto, Japan). The analytes were quantified by the multiple reaction monitoring (MRM) mode. The analysis was performed following one or two transitions per compound, the first one for quantitative purposes and the second one for confirmation. The optimum ESI conditions were determined as interface
2.3. Total phenolic and flavonoid contents The solutions of the extracts were prepared with ethanol at 1000 mg/L. For the determination of the total phenolic content, 180 μL of distilled water, 4.0 μL Folin-Ciocalteu reagent and 12.0 μL of 2% Na2CO3 solution were added to 4.0 μL of sample solution and the solution was kept at ambient temperature for 2 h. Following that, the absorbance of the sample was measured at 760 nm. The calibration curve of pyrocatechol was prepared with the standard solutions in the concentration range of 0.5–4.0 μg/mL and the total phenolic content results were calculated in terms of pyrocatechol equivalents (PE) (Slinkard and Singleton, 1977). For the determination of total flavonoid content, 172 μL of 80% ethanol and 4 μL of 1.0 mol/L KCH3COO were added to 20 μL of sample solution. After 1 min, 4 μL of 10% Al(NO3)3 solution was added to the mixture and the absorbance was measured at 415 nm after 40 min. The calibration curve of quercetin was prepared in the concentration range of 5–40 μg/mL and the total flavonoid contents 2
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The fatty acid analysis of the species was achived by a HeadspaceGC–MS/FID (Agilent) GC–MS system with a nonpolar Phenomenex DB5 column (30 m - 0.32 mm, 0.25 μm film thickness). The flow rate of helium was 1 L/min (20 psi). A temperature program was applied. The column temperature was hold at 40°C for 5 min, then increased to 280°C with a rate of 5°C/min and hold for 10 min. The split ratio was adjusted to 1:20 and the injection volume was 0.1 μL. The ionization energy was 70 eV. The scanning range was set as m/z to 35–450 atomic mass unit (amu). The GC–MS libraries of NIST and Wiley were used to identify the components. The relative percentages of the separated components were calculated by computerized integrator using total ion chromatography.
0.02 0.03 0.01 0.02 0.01 0.01 0.06 0.02 0.02 23.74/31.66 17.05/21.16 24.33/33.73 12.17/16.64 13.09/19.51 24.39/34.35 13.52/21.07 13.29/20.07 13.55/20.74
0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01
0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01
1.00 0.99 1.01 1.00 1.01 1.00 1.00 1.00 1.00
2.7. GC–MS analysis
100–3200 75–2400 100–3200 50–1600 50–1600 100–3200 50–1600 50–1600 50–1600 0.991 0.991 0.990 0.994 0.990 0.990 0.996 0.990 0.991
In the present study, n-hexane (CAH), acetone (CAA) and methanol (CAM) extracts of C. album subsp. album var. micropyhllum were obtained with yields of 1.00%, 0.84% and 0.98%, respectively. 3.1. Total phenolic and flavonoid contents Total phenolic (TPC) and flavonoid contents (TFC) of CAH, CAA and CAM were determined (Table 2). The highest TPC was found for CAA as 64.37 μg PEs/mg extract. The richest sample in flavonoids was also the acetone extract (CAA, 126.67 μg QEs/mg extract). Since acetone is a good solvent for phenolics and flavonoids, the results obtained were reasonable. Adedapo et al. (2011) also determined that the TPC and TFC of the acetone extracts of C. album leaves were higher than the ones of ethanol and water extracts (Adedapo et al., 2011). 3.2. Antioxidative effect The antioxidant abilities of CAH, CAA and CAM were investigated in terms of free radical scavenging activity and cupric ion reducing antioxidant capacity (Table 2). CAA and CAM possessed similar moderate DPPH free radical scavenging activities as 0.68 mmol TR/g extract. CAA had the highest cupric ion reducing antioxidant capacity (0.41 mmol TR/g extract) among all the extracts analyzed. Balpetek Külcü et al. (2019) investigated the antioxidant activities of methanol, chloroform and hexane extracts of C. album by DPPH free radical activity scavenging and CUPRAC assays and determined that the acetone extract had a higher DPPH scavenging activity, whereas the methanol extract exhibited a higher cupric ion reducing capacity (Balpetek Külcü et al., 2019). The antioxidant activity of CAA might be caused by its higher phenolic and flavonoid contents. It is usually complicated to compare the results of various antioxidant activity studies because of different conditions and expressions. However, the results given in terms of trolox equivalents in the present work could indicate the importance of this species among other antioxidants and plants. 3.3. Anticholinesterase activity β-amyloid, the abnormal protein, might be one of the key factors in the progress of Alzheimer’s disease and the cholinesterases promote the aggregation of β-peptides and amyloid formation (Köseoğlu Yilmaz et al., 2014). The anticholinesterase activity of the C. album subsp. album var. micropyhllum extracts were determined by the Ellman method (Table 2). The method is based on the measurement of the
d
c
tR – Retention time. RSD – Relative standard deviation. RME – Relative mean error. U – Relative standard uncertainity at 95% confidence level (k = 2). b
a
153.40 353.30 609.10 611.10 431.00 447.00 301.20 285.20 269.20 7.00 8.03 13.67 13.68 14.54 15.13 17.09 17.78 19.20 Protocatechuic acid Chlorogenic acid Rutin Hesperidin Apigetrin Astragalin Quercetin Luteolin Apigenin
109.10-108.00 191.20-85.00 300.10-301.10 303.00-449.30 268.10-269.10 284.10-227.10 151.10-179.10 133.10-151.00 117.00-151.10
Neg Neg Neg Pos Neg Neg Neg Neg Neg
y = 120,226 + 590.460x y = 87,418.5 + 697.935x y = 30,144.8 + 469.333x y = 123,981 + 2539.52x y = 146,897 + 1052.01x y = 44,598.6 + 329.506x y=-146948 + 1826.89x y = 495,252 + 3166.03x y = 483,037 + 3115.89x
Intraday
Linear range (μg/L) r2 Ion mode Molecular ions (m/z) Parent ion (m/z) tRa Analytes
Table 1 Analytical performance of the LC–MS/MS method.
temperature; 350°C, desolvation line temperature; 250°C, heat block temperature; 400°C, nebulizing gas flow (nitrogen); 3 L/min and drying gas flow (nitrogen); 15 L/min. The analytical performance of the LC–MS/MS method was given in Table 1.
3. Results and discussion
Calibration equation
LOD/LOQ (μg/L)
RSD (%)b
Interday
Ud RME (%)c
P. Köseoğlu Yılmaz, et al.
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Table 2 Total phenolic and flavonoid contents, and biological activities of C. album subsp. album var. microphyllum extractsa. Extract
CAH CAA CAM
Total phenolic content (μg PEs/mg)d
44.46 ± 1.26 62.32 ± 1.30 40.89 ± 1.36
Total flavonoid content (μg QEs/mg)e
DPPHb (mmol TrEs/g)f
52.99 ± 3.26 126.67 ± 3.08 30.22 ± 2.77
0.03 ± 0.00 0.68 ± 0.07 0.68 ± 0.05
CUPRACc (mmol TrEs/g)
0.16 ± 0.06 0.41 ± 0.05 0.30 ± 0.01
Cholinesterase inhibition (%) AchEg
BchEh
NAi NA NA
44.31 ± 2.13 65.29 ± 1.56 52.64 ± 2.78
Calibration curve equation of total phenolic content analysis – y = 0.0336 + 0.0280x, r2 = 0.9957. Calibration curve equation of total flavonoid content analysis – y = 0.0404 + 0.0366x, r2 = 0.9946. Calibration curve equation of DPPH analysis – y = 0.0089 + 0.0232x, r2 = 0.9950. Calibration curve equation of CUPRAC analysis – y = 0.0047 + 0.0184x, r2 = 0.9975. a The results are average of three measurements ± standard deviation. b DPPH – 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay. c CUPRAC – cupric ion reducing antioxidant capacity assay. d PEs – pyrocatechol equivalents. e QEs – quercetin equivalents. f TrEs – trolox equivalents. g AChE – Acetylcholinesterase. h BChE – Butyrylcholinesterase. i NA – not active.
of whole plant methanolic extract was found as hesperidin. In another report, the polyphenolic profiles of six quinoa (C. quinoa) flours were determined by UHPLC-ES-MS/MS and phenolics such as rutin, hesperidin, quercetin and chlorogenic acid, which were also detected in this study, were quantified in all of the samples (Pellegrini et al., 2018). Ozer et al. (2016) determined the phenolic phytochemicals of ethanol and water extracts of C. botrys including chlorogenic acid, luteolin and apigenin. Apigenin was not detected and chlorogenic acid was quantified in the water extract (118.83 μg/g extract) similar to the present report, whereas luteolin was also determined in the methanol extract (11.28 μg/g extract) in this study, differently. In another report, astragalin (below quantification limit), apigetrin (below quantification limit) and rutin (2.80 μg/g extract in leaves, 1.95 μg/g extract in stems) were detected in the methanol extract of C. hybridum (Podolak et al., 2016). In the present study, apigetrin and astragalin were also quantified at concentrations of 7.94 μg/g extract and 40.58 μg/g extract, respectively. Repo-Carrasco-Valencia et al. (2010) investigated flavonoids and other phenolic compounds in cultivated C. quinoa samples and among the analyzed phenolics quercetin was determined in all of the samples with the highest concentration of 55.5 mg/100 g. Protocatechuic acid was not detected in any of the quinoa. On the other hand Laghari et al. (2011) determined protocatechuic acid in the methanolic extract of the fruits of C. album. In the present study quercetin and protocatechuic acid were quantified in the methanol extract at 112.21 μg/g extract and 13.79 μg/g extract, respectively. Laghari et al. also determined gallic acid, protocatechuic aldehyde, vanillic acid, caffeic acid, syringic acid, syringe aldehyde, p-coumaric acid, ferulic acid, m-coumaric acid, and o-coumaric acid, differently (Laghari et al., 2011).
absorbance of yellow-colored solution occured as a result of the thio anion produced by the enzymatic hydrolysis of the substrate (AcI or BuI) reacting with DTNB (Ellman et al., 1961). None of the extracts exhibited acetylcholinesterase inhibition. On the other hand, CAA and CAM had moderate anti-butyrylcholinesterase activity as 65.29% and 52.64% inhibitions, respectively. To the best of our knowledge, this study is the first report on the in vitro anticholinesterase activity of a Chenopodium species. 3.4. Quantification of phenolics by LC–MS/MS The phenolic compounds from natural sources may have antioxidant properties which can be counteracting for the reactive oxygen species associated diseases. Considering this, the phenolic content of CAM was investigated by LC–MS/MS. The analyzed phenolics as protocatechuic acid, chlorogenic acid, rutin, hesperidin, apigetrin, astragalin, quercetin, luteolin and apigenin were selected considering the reports on phytochemical studies on other Chenopodium species in the literature. It was found that CAM had significant amounts of hesperidin (9769.13 μg/g extract) and rutin (2935.19 μg/g extract). Hesperidin (Wilmsen et al., 2005) and rutin (Yang et al., 2008) have high antioxidant properties similar to synthetics, which might be the reason of the activity of CAM. Also protocatechuic acid, chlorogenic acid, apigetrin, astragalin, and quercetin were quantified (Table 3). Similar to the present work, Paśko et al. (2008) quantified hesperidin (1.86 mg/kg extract) and rutin (360 mg/kg extract) in methanolhydrochloric acid-water extract (8:1:1, v/v) of C. quinoa seeds in which rutin was the main flavonoid. In the present study, the major flavonoid Table 3 Phenolic content of C. album subsp. album var. microphyllum methanolic extracta. Analyte
Phenolic content (μg/g)
Protocatechuic acid Chlorogenic acid Rutin Hesperidin Apigetrin Astragalin Quercetin Luteolin Apigenin
13.79 ± 0.30 20.34 ± 0.61 2935.19 ± 39.92 9769.13 ± 158.26 7.94 ± 0.10 40.58 ± 0.62 112.31 ± 6.44 ND ND
3.5. Fatty acid analysis by GC–MS The fatty acid composition of CAH was analyzed by GC–MS (Table 4). Nine components were identified including 100% of CAH. The main constituents of the fatty acid composition were identified as myristic acid (C14:0) (18.26%) and cis-10-pentadecanoic acid (C15:1) (15.39%). Differently, the main component of the fatty acid composition of C. album was determined as C18:3w3 (α-lineolenic acid) in another study (Guerrero and Torija Isasa, 1997). 4. Conclusions Plants have been used as folk medicine and with nutritional purposes for centruies. Today biologically active compounds isolated from
a The results are average of three measurements ± standard deviation.
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Table 4 Fatty acid composition of C. album subsp. album var. microphyllum n-hexane extracta. tR (min)b
Components
Fatty acid composition (%)
18.95 22.95 27.72 28.73 29.42 30.48 31.38 32.19 33.35
Myristic acid (C14:0) cis-10-Pentadecanoic (C15:1) Elaidic acid (C18:1n9t) Linoleaidic acid (C18:2n6t) Linoleic acid (C18:2n6c) Arachidic acid (C20:0) cis-11-Eicosenoic acid (C20:1) Heneicosanoic acid (C21:0) cis-11,14-Eicosadienoic acid (C20:2) Total:
18.26 ± 0.96 15.39 ± 0.54 11.86 ± 0.71 3.97 ± 0.09 14.78 ± 0.18 9.85 ± 0.59 8.99 ± 0.48 11.27 ± 0.12 5.63 ± 0.06 100.0
a b
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The results are average of three measurements ± standard deviation. tR – Retention time.
plants are still important sources for modern drug formulations and nutraceuticals. The present study provided information on chemical composition and biological activities of C. album subsp. album var. micropyhllum for the first time. For years, the seeds and the leaves of C. album have been used for nutriton and as folk medicine around the world. This report could be the first step for this species to be utilized similar to C. album. On the other hand, further nutritional, toxicological and pharmacological studies must be performed for nutritional and pharmaceutical purposes. References Adedapo, A., Jimoh, F., Afolayan, A., 2011. Comparison of the nutritive value and biological activities of the acetone, methanol and water extracts of the leaves of Bidens pilosa and Chenopodium album. Acta Pol. Pharm. 68, 83–92. Ahmed, A.A., 2000. Highly oxygenated monoterpenes from Chenopodium ambrosioides. J. Nat. Prod. 63, 989–991. https://doi.org/10.1021/np990376u. Apak, R., Guclu, K., Ozyurek, M., Karademir, S.E., 2004. Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. J. Agric. Food Chem. 52, 7970–7981. 7970-7981. https://doi.org/10.1021/jf048741x. Balpetek Külcü, D., Demir Gökışık, C., Aydın, S., 2019. An investigation of antibacterial and antioxidant activity of nettle (Urtica dioicaL.), mint (Mentha piperita), thyme (Thyme serpyllum) and Chenopodium album L. plants from Yaylacık plateau, Giresun. Turkey J. Agric. Food Sci. Technol. 7, 73–80. https://doi.org/10.24925/turjaf.v7i1. 73-80.2123. Bhargava, A., Shukla, S., Ohri, D., 2006. Chenopodium quinoa—an Indian perspective. Ind. Crops Prod. 23, 73–87. https://doi.org/10.1016/j.indcrop.2017.10.006. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. https://doi.org/10.1038/1811199a0. Corio-Costet, M.F., Chapuis, L., Delbecque, J.P., 1998. Chenopodium album L. (fat hen): in vitro cell culture, and production of secondary metabolites (phytosterols and ecdysteroids). In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry 41: Medicinal and Aromatic Plants X. Springer-Verlag, Berlin, Heidelberg, New York, pp. 97–112. Cutillo, F., D’Abrosca, B., DellaGreca, M., Zarrelli, A., 2004. Chenoalbicin, a novel cinnamic acid amide alkaloid from Chenopodium album. Chem. Biodivers. 1, 1579–1583. https://doi.org/10.1002/cbdv.200490118. Cutillo, F., DellaGreca, M., Gionti, M., Previtera, L., Zarrelli, A., 2006. Phenols and lignans from Chenopodium album. Phytochem. Anal. 17, 344–349. https://doi.org/10.1002/ pca.924. Davis, P.H., 1988. Flora of Turkey and the East Aegean Island 2 University Press, Edinburgh. Dini, I., Schettino, O., Simioli, T., Dini, A., 2001. Studies on the constituents of Chenopodium quinoa seeds: isolation and characterization of new triterpene saponins. J. Agric. Food Chem. 49, 741–746. https://doi.org/10.1021/jf000971y. Elif Korcan, S., Aksoy, O., Erdoğmuş, S.F., Ciğerci, İ.H., Konuk, M., 2013. Evaluation of antibacterial, antioxidant and DNA protective capacity of Chenopodium album’s ethanolic leaf extract. Chemosphere 90, 374–379. https://doi.org/10.1016/j.
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