Journal of Food Measurement and Characterization https://doi.org/10.1007/s11694-020-00562-6 ORIGINAL PAPER Method validation of 15 phytochemicals in Hypericum lysimachioides var. spathulatum by LC–MS/MS, and fatty acid, essential oil, and aroma profiles with biological activities Mehmet Akdeniz1 · Mustafa Abdullah Yilmaz2 · Abdulselam Ertas3 Ufuk Kolak7 · Ismail Yener4 · Mehmet Firat5 · Firat Aydin6 · Received: 18 April 2020 / Accepted: 11 July 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020 Abstract The aim of the present study was to develop and validate a LC–MS/MS method for quantification of 15 phytochemicals in Hypericum species. The developed method was fully validated in terms of repeatability (inter-day and intra-day precision), limits of detection and quantification, linearity, recovery and relative standard uncertainty. The developed and validated LC–MS/MS method was applied to determine 15 phytochemicals in the ethanol extracts of H. lysimachioides var. spathulatum aerial parts (HLS-A) and roots (HLS-R). Hyperoside was found to be the major compound in HLS-A and HLS-R ethanol extracts (16,560.3 and 3561.6 µg analyte/g extract, respectively). According to the results of GC–MS analyses, cis-13,16-docosadienoic acid (35.0%), caryophyllene oxide (24.33%) and undecane (28.21%) were determined as the major components in fatty acid, essential oil and aroma compositions of H. lysimachioides var. spathulatum, respectively. HLS-A and HLS-R ethanol extracts showed moderate activity in ABTS cation radical decolorization assay. The major components (hyperoside, astragalin, and quercetin) present in HLS-A and HLS-R ethanol extracts were found to have also the highest antioxidant effect in ABTS cation radical scavenging method. The extracts, main constituents in the extracts and essential oil had no toxic-cytotoxic potential against PDF, MCF-7 and HT-29 cell lines. HLS-A ethanol extract and essential oil exhibited high butyrylcholinesterase inhibitory activity. Quercetin showed the highest inhibitory effect against acetyl- and butyrylcholinesterase, urease and tyrosinase among the tested samples. According to biological activity studies, H. lysimachioides var. spathulatum and their major components might be promoted as promising sources of natural agents and used in the development of nutraceuticals, functional food ingredients and pharmaceutical industry. Keywords Hypericum · LC–MS/MS method validation · Fatty acid · Essential oil · Biological activities Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11694-020-00562-6) contains supplementary material, which is available to authorized users. 4 Faculty of Pharmacy, Department of Analytical Chemistry, Dicle University, 21280 Diyarbakir, Turkey 5 The Council of Forensic Medicine, Diyarbakir Group Chairmanship, 21070 Diyarbakir, Turkey Faculty of Education, Department of Biology, Van Yüzüncü Yil University, 65080 Van, Turkey 6 Dicle University Science and Technology Research and Application Center, 21280 Diyarbakir, Turkey Faculty of Science, Department of Analytical Chemistry, Dicle University, 21280 Diyarbakir, Turkey 7 Faculty of Pharmacy, Department of Analytical Chemistry, Istanbul University, 34116 Istanbul, Turkey * Abdulselam Ertas abdulselamertas@hotmail.com; abdulselam.ertas@dicle.edu.tr 1 2 3 The genus Hypericum L. belonging to Hypericaceae family (Guttiferae, Clusiaceae) and distributed all around the world comprises more than 500 species [1]. Since ancient times, Faculty of Pharmacy, Department of Pharmacognosy, Dicle University, 21280 Diyarbakir, Turkey 13 Vol.:(0123456789) M. Akdeniz et al. Hypericum species, especially H. perforatum L. (St. John’s wort), has been used in traditional medicine externally for the treatment of wounds, inflammation of skin, and nerve pain, and internally as antidepressant [2]. Phytochemical researches showed that Hypericum species possessed several types of secondary metabolites such as phloroglucinols (adhyperforin, and hyperforin), flavonoids (quercitrine, hyperoside, and quercetin), naphthodianthrones (pseudohypericin, and hypericin), tannins, xanthones, phenolic acids, and the extracts prepared from Hypericum species exhibited many pharmacological and biological activities [2–5]. Hypericum extracts, especially H. perforatum extract, have been commonly used as antidepressant in Germany and other European countries, also in USA since these extracts have less side effects than those of synthetic antidepressant drugs [6]. Hypericin and hyperforin in Hypericum species are responsible for the antidepressant activity [5]. In Turkey, Hypericum L. is represented by 106 taxa under 20 sections and 48 taxa of them are endemic (45%) [7], and botanical, phytochemical and biological activity studies were carried out on some Hypericum species [5, 8–10]. In Turkey, H. lysimachioides is represented by two varieties; H. lysimachioides Boiss. et Noe var. lysimachioides Boiss. et Noe and H. lysimachioides Boiss. et Noe var. spathulatum Robson [11]. There is a limited study on H. lysimachioides var. lysimachioides. Ozen et al. investigated the fatty acid composition of H. lysimachioides var. lysimachioides leaves and flowers, and reported that linolenic and palmitic acids were the main components in both leaves and flowers, unusual 3-hydroxy fatty acids were also obtained from the flowers [12]. H. lysimachioides var. lysimachioides essential oil exhibiting antimicrobial activity against nine microorganisms was analysed by Toker et al. and caryophyllene oxide was found to be the major constituent [13]. Baris et al. studied the antimicrobial, antifungal and antioxidant (reducing power, free radical scavenging, deoxyribose, metal chelating assays) effect of H. lysimachioides var. lysimachioides ethanol extract [14]. The significant mutagenicity of the methanol, ethyl acetate, hexane and petroleum ether extracts of H. lysimachioides var. lysimachioides was determined in vitro [15]. Hakimoglu et al. reported high effect of H. lysimachioides var. lysimachioides ethanol extract on lipid profile in hypercholesterolemic rabbits and its antioxidant activity [16]. This is the first phytochemical and biological report on H. lysimachioides var. spathulatum, except for a botanical research [17]. Within this context, it is important to determine the content of Hypericum genus, which is widely used in our country and in the world, in terms of components with pharmacological potential such as hypericin, hyperforin, pseudohypericin and hyperoside. Considering the use of extracts of these species in the world, determining the amount of major components and effective components is of particular importance for estimating the pharmacological effect in these extracts. The aim of this study was to develop and validate a LC–MS/MS method for the quantification of 15 compounds for Hypericum species, and also to evaluate the fatty acid, essential oil and aroma compositions of H. lysimachioides var. spathulatum using GC–MS and GC-FID, 15 phytochemicals by a new developed and validated LC–MS/ MS method, and the antioxidant, anticholinesterase, antiurease, antityrosinase and cytotoxic activities of its essential oil and the ethanol extracts prepared from its aerial parts and roots. The developed method was validated concerning linearity, limit of detection, limit of quantification, repeatability, recovery and relative standard uncertainty (Fig. S1 and Tables S1-46). Materials and methods Plant material Hypericum lysimachioides Boiss. et Noe var. spathulatum Robson aerial parts and roots were collected and identified by M. Firat (Department of Biology, Faculty of Education, Yuzuncu Yil University). A voucher specimen was kept in the Herbarium of Yuzuncu Yil University (Table 1). Extraction of plant materials for biological activities and LC–MS/MS analyses The aerial parts (100 g) and roots (100 g) of H. lysimachioides var. spathulatum were air-dried at shade. After powdering the plant materials, they were macerated three times with ethanol Table 1 Information about of Hypericum lysimachioides var. spathulatum Samples Abbreviations Yield (%) Collection location Collection time Herbarium number H. lysimachioides var. spathulatum aerial part ethanol extract H. lysimachioides var. spathulatum root ethanol extract H. lysimachioides var. spathulatum essential oil H. lysimachioides var. spathulatum aroma HLS-A 15.5 HLS-R HLS-E HLS-Ar 8.1 0.5 – 13 Van/ Bahçesaray July 2016 M. Firat 32875 (VANF) Method validation of 15 phytochemicals in Hypericum lysimachioides var. spathulatum by… (24 h) (50 mL) at room temperature. The yields of extracts were given in Table 1 with their abbreviations. LC–MS/MS method development and validation Quantitative investigation of 15 phytochemicals in the ethanol extracts of H. lysimachioides var. spathulatum aerial parts and roots was performed by using high performance liquid chromatography tandem mass spectrometry technique. The system used was a Shimadzu brand LC–MS-8040 instrument. The column parameters were; 150 mm × 4.6 mm, 2.7 µm, The column temperature; 40 °C, Mobile phases; A: water, 5 mM ammonium formate and 0.15% formic acid and B: methanol, 5 mM ammonium formate and 0.15% formic acid, The gradient program; 20–100% B (0–25 min), 100% B (25–35 min), 20% B (35–45 min). Linearity (linearity ranges; 100–3200, 75–2400, 50–1600 and 10–320), correlation of determination (R2 ≥ 0.990), repeatability, limit of quantification (between 0.51 and 11.65 mg/L), limit of detection (between 0.32 and 10.51 mg/L), interday and intraday RSD (smaller than 0.0218 and 0.0227, respectively), relative standard uncertainty and recovery (ranged from 99.41% to 101.04%) were the parameters investigated in the analytical validation of the developed method [18–20]. The detailed information on the validation parameters, chromatography and mass spectrometry conditions were presented as supplementary material. Validation parameters were given in Fig. S1, Table 2 and Tables S1-46. GC–MS analysis The aerial parts (100 g) and roots (100 g) were macerated three times with petroleum ether (50 mL) (24 h) at room temperature. Information related to esterification studies and GC–MS conditions were given as supplementary material [21]. Essential oil was obtained using a Clevenger apparatus from the dried aerial parts of H. lysimachioides var. spathulatum (100 g) which were crumbled into small pieces in distilled water (500 mL) for 3 h. One gram of dried plant sample was taken into a vial and incubated for 15 min at 40 °C. Aroma compounds were trapped in SPME cartridges (StableFlex carboxen-polydimethylsiloxane, Supelco) before analysis. GC–MS conditions related to the essential oil and aroma studies were presented as supplementary material [21]. Biological activities Total flavonoid‑phenolic content and antioxidant activity of samples respectively [22, 23]. The following equations were used to calculate total flavonoid and phenolic contents of the extracts: Absorbance = 0.0334 + 0.1961 quercetin (μg) (r2 = 0.9957). Absorbance = 0.0564 + 0.0371 pyrocatechol (μg) (r2 = 0.9936). ABTS cation radical and DPPH free radical scavenging activities, and cupric reducing antioxidant capacity (CUPRAC) methods were used to determine the antioxidant capacity [24–26]. Anticholinesterase inhibitory enzyme activity of samples Anticholinesterase inhibition activities of the samples were evaluated by the method Ellman et al. with slight modifications [27]. Acetyl- and butyryl-cholinesterase enzymes were employed for the enzyme inhibition activity whereas acetylthiocholine iodide and butyrylthiocholine iodide were substrates. Briefly, 10 μL sample solution, 130 µL buffer (100 mM, Na3PO4 pH 8) and 20 μL enzyme were mixed and incubated in room temperature for 15 min. Twenty microliters DTNB and 20 μL substrate were added to the mixture, absorbances were recorded after 10 min of incubation at 412 nm. Urease and tyrosinase inhibitory enzyme activities of samples Urease inhibitory activity of the samples was determined according to the protocol that Hina et al. developed [28]. Ten microliters of sample solution were mixed with 25 μL of urease enzyme solution. First absorbance reading was made after the addition of 100 mM 50 μL urea at 630 nm. Forty-five microliters of phenol and 70 μL of alkaline solution were added to the mixture and final absorbance reading at 630 nm was recorded after 20 min of incubation. Tyrosinase inhibitory activity was tested according to method that Hearing and Jimenez [29] described with modifications. Ten microliters of sample solution, 150 μL of buffer solution (phospahate, pH: 8) and 20 μL of tyrosinase enzyme solution were mixed and incubated for 10 min prior to first absorbance recording at 475 nm. Twenty microliters of L-DOPA solution was then added and final absorbance was recorded [30]. Urease and tyrosinase inhibition(%) = 100 − (OD test well /OD control) × 100 Tiourea and kojic acid were used as a standard inhibitor for urease and tyrosinase inhibition, respectively. Total flavonoid and phenolic contents of the samples were calculated as equivalent to quercetin and pyrocatechol, 13 13 Table 2 Analytical parameters of LC–MS/MS method for 15 phytochemicals No Analytes RTa Mother ion Fragment ions (m/z)2 Ion mode Equation Rb, c RSD%d Linear Range (µg/L) Interday Intraday LOD/LOQ (µg/L)e U%f Recovery (%) Interday Intraday 1 Protocatechuic acid 7.00 153.4 109.1–108.0 Neg y = 590.460x + 120,226 0.9909 0.60 0.60 100–3200 4.26/5.32 100.96 99.88 0.0215 2 Chlorogenic acid 8.03 353.3 191.2–85.0 Neg y = 697.935x + 87,418.5 0.9910 0.74 0.55 75–2400 2.44/3.36 99.41 99.99 0.0299 3 Luteolin-7-glucoside 13.20 447.0 285.1–284.1 Neg y = 215.412x + 36,852.1 0.9939 0.52 0.37 75–2400 2.30/3.02 100.14 100.72 0.0086 4 Rutin 13.67 609.1 300.1–301.1 Neg y = 469.333x + 30,144.8 0.9902 0.63 0.70 100–3200 1.283/1.90 100.49 100.37 0.0136 5 Hesperidin 13.68 611.1 303.0–449.3 Pos y = 2539.52x + 123,981 0.9942 0.81 0.73 50–1600 0.96/1.44 100.53 99.94 0.0162 6 Hyperoside 13.69 463.0 300.1–271.0 Neg y = 185.593x + 8126.67 0.9905 0.74 0.56 100–3200 5.48/6.50 100.39 100.15 0.0126 7 Apigetrin 14.54 431.0 268.1–269.1 Neg y = 1052.01x + 146,897 0.9902 0.47 0.67 50–1600 1.23/1.75 100.60 100.47 0.0132 8 Quercitrine 14.98 447.0 300.0–301.1 Neg y = 175.298x + 33,626.6 0.9918 0.79 0.63 100–3200 10.51/11.65 99.99 100.02 0.0133 9 Astragalin 15.13 447.0 284.1–227.1 Neg y = 329.506x + 44,598.6 0.9900 0.86 0.77 100–3200 5.52/6.77 100.02 100.17 0.0153 10 Quercetin 17.10 301.2 151.1–179.1 Neg y = 1826.89x-146948 0.9962 1.77 2.27 50–1600 1.25/1.81 100.10 100.12 0.0573 11 Luteolin 17.78 285.2 133.1–151.0 Neg y = 3166.03x + 495,252 0.9901 1.19 0.79 50–1600 0.61/0.87 99.61 100.07 0.0188 12 Apigenin 19.20 269.2 117.0–151.1 Neg y = 3115.89x + 483,037 0.9910 0.87 0.90 50–1600 0.32/0.52 99.85 100.22 0.0181 13 Pseudohypericin 26.34 519.0 487.1–475.1 Neg y = 2548.96x + 468,900 0.9908 0.61 0.89 50–1600 2.15/2.55 100.33 100.34 0.0172 14 Hyperforin 28.97 535.3 383.3–315.2 Neg y = 44,260.6x + 203,394 0.9901 2.18 1.64 10–320 0.32/0.51 100.76 100.61 0.0418 15 Hypericin 30.18 503.0 405.1–433.1 Neg y = 7676.03x + 605,593 0.9925 0.93 0.95 50–1600 1.27/1.88 101.04 100.34 0.0189 a RT: retention time (min) b Mother ion (m/z): molecular ions of the standard compounds (m/z ratio) c R2: coefficient of determination d RSD: relative standard deviation e LOD/LOQ (µg/L): limit of detection/quantification f U: relative uncertainty at 95% confidence level (k = 2) M. Akdeniz et al. Method validation of 15 phytochemicals in Hypericum lysimachioides var. spathulatum by… Toxic‑cytotoxic activity of samples Cytotoxic activities were evaluated by using MTT colorimetric method described elsewhere. Breast cancer cell lines (MCF-7) and colon cancer cell lines (HT-29) were used for cytotoxicity while primary dermal fibroblast cell lines (PDF) for toxicity. 22.000, 20.000 and 12.000 cells seeded into the plate for MCF-7, HT-29 and PDF, respectively, and incubated for 24 h. Ten microliters of sample solutions varying concentrations (10, 25, 50, 100, 200 μg/mL) were added after culture medium replaced then incubated for 48 h. Ten microliters of MTT reagent were added and incubated for 4 h and generation of purple color was observed. After addition of 100 μL detergent solution, final incubation was performed overnight in humidified atmosphere of 5% CO2 at 37 °C. Final absorbance recordings were made at 570 and 690 nm [31, 32]. Statistical analysis Table 3 Quantitative determination (µg analyte/g extract) of 15 phytochemicals in the ethanol extracts of H. lysimachioides var. spathulatum aerial parts and roots by LC–MS/MS Samples HLS-A HLS-R Protocatechuic acid Chlorogenic acid Luteolin-7-glucoside Rutin Hesperidin Hyperoside Apigetrin Quercitrine Astragalin Quercetin Luteolin Apigenin Pseudohypericin Hyperforin Hypericin 182.6 158.2 13.7 203.0 85.7 16,560.3 5.2 N.D 1091.5 422.3 6.3 5.9 137.6 N.D 15.6 219.3 11.0 14.8 137.3 66.0 3561.6 2.3 N.D 196.4 49.1 32.3 22.8 0.8 N.D N.D N.D not detected All statistical calculations were performed using the Minitab 16.2.1 statistical software (Minitab Inc. 2010). Results and discussion The results of LC–MS/MS In recent years, phenolic acids have been a focus of interest by food producers and researchers owing to their use in the human diet, their notable antioxidant activities, and their role in the prevention of oxidative stress induced diseases [32, 33]. Many scientific reports have been published on the determination of plant components by LC–MS/MS technique [19, 34, 35]. The developed and validated method was used to determine 15 analytes (pseudohypericin, rutin, chlorogenic acid, hesperidin, luteolin 7-glucoside, protocatechuic acid, hyperoside, apigetrin, hypericin, quercitrine, astragalin, luteolin, apigenin, quercetin, and hyperforin,) in the ethanol extracts of H. lysimachioides var. spathulatum aerial parts (HLS-A) and roots (HLS-R) (Table 3; Fig. 1). Hyperoside (quercetin-3-D-galactoside) was found to be the major compound in HLS-A and HLS-R extracts (16,560.3 and 3561.6 µg analyte/g extract, respectively). Quercitrine and hyperforin were not determined in HLS-A and HLS-R extracts, and hypericin in HLS-R extract. It was determined that HLS-A possessed pseudohypericin (137.6 µg analyte/g extract) and hypericin (15.6 µg analyte/g extract) which were specific to Hypericum species. The results of this study and those in the literature were well-matched; the amounts of phyochemicals specific to Hypericum species such as hypericin and pseudohypericin differ from each other depending on Hypericum species and their parts. Ayan et al. [36] determined hypericin and pseudohypericin using HPLC in the stems, leaves and flowers of all studied Hypericum species which grow in Turkey (H. triquetrifolium Turra, H. linarioides Bosse, H. origanifolium Willd., H. orientale L., H. hyssopifolium L., H. perforatum L., H. scabrum L., and H. montbretii Spach), except for H. heterophyllum Vent. The lowest hypericin and pseudohypericin contents were determined in H. hyssopifolium leaves (0.030 and 0.051 mg/g dried plant, respectively), and the highest ones in H. montbretii flowers (2.52 and 3.58 mg/g dried plant, respectively). In a study conducted by Cirak et al. [37], a variety of bioactive compounds such as hyperforin, pseudohypericin, hyperoside, adhiperforin, hypericin, chlorogenic acid, neochlorogenic acid, quercetin, caffeic acid, 2,4-dihydroxybenzoic acid, isoquercitrin, avicularin, rutin, quercitrine, epicatechin, catechin, mangiferin, and amentoflavone were investigated for the first time in the aerial parts of eight Hypericum species endemic to Turkey. In another study reported by Zorzetto et al. [38], H. grandifolium Choisy, H. canariense L., and H. reflexum L., traditionally used as diuretic, wound healing, worm medication, sedative, and antidepressive, were examined phytochemically and biologically. Hypericin, hyperforin, chlorogenic acid, rutin, hyperoside, isoquercitrin, quercitrine, and quercetin contents in methanol extracts of these species were analyzed by HPLC–DAD and HPLC–MS and the composition of their essential oils obtained by hydrodistilation method were analyzed by GC-FID and GC–MS techniques. It was found that 13 M. Akdeniz et al. Fig. 1 LC–MS/MS chromatograms a TIC chromatogram of standard chemicals analysed by LC–MS/MS method. 1 Protocatechuic acid. 2 Chlorogenic acid. 3 Luteolin-7-glucoside. 4 Rutin. 5 Hesperidin. 6 13 Hyperoside. 7 Apigetrin. 8 Quercitrine. 9 Astragalin. 10 Quercetin. 11 Luteolin. 12 Apigenin. 13 Pseudohypericin. 14 Hyperforin. 15 Hypericin. b HLS-A, c HLS-R Method validation of 15 phytochemicals in Hypericum lysimachioides var. spathulatum by… three studied Hypericum species showed differences in both polar extracts and essential oil components. It was found that H. reflexum contained small amounts of naphtodiantrone compounds (hypericin and pseudohypericin) and possessed high amounts of chlorogenic acid and rutin. As a result, the method was developed and validated in LC–MS / MS to identify components responsible for the pharmacological effects of Hypericum species used both as raw drugs and extract. While developing the method, the H. lysimachioides var. spathulatum species which was not studied previously was selected and the method was validated over the ethanol extract of this species. Moreover, the essential oil, aroma and fatty acid compositions of this species have been investigated. The results of GC–MS analyses After esterification of the petroleum ether extracts prepared from the aerial parts and roots of H. lysimachioides var. spathulatum, their fatty acid compositions were determined by GC–MS and GC-FID (Table 4). Six components were identified, constituting 99.9% of HLS-A petroleum ether extract, its main components were identified to be cis13,16-docosadienoic acid (C22:2) (35.0%) and nervonic acid (C24:1) (17.8%). Four components were identified, including 100% of HLS-R petroleum ether extract, its major constituents were identified to be stearic acid (C18:0) (50.3%) and arachidic acid (C20:0) (29.2%). The fatty acid profiles of HLS-A and HLS-R were different from each other. HLS-A petroleum ether extract consisted of unsaturated fatty acid while the percentage of saturated fatty acids in HLS-R was found to be higher than the unsaturated ones. There is no study in literature on the fatty acid content of this species. Ozen et al. [12], examined the fatty acid Table 4 Fatty acid compositions of the ethanol extracts of H. lysimachioides var. spathulatum aerial parts and roots by GC–MS RT (min)a Constituentsb Composition (%)c HLS-A HLS-R 25.76 27.32 27.97 29.26 33.86 35.63 35.99 a Stearic acid (C18:0) Linoleic acid (C18:2n6c) γ-Linolenic acid (C18:3n6) Arachidic acid (C20:0) cis-13,16-Docosadienoic acid (C22:2) Lignoceric acid (C24:0) Nervonic aid (C24:1) Total identified (%) Retention time (as minute) b A nonpolar Restek Rt-2560 fused silica column c Relative weight percent 9.2 15.1 – 6.0 35.0 16.8 17.8 99.9 50.3 4.5 16.0 29.2 – – – 100.0 composition of H. lysimachioides var. lysimachioides leaves and flowers and found that its flower parts contained unusual 3-hydroxy fatty acids. Ozen et al. [39], investigated the fatty acid compositions of some Hypericum species, and reported that the major components of H. perforatum were palmitic acid (C16:0; 24.87%) and linolenic acid (C18:3 n-3; 21.94%), while the major components of H. retusum were 3-hydroxymyristate (C14:0; 28.29%) and stearic acid (C18:0; 16.47%). These results indicated that fatty acid profile of Hypericum species differ from each other depending on Hypericum species and their parts as mentioned in its phytochemical contents. The yield of H. lysimachioides var. spathulatum essential oil (HLS-E) was 0.5% (v/w) on a dry weight basis. This is the first study on the essential oil composition of H. lysimachioides var. spathulatum. Thirty-six compounds were identified in HLS-E, and accounted for 91.24% of HLS-E (Table 5; Fig. 2). Monoterpene hydrocarbons of HLS-E account for 27.28%, oxygenated monoterpenes 9.37%, sesquiterpene hydrocarbons 16.65%, oxygenated sesquiterpenes 26.08%, and others 11.86%. The result of this GC–MS analysis showed that the main constituents of HLS-E were identified as caryophyllene oxide (24.33%), α-pinene (14.82%) and caryophyllene (11.41%). Toker et al. [13], reported that caryophyllene oxide (30.8%) was also the major component in H. lysimachioides var. lysimachioides. α-Pinene was determined as the main constituent in several Hypericum species such as H. salsolifolium Hand.-Mazz., H. retusum Aucher and H. thymbrifolium Boiss. & Noe (34.92%, 35.02%, and 51.31%, respectively) while transcaryophyllene (23.92%) was in H. pseudolaeve Robson [40, 41]. Erken et al. analyzed the essential oil of five Hypericum species grown in Turkey, and indicated that H. perforatum L., H. montbretii Spach, H. cerastoides (Spach) Robson and H. calycinum L. contained α-pinene (50%, 26%, 58%, 24%, respectively) and H. adenotrichum Spach (endemic) possessed germacrene D (38%) [42]. This is the first study on the aroma composition of H. lysimachioides var. spathulatum (HLS-Ar). Twentyseven compounds were identified in, and accounted for 92.09% of HLS-Ar (Table 5; Fig. 2). HLS aroma was rich in monoterpene hydrocarbons (47.26%) comprising mainly by undecane (28.21%). The percentages of oxygenated monoterpenes in HLS-Ar was low (0.96%), and oxygenated sesquiterpenes were absent. The sesquiterpene hydrocarbons and others consisted of HLS-Ar (8.36% and 35.51%, respectively). There are few studies on the aroma contents of Hypericum species [43, 44], Caprioli et al. [44], reported that the essential oil and aroma contents of H. androsaemum L. were similar, and its major components were β-pinene (12.3–20.7%), limonene (22.6–40.0%), and α-pinene (7.1–15.1%) [44]. Contrary to the literature, in the present study, it was determined 13 M. Akdeniz et al. Table 5 Chemical compositions of the essential oil and aroma of H. lysimachioides var. spathulatum No RIa Constituentsb 1 848 2-Hexenal 0.17 0.43 Co-GC, MS, RI 2 859 Octane. 2-methyl 0.14 0.23 Co-GC, MS, RI 3 899 Nonane 0.41 5.45 Co-GC, MS, RI 4 914 Butyrolactone – 0.60 Co-GC, MS, RI HLS-essential oilc HLS-aromac Identification methods 5 935 α-Pinene 14.82 19.72 Co-GC, MS, RI 6 950 Camphene 0.13 0.11 Co-GC, MS, RI 7 961 Benzaldehyde – 0.13 Co-GC, MS, RI 8 967 Nonane. 3-methyl 0.13 0.41 Co-GC, MS, RI 9 981 β-Pinene 0.35 0.30 Co-GC, MS, RI 10 991 β-Myrcene 0.54 2.53 Co-GC, MS, RI 11 1018 α-Terpinen – 0.14 Co-GC, MS, RI 12 1026 p-Cymene 0.52 1.26 Co-GC, MS, RI 13 1030 Limonene 0.87 4.09 Co-GC, MS, RI 14 1036 trans-β-Ocimene 0.41 0.84 Co-GC, MS, RI 15 1047 cis-β-Ocimene 8.94 17.13 Co-GC, MS, RI 16 1059 γ-Terpinene 0.23 0.35 Co-GC, MS, RI 17 1062 Decane. 2-methyl – 0.25 Co-GC, MS, RI 18 1091 Terpinolen 0.34 0.38 Co-GC, MS, RI 19 1099 Undecane – 28.21 Co-GC, MS, RI 20 1100 Linalool 7.66 tr Co-GC, MS, RI 21 1128 α-Campholenal – 0.96 Co-GC, MS, RI 22 1180 Terpinen-4-ol 0.20 – Co-GC, MS, RI Co-GC, MS, RI 23 1193 α-Terpineol 0.70 tr 24 1299 Tridecane – 0.21 Co-GC, MS, RI 25 1301 Thymol 0.81 – Co-GC, MS, RI 26 1358 α-Longipinene – 0.36 Co-GC, MS, RI 27 1382 α-Copaene 0.13 0.36 Co-GC, MS, RI 28 1391 β-Bourbonene 0.10 – Co-GC, MS, RI 29 1427 Caryophyllene 11.41 6.29 Co-GC, MS, RI 30 1434 Himachala-2.4-diene 0.95 – Co-GC, MS, RI 31 1446 Aromadendrene 1.51 – Co-GC, MS, RI 32 1462 Humulene 0.95 0.21 Co-GC, MS, RI 33 1483 γ-Muurolene 0.46 0.73 Co-GC, MS, RI 34 1490 Germacrene D 0.12 0.41 Co-GC, MS, RI 35 1506 α-Muurolene 0.80 – Co-GC, MS, RI 36 1521 γ-Cadinene 0.11 – Co-GC, MS, RI 37 1530 δ-Cadinene 0.94 tr Co-GC, MS, RI 38 1551 α-Calacorene 0.12 – Co-GC, MS, RI 39 1587 Spathulenol 0.74 – Co-GC, MS, RI 40 1594 Caryophyllene oxide 24.33 tr Co-GC, MS, RI 41 1602 Viridiflorol 0.30 – Co-GC, MS, RI 42 1620 Bisabolene epoxide 0.71 – Co-GC, MS, RI 43 1846 Hexahydrofarnesyl acetone 0.77 – Co-GC, MS, RI 44 2697 Heptacosane Co-GC, MS, RI 9.42 – Total identified (%) 91.24 92.09 Monoterpene hydrocarbons (%) 27.28 47.26 Oxygenated monoterpenes (%) 9.37 0.96 Sesquiterpene hydocarbons (%) 16.65 8.36 Oxygenated sesquiterpenes (%) 26.08 – Others (%) 11.86 35.51 Co-GC co-injection with authentic compounds, RI retention index literature comparison, tr trace a Kovats index on HP–5MS fused silica column 13 Method validation of 15 phytochemicals in Hypericum lysimachioides var. spathulatum by… Table 5 (continued) b A nonpolar Agilent HP-5MS fused silica column c Percentage concentration Fig. 2 Essential oil and aroma chromatograms of H. lysimachiodies var. spathulatum by GC–MS, a Essential oil TIC chromatogram b Aroma TIC chromatogram that the aroma and essential oil compositions of H. lysimachioides var. spathulatum were not similar and the aroma components were mostly in monoterpene and the essential oil components were mostly in sesquiterpene structure. While the LC–MS/MS method was developed for determining the chemical contents of the species belonging to Hypericum genus, the chemical contents of H. lysimachioides var. spathulatum species, whose essential oil, aroma and fatty acid content were not determined before, were also determined for the first time with this study. The results of biological activities Total phenolic and flavonoid content Total phenolic and flavonoid contents of HLS-A and HLS-R ethanol extracts were determined as equivalent to pyrocatechol and quercetin, respectively (Table 6). HLS-A ethanol extract was found to be more rich in phenolic and flavonoid contents than those of HLS-R extract. 13 M. Akdeniz et al. Antioxidant and toxic‑cytotoxic activities Cupric reducing antioxidant capacity (CUPRAC), DPPH free radical scavenging and ABTS cation radical decolorisation assays were used to determine the antioxidant capacities of HLS-A and HLS-R ethanol extracts, HLS-E, and three compounds (hyperoside, astragalin, and quercetin) which were the main constituents of HLS-A (Table 6). HLS-E was found to be not active in CUPRAC, DPPH free radical and ABTS cation radical scavenging methods. HLS-A, HLS-R ethanol extracts, hyperoside, astragalin, and quercetin (IC50: 30.78 ± 1.08 and 31.63 ± 0.98 μg/mL, 1.94 ± 0.16, 4.12 ± 0.12 and 1.09 ± 0.06 μg/mL, respectively), exhibited the best antioxidant effect in ABTS cation radical scavenging method among the mentioned antioxidant assays. HLS-A and HLS-R ethanol extracts showed moderate antioxidant activity in ABTS cation radical scavenging method, hyperoside and quercetin indicated higher antioxidant potential than the standards (α-TOC and BHT) in CUPRAC, DPPH free radical and ABTS cation radical scavenging methods (Table 6). As indicated in Table 6, HLS-A and HLS-R ethanol extracts, HLS-E, and three compounds (hyperoside, astragalin, quercetin) did not have a toxic effect on the Primary Dermal Fibroblast cell line (PDF, healthy cell). HLS-A and HLS-R ethanol extracts did not show cytotoxic effect against primary dermal fibroblast cell lines (PDF), breast cancer cell line (MCF-7) and colon cancer cell lines (HT-29) while HLS-E exhibited low cytotoxicity against MCF-7 and HT-29 cell lines. Hyperoside, astragalin and quercetin were found to be not cytotoxic against MCF-7 and HT-29 cell lines. In the literature, there are no studies on the total phenolicflavonoid content, antioxidant, toxic and cytotoxic effect of H. lysimachioides var. spathulatum. According to the previous studies on other Hypericum species, it can be said that this species have generally high antioxidant potential as indicated in this study. In a study conducted by Karatoprak et al. on H. scabrum L., its total phenolic content was determined to be 186.94 ± 2.66 mg GAE/g extract [10]. Additionally, it was determined that H. scabrum showed high antioxidant activity in ABTS, DPPH and β-carotene methods, and have cytotoxic effects on A549, COLO 205 and L929 cell lines. In another study conducted by Eruygur et al. [45], it was found that H. lydium Boiss. showed high antioxidant potential. Literature survey revealed that Hypericum species possess good antioxidant potential in addition to other pharmacological effects. Enzyme inhibitory activities This is the first study on the enzyme activity of H. lysimachioides var. spathulatum. The results of acetyl- and butyrylcholinesterase inhibitory activity of HLS-A and HLS-R 13 ethanol extracts, HLS-E, and three compounds (hyperoside, astragalin, quercetin) were shown in Table 6. None of the tested samples have inhibitory activity against acetylcholinesterase at 200 µg/mL, except for quercetin. Quercetin was found to be highly active against acetylcholinesterase (75.34 ± 3.12 inhibition%), and more active against butyrylcholinesterase (95.67 ± 1.37 inhibition%) than the standard compound (galanthamine). HLS-A (62.06 ± 1.06 inhibition%) and HLS-E (56.18 ± 1.12% inhibition) exhibited almost the same butyrylcholinesterase inhibitory activity with galanthamine (76.10 ± 0.23 inhibition%). Hyperoside (77.60 ± 1.65 inhibition%) showed the same inhibitory activity against butyrylcholinesterase with galanthamine. The antiurease and antityrosinase capacities of HLS-A and HLS-R ethanol extracts, HLS-E, and three compounds (hyperoside, astragalin, quercetin) were determined at 200 µg/mL (Table 6). Hyperoside (51.29 ± 1.05 inhibition%) and quercetin (65.25 ± 0.93 inhibition%) showed the highest antiurease activity among the tested samples. Quercetin (83.43 ± 1.65 inhibition%) exhibited the best antityrosinase effect among the tested samples. HLS-A and HLS-R ethanol extracts, and HLS-E showed very low antiurease and antityrosinase activities. Eruygur et al. found that H. lydium showed good anticholinesterase, α-amylase and antityrosinase inhibition activity [45]. Mahomoodally et al. [46] reported that H. lanuginosum Lam. exhibited moderate anticholinesterase and antityrosinase activities [46]. Conclusion A new LC–MS/MS method was developed to determine the chemical content of Hypericum species and this method was validated using the ethanol extract of the H. lysimachioides var. spathulatum species. Furthermore, the essential oil, aroma, fatty acid contents, and antioxidant, toxic-cytotoxic, anticholinesterase, antiurease and antityrosinase activities of this species were determined for the first time in the literature. The results of LC–MS/MS method developed and validated for Hypericum species indicated that the amounts of hyperoside, astragalin and quercetin were high in the ethanol extract of H. lysimachioides var. spathulatum aerial parts. GC–MS analyses showed that the main fatty acid was found to be cis-13,16-docosadienoic acid in HLS-A petroleum ether extract and stearic acid in that of HLS-R, the major component was caryophyllene oxide in HLS-E, and undecane in HLS-Ar. Among the tested antioxidant methods in this study, HLS-A and HLS-R exhibited moderate antioxidant activity in ABTS cation radical scavenging assay, and HLS-E was found to be not active. Hyperoside, astragalin and quercetin showed also the highest antioxidant effect in ABTS cation radical scavenging assay. None of the tested Antioxidant activity A0.5 (µg/mL) IC50 (µg/mL) Samples CUPRAC DPPH HLS-A 57.02 ± 1.02a 73.86 ± 1.31a HLS-R a 56.61 ± 1.01 b 80.21 ± 1.34 31.63 ± 0.98 38.49 ± 2.08 6.74 ± 0.07 HLS-E 177.84 ± 5.03b ≥ 200c 60.79 ± 0.92b – – ABTS Enzyme activity (200 µg/mL) Cytotoxic activity Inhibition% IC50 (µg/mL) Phenolic content (μg PEs/mg extract)a Flavonoid con- AChE tent (μg QEs/mg extract)b 30.78 ± 1.08a 59.60 ± 2.52a 30.75 ± 1.79a a b b BChE 16.04 ± 2.62a b Urease 62.06 ± 1.06a b 5.34 ± 2.20 38.84 ± 0.66 4.12 ± 2.00c 56.18 ± 1.12c Tyrosinase 2.72 ± 1.22a PDF 20.40 ± 1.92a b b HT-29 > 200a a 22.28 ± 1.04 26.80 ± 0.86 > 200 NA 1.73 ± 1.12c > 200 a MCF-7 > 200 a > 200 a 176.14 ± 2.53b > 200 a > 200 a 152.75 ± 3.29b Hyperoside 5.32 ± 0.01c 5.23 ± 0.32d 1.94 ± 0.16c – – NA 77.60 ± 1.65e 51.29 ± 1.05c NA > 200 a > 200 a > 200 a Astragalin 29.43 ± 0.76d > 200c 4.12 ± 0.12d – – NA NA NA NA > 200 a > 200 a > 200 a Quercetin 3.21 ± 0.02e 2.39 ± 0.37e 4 f 1.09 ± 0.06c f – – 75.34 ± 3.12d 95.67 ± 1.37f 65.25 ± 0.93d 83.43 ± 1.65d > 200 a > 200 a > 200 a e α-TOC 22.42 ± 0.01 13.10 ± 0.52 12.48 ± 0.63 – – – – – – – – – BHT4 7.23 ± 0.02 g 62.15 ± 0.35 g 12.62 ± 0.28e – – – – – – – – – Galanthamine4 – – – – – 89.12 ± 0.64e 76.10 ± 0.23e – – – – – Kojic acid4 – – – – – – – – 91.64 ± 0.23e – – – Thioureac – – – – – – – 94.64 ± 0.16e – – – – Values expressed are means ± S.D. of three parallel measurements and values were calculated according to negative control, Values with different letters in the same column were significantly different (p < 0.05) NA not active a PEs, pyrocatechol equivalents (y = 0.0435 + 0.0648 pyrocatechol (μg) (r2 = 0.9911) b QEs, quercetin equivalents (y = 0.0333 + 0.2016 quercetin (μg) (r2 = 0.9961) c Standard compound Method validation of 15 phytochemicals in Hypericum lysimachioides var. spathulatum by… Table 6 Antioxidant activities with total phenolic-flavonoid contents, anticholinesterase, urease, tyrosinase enzyme inhibition and cytotoxic activities of H. lysimachioides var. spathulatum ethanol extracts, essential oil and major compounds 13 M. Akdeniz et al. samples have cytotoxic capacity against PDF, MCF-7 and HT-29. Quercetin exhibited the highest inhibitory effect against acetyl- and butyryl-cholinesterase, urease and tyrosinase among the tested samples. Hypericum species consumed for medicinal purposes all around the world, and since phytochemical contents of Hypericum species differ from each other and their parts, it is important to determine qualification and quantification of their bioactive phytochemicals. Thus, in the current study a comprehensive LC–MS/MS method was developed to identify fingerprint phytochemicals in H. lysimachioides var. spathulatum and the analytical method was validated. The developed LC–MS/MS method is a method that can be used not only for the studied species but also for other Hypericum species. 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