{Phenolic profiling and bioactivities of fresh fruits and jam of Sorbus species} J. Serb. Chem. Soc. 82 (6) 651–664 (2017) UDC 634.18:576+615.9:615.279: JSCS–4994 615.281:577.164.2 Original scientific paper 651 Phenolic profiling and bioactivities of fresh fruits and jam of Sorbus species ZORICA O. MRKONJIĆ1*, JELENA D. NAĐPAL1#, IVANA N. BEARA1#, VERICA S. ALEKSIĆ SABO2, DRAGANA D. ČETOJEVIĆ-SIMIN3, NEDA M. MIMICA-DUKIĆ1# and MARIJA M. LESJAK1# 1Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia, 2Department of Biology and Ecology, Faculty of Sciences University of Novi Sad, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia and 3Oncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Dr Goldmana 4, 21204 Sremska Kamenica, Serbia (Received 2 February, revised 6 March, accepted 10 April 2017) Abstract: The purpose of this study was a comprehensive examination of the phenolic profile, the vitamin C content, and the antioxidant, anti-acetylcholine- sterase, cytotoxic and antimicrobial activities of extracts and jams of fruits of two edible Sorbus species: well characterized S. aucuparia and two insuf- ficiently explored forms of S. torminalis (torminalis and semitorminalis). Char- acterisation of 44 phenolics was realized using LC–MS/MS and 15 compounds were confirmed, with chlorogenic acid being the most dominant in S. aucup- aria and ferulic acid in both S. torminalis forms. S. aucuparia demonstrated potent antioxidant activity, while that of both S. torminalis forms was mod- erate. Jam extracts had the highest content of vitamin C. S. aucuparia exhibited some anti-acetylcholinesterase activity, while S. torminalis f. torminalis showed the best antimicrobial activity against Staphylococcus aureus and both forms (torminalis and semitorminalis) possessed the highest activity against Escher- ichia. coli. The results obtained herein are a great base for further research of edible Sorbus species with the aim of promoting their better usage as nutra- ceuticals. Keywords: phenolics; antioxidant; anti-acetylcholinesterase activity; cytotoxic activity; antimicrobial activity. INTRODUCTION In spite of their widespread use in diet, preservatives preparation, beverage manufacture and traditional medicine (the details are presented in the Supple- * Corresponding author. E-mail: zoricamrkonjic@gmail.com # Serbian Chemical Society member. https://doi.org/10.2298/JSC170202049M 652 MRKONJIĆ et al. mentary material to this paper), there are still very few reports concerning the detailed chemical composition and biological activities of fruits of different Sor- bus species. Bearing this in mind, in this paper, the fruits of the well-known S. aucuparia and the unexplored S. torminalis f. torminalis and S. torminalis f. semitorminalis were the subjects of extensive study. Namely, within the Sorbus genus, the most renowned species is S. aucuparia (rowan), which is edible for humans and domestic animals. It was confirmed that rowan fruits contain a high content of natural products, such as phenolics,1–6 vitamins C, B1, B2, E and K,5,7 carotenoids,8 carboxylic acids9 and sugars,7 which surely contribute to their salubrity. S. aucuparia is a very variable species and occurs in nature as five subspecies: S. aucuparia subsp. aucuparia, S. aucu- paria subsp. fenenkiana, S. aucuparia subsp. glabrata, S. aucuparia subsp. prae- morsa and S. aucuparia subsp. sibirica. In this study, S. aucuparia subsp. aucu- paria var. aucuparia fruits were the subject of in-depth investigation whereby their detailed phenolic profile was elucidated for the first time. Herein, fresh fruit extracts and jam were investigated, as those are the forms in which the fruits of Sorbus species are usually consumed. On the other hand, other species from Sorbus genus, such as S. torminalis (wild service or chequer tree), have been poorly studied to date. As the fruits of S. aucuparia, the fruits of S. torminalis are commonly present in human and animal diet. This species occur in wide variety of forms, mostly differing in leaf shape. In this paper, two forms of S. torminalis, f. torminalis and f. semitormin- alis, were studied. The morphological differences between leaves of these two forms are barely noticeable, while their colour and fruits are identical. The only variations between leaves are hairs on underside, which are hard to observe. Specifically, leaves of f. torminalis are hairy only during a short period when young, while those of f. semitorminalis are permanently covered with hairs.10 Thus, due to their strong morphological similarities, these two forms could be easily mistaken for each other if they were not harvested by professional plant collectors. Consequently, it was considered worthwhile to determine whether their fruits have a similar chemical composition and biological potency, and con- clude if substituting one of these species with the other in functional foods could result in quality differences. While the antioxidant and anti-acetylcholinesterase (AChE) activities, as well as total phenolic and flavonoid contents of S. tormin- alis fruits have only been poorly investigated,11,12 no literature data related to the detailed phenolic profile, vitamin C content and other biological activities of these fruits has hitherto been reported. For this reason, the present research was focused on the examination and comparison of the phenolic profile, vitamin C content, and antioxidant, anti-AChE, cytotoxic and antimicrobial activities of water and methanol extracts of the edible fresh fruits, and traditionally prepared Sorbus – CHEMICAL AND BIOACTIVITIES PROFILING 653 jams of S. aucuparia and of the uninvestigated S. torminalis f. torminalis and S. torminalis f. semitorminalis plant species. EXPERIMENTAL Experimental details related to the employed chemicals and reagents, plant material col- lection and extracts preparation, are given in the Supplementary material to this paper. LC–MS/MS analysis of single phenolic compounds Determination of selected phenolic compounds in the extracts of S. aucuparia and both S. torminalis forms, torminalis and semitorminalis, was performed according to a previously reported procedure.13 Additional brief details are given in the Supplementary material. Vitamin C content The vitamin C content was determined by a method given in the literature14 adapted for 96-well microplates.15 Briefly, each sample was evaporated in vacuo at 40 °C and mixed with metaphosphoric acid (0.1 g mL-1) to obtain final concentrations of 60, 90 and 120 mg mL-1 for all extracts, except for the extracts of jam that were prepared in concentrations of 40, 60 and 80 mg mL-1. The mixtures were stirred for 45 min at room temperature. The prepared extracts in metaphosphoric acid (30 µL) were mixed with 270 µL of 2,6-dichlorophenolindophenol (72 mg mL-1) and the absorbance at 515 nm was measured within 5 min. The vitamin C content was determined using a standard calibration curve of vitamin C (ranging 0‒320 μg mL-1) and the results are presented as the mean value of three measurements. Antioxidant and anti-AChE activities The scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion (O2•-), nitric oxide (•NO) and hydroxyl (HO•) radical, reducing power (FRAP assay) and inhibition of lipid peroxidation (LP) were tested according to previously published methods.16 To evaluate the inhibitory activity of the extracts, the AChE Ellman method17 with numerous modific- ations was used. Details of these experiments are given in the Supplementary material. Cell growth activity Preparation of the samples and standards, experimental conditions for maintenance of the cell lines, and the sulforhodamine B (SRB) assay procedure were performed according to previously published procedures.16 Each experimental method is briefly explained in the text below. Preparation of samples and standards. For the evaluation of the cell growth activity, aqueous stock solutions of the extracts and podophyllotoxin (10 mg mL-1 in dimethyl sulfox- ide (DMSO)) were diluted in NaCl (9 mg mL-1) to obtain ranges of concentrations 0.1‒1000 and 0.00001‒1 μg mL-1, respectively. Maintenance of cell lines. Cell growth activity was evaluated in vitro using human cell lines: HeLa (cervix epitheloid carcinoma, European Collection of Authenticated Cell Cultures (ECACC), No. 93021013), MCF7 (breast adenocarcinoma, ECACC No. 86012803), HT-29 (colon adenocarcinoma, ECACC No. 91072201) and MRC-5 (human foetal lung, ECACC 84101801). The cell lines were grown in Dulbecco-modified Eagle's medium with 4.5 % glucose, supplemented with 10 % heat-inactivated foetal calf serum (FCS), 100 IU mL-1 penicillin and 100 μg mL-1 streptomycin. The cells were cultured in 25-cm2 flasks at 37 °C, in a 5 % CO2 atmosphere of high humidity, and sub-cultured twice a week. A single cell suspension was obtained using 0.1 % trypsin with 0.04 % EDTA. 654 MRKONJIĆ et al. SRB assay. The cell lines were harvested and plated into 96-well microtitre plates at seeding density of (3–5)×103 cells well-1 in a volume of 199 or 180 μL, and pre-incubated in complete medium supplemented with 5 % FCS at 37 °C for 24 h. Serial dilutions of the ext- racts (1 μL) were added into 199 μL of medium, while serial dilutions of podophyllotoxin (20 μL) were added into 180 μL of medium, in order to achieve the required final concentrations. All samples were filtered through 0.22 μm microfilters to obtain sterility. Equal volumes of water and DMSO were added in the control wells. The concentration of DMSO in the cell cultures was ≤ 5 μL mL-1. After adding the dilutions, the microplates were incubated at 37 °C for 48 h. The cell growth was evaluated by the colorimetric SRB assay.18 Colour development was measured using photometer at 540 nm against 620 nm as the background. Antimicrobial activity To evaluate minimum inhibitory concentration (MIC) of the extracts, the method for the determination of the antimicrobial activity of antimicrobial agents was used.19 First, two-fold dilutions of the tested plant extracts were prepared in microtitre plates. The final concentration of each extract ranged 1‒128 mg mL-1. Subsequently, a bacterial suspension, previously adjusted to match a turbidity of a 0.5 McFarland nephelometer standard, was diluted in double-strength Mueller–Hinton medium (1:100 volume ratio). Into each well, inoculated double-strength medium were added to the same volume as each extract (1:1 volume ratio). The number of the bacteria in the assay was about 1×106 colony forming units (CFU) mL-1. Antimicrobial activity of the extracts was tested against Gram negative bacteria, Escherichia coli American Type Culture Collection (ATCC) 25922 and Gram-positive bacteria, Staphylo- coccus aureus ATCC 25923. The microtitre plates were incubated overnight at 37 °C, without shaking, amended with a 10 μL of a 1 % solution of 2,3,5-triphenyltetrazolium chloride and incubated additionally for 2 h until development of the red colour. Controls for plate sterility and bacterial growth without extracts were also included. The lowest concentration of extracts that inhibited bacterial growth, which was identified by the absence of red formazane, was considered as the MIC. The MIC determination was performed in three replicates and three independent experiments. RESULTS AND DISCUSSION Phenolic profile Qualitative and quantitative analyses of 44 phenolics in all extracts were performed using the LC–MS/MS technique. The content of the 12 determined phenolics are presented in Table I and differences between the species and two S. torminalis forms are evident. Concerning phenolic acids, chlorogenic acid was the most abundant compound in S. aucuparia extracts, while ferulic acid was the most dominant in extracts of both S. torminalis forms. Analyses of the selected flavonoids showed that all extracts contain amentoflavone, which was dominant flavonoid in both S. torminalis forms, especially in semitorminalis form. Addit- ionally, rutin, quercetin-3-O-glucoside and hyperoside were characteristic for extracts of S. aucuparia. Furthermore, coumarins and lignans were not detected, with the exception of aesculetin, which was present only in S. torminalis f. semitorminalis. Sorbus – CHEMICAL AND BIOACTIVITIES PROFILING 655 It is possible to see a common trend among the extracts concerning deter- mined phenolic amounts was recognizable. Namely, aqueous methanol was more efficient than the extraction in water, while jam extracts contained significantly lower amounts of most phenolics, than did the fresh fruits. TABLE I. Contents of detected phenolics (µg g-1 dw) in S. aucuparia and S. torminalis (forms torminalis and semitorminalis) fruit extracts; means within each row with different letters (a‒i) differ significantly (p ≤ 0.05); compounds analyzed, but the peak was below the limit of quantification (LOQ):13 p-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, vanillic acid, cin- namic acid, caffeic acid, syringic acid, o-coumaric acid, p-coumaric acid, 3,4-dimethoxycin- namic acid, sinapic acid (phenolic acids); apigenin, apigenin-7-O-glucoside, baicalin, baica- lein, apiin, daidzein, naringenin, vitexin, genistein, isorhamnetin, luteolin, luteolin-7-O-gluco- side, myricetin, kaempferol, epigallocatechin gallate, chrysoeriol, quercetin, epicatechin (fla- vonoids); umbelliferone, scopoletin (coumarins); matairesinol, secoisolariciresinol (lignans) W – water extract, M – methanol extract, J – jam Compound Species S. aucuparia S. torminalis f. torminalis S. torminalis f. semitorminalis W M J W M J W M J Phenolic acids Gallic acid > /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. 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