Hindawi BioMed Research International Volume 2019, Article ID 3692605, 10 pages https://doi.org/10.1155/2019/3692605

Research Article Chemical and Bioactivity Evaluation of planum and Cnicus benedictus Polyphenolic-Rich Extracts

Gabriela Paun,1 Elena Neagu,1 Veronica Moroeanu,1 Camelia Albu,1 Simona Savin,1 and Gabriel Lucian Radu 1,2

 National Institute for Research-Development of Biological Sciences, Bucharest , , Romania Faculty of Applied Chemistry and Materials Science, Politehnica University of Bucharest, Bucharest, Romania

Correspondence should be addressed to Gabriel Lucian Radu; [email protected]

Received 25 January 2019; Accepted 26 February 2019; Published 12 March 2019

Academic Editor: Min-Hui Li

Copyright © 2019 Gabriela Paun et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Tis study evaluated the biological activities of Eryngium planum and of Cnicus benedictus extracts enriched in polyphenols obtained by nanofltration. Te HPLC-MS analysis showed that E. planum contains mainly favonoids, especially rutin, while in C. benedictus extracts show the high concentration of the phenolic acids, principally the chlorogenic acid and sinapic acid. Herein, there is the frst report of ursolic acid, genistin, and isorhamnetin in E. planum and C. benedictus. C. benedictus polyphenolic-rich extract showed high scavenging activity (IC50=0.0081 mg/mL) comparable to that of standard compound (ascorbic acid) and a higher reducing power (IC50=0.082mg/mL),withIC50 having a signifcantly lower value than IC50 for ascorbic acid. Both extracts were nontoxic to NCTC cell line. Among the investigated herbs, E. planum polyphenolic-rich extract showed the highest inhibitory activities with the IC50 value of 31.3 �g/mL for lipoxygenase and 24.6 �g/mL for hyaluronidase. Both polyphenolic-rich extracts had a higher inhibitory efect on �-amylase and �-glucosidase than that of the acarbose. Te synergistic efect of ursolic acid, rutin, chlorogenic acid, rosmarinic acid, genistin, and daidzein identifed in polyphenolic-rich extracts could be mainly responsible for the pharmacological potentials of the studied extracts used in managing infammation and diabetes.

1. Introduction Lipoxygenases, which catalyse the addition of oxygen to arachidonic acid and other polyunsaturated fatty acids, Recently, the research is more and more focused on the and hyaluronidase, which hydrolyses hyaluronic acid, are polyphenol’s efect on the human health and on the natural involved in the infammatory process [6, 7]. antioxidants sources with low or no side efects to be used Development and use of efcient technologies for the in medicine and in the food industry [1]. Data from literature bioactive compound’sextraction and concentration represent frmly support the role of phenolic compounds in the preven- a current priority. Nowadays, ultrasonic-assisted extraction tion of degenerative diseases, such as neurodegenerative dis- (UAE) and membrane technology have become the most eases and diabetes mellitus [2–4]. Several studies showed that important and efcient technique for extraction [7, 8], sep- the �-amylases and �-glucosidases have signifcant activities aration, and concentration of the phenolic compounds from in the metabolism of polysaccharides. Dietary interventions various sources [8–13]. based on plant natural inhibitors could successfully replace Eryngium planum L. ( family) is a medicinal commercially drugs, such as acarbose, in the management of plantusedinEuropeanandAsiantraditionalmedicine, hyperglycaemia. Te beneft stays in that there are no adverse especially for various infammatory disorders treatment. Te side efects [5]. Eryngium aerial part bioactivity is mainly generated Te frequent use of nonsteroidal anti-infammatory drugs by polyphenols and [14–16]. (NSAIDs) with various side efects determined lately a new Cnicus benedictus L. (blessed thistle) belonging to the direction in research toward the natural sources, efective family Asteraceae is mainly distributed from Asia Minor against infammatory disorders and with few side efects. through southern Europe. Tis medicinal plant contains 2 BioMed Research International alkaloids, cnicin, benedictin, mucilage, polyacetylene, triter- solvents and reference compounds (p-coumaric acid, cafeic penoide, lignans, favonoids, tannin, phytosterines, and acid, ferulic acid, gallic acid, chlorogenic acid, rosmarinic volatile oils [17–19]. Tis is a plant with antidepressive, anti- acid, ellagic acid, rutin, luteolin, quercetin, quercetin 3- infammatory, antiseptic, cardiac, and antimicrobial prop- �-D-glucoside, apigenin, kaempferol, daidzein, genistein, erties and cnicin is the compound responsible for blessed and genistin) were purchased from Sigma (Sigma Aldrich, thistle’s medicinal properties [20]. Germany), Fluka (Switzerland), and Roth (Carl Roth GmbH, Tere are only several studies on the chemical composi- Germany). tion of these herbs, yet very few phytopharmacological stud- Te liquid chromatography analysis was performed using ies have been exhaustively investigated up till now. Taking a HPLC SHIMADZU system coupled to a MS detector, into account the considerations mentioned above, the present LCMS-2010 detector (liquid chromatography mass spec- study was undertaken to investigate the anti-infammatory trometer), and equipped with an electrospray ionization and the antidiabetic activities of Eryngium planum and (ESI) interface. For analysis, a C18 Kromasil 3.5, 2.1x100mm Cnicus benedictus polyphenolic-rich extracts. In addition, column was used. Te mobile phase was composed of solvent the antioxidant activity and cytotoxicity of polyphenolic-rich A (formic acid in water, pH=3.0) and solvent B (formic extracts of selected herbs were also investigated. acid in acetonitrile, pH=3.0). Te polyphenolic compound’s However, up to date, these characteristics of E. planum separation was performed using binary gradient elution: 0 and C. benedictus polyphenolic-rich extracts have not yet min 5% solvent B; 0.01-20 min 5-30% solvent B; 20-40 min been studied; therefore, no reference is available regarding 30% solvent B; 40.01-50 min 30-50% solvent B; and 50.01-52 the UAE and the membrane concentration of polyphenolic min 50-5% solvent B. Te fow rate was 0-5 min 0.1 mL/min; compounds from these herbs. 5.01-15 min 0.2 mL/min; 15.01-35 min 0.1 mL/min; 35.01-50 min 0.2 mL/min; and 50-52 min 0.1 mL/min. Te quantitative 2. Materials and Methods analysis of the components was achieved with reference to standards. .. Plant Material. Eryngium planum herb was collected For ursolic acid HPLC-MS determination, a C18 Nucle- from Valcea district (Romania) and Cnicus benedictus herb osil 100-3.5, 100x2.1 mm, KROMASIL column was used. was collected from Razhdak district (Bulgaria). Te voucher Mobile phase was formic acid in water (solvent A) and specimen number 657490 for Eryngium planum and voucher methanol (solvent B). Te fow rate for solvent A was 0.042 specimen number 665998 for Cnicus benedictus were pre- mL/min and for solvent B was 0.258 mL/min. Te injection served in the Botanic Garden of Cluj-Napoca, Romania. volume was 20 �L and the temperature of the column was ∘ kept constant at 20 C. ESI source and negative ionization .. Extraction and Processing. Dried aerial parts were mode were used. grounded (10 g), mixed with the 50% (v/v) ethanol in water as solvent, and shaken for 5 minutes for the extraction process. .. Antioxidant Activity Next, the extraction was performed by ultrasonic-assisted extraction (UAE) using a sonication water bath (Elma ... DPPH Radical Scavenging Activity. Te 2,2-diphenyl- Transsonic T 460, Germany) at 35 kHz for 90 min. Afer these 1-picrylhydrazyl (DPPH) radical scavenging activity of the steps, extracts were fltered through the flter paper under polyphenolic-rich extracts was performed according to the vacuum and microfltered through 0.45 �mporesizemicro- method described by Bondet with some modifcation [24]. fltration membrane (Millipore). Nanofltration was applied 100�l sample extract or standard (ascorbic acid) with varying to concentrate biologically valuable components and obtain concentrations was mixed with 2900 mL of DPPH∙ methano- polyphenolic-rich extracts. Te nanofltration was performed lic solution and the absorbance was measured at 519 nm afer with a tangential fltration KOCH Membrane laboratory unit 30 min of reaction. equipped with an NF90 (Dow Filmtec, Sterlitech Company, 3+ 2+ USA), with a molecular weight cut-of of 200–300 Da, made ... Reducing Power. Te reduction of Fe into the Fe of polyamide material. potential of the extracts was determined according to the method of Baba and Malik [25]. In brief, 100 �Lofextract,2.5 .. Total Phenolic and Content. Te total phenolic mL phosphate bufer (0.2M, pH 6.6), and 2.5 mL potassium ∘ content was determined using Folin-Ciocalteu assay [21]. ferricyanide (1%) were mixed and incubated at 50 Cfor20 Te total phenolic content was calculated on the basis of 2 min. Ten, 2.5 mL trichloroacetic acid (10%) was added, the a calibration curve of gallic acid (y=0.0027x+0.017, R = mixture was centrifuged, and the upper layer (2.5 mL) was 0.9965)andexpressedasmggallicacidequivalentsperlitter mixed with 2.5 mL of deionized water and 0.5 mL of 0.1% of extracts (mg GAE/L). ferric chloride; then the absorbance was measured at 700 nm; Te total favonoid content was measured using the AlCl3 ascorbic acid was used as standard. colorimetric method as described by Lin [22]. Results were expressed as quercetin (QE) mg/mL using the equation based 2 .. In Vitro Anti-Inflammatory Assay on the calibration curve: y = 0.004x + 0.0535; R = 0.9934. ... Lipoxygenase (LOX) Inhibition. Te LOX inhibition .. HPLC-MS Analysis. Te analysis of polyphenols was was performed by the spectrophotometric assay [26]. Te performed using a validated HPLC-MS method [23]. All increase in absorbance at 234 nm was due to the formation of BioMed Research International 3

the product 13-hydroperoxyocta-decadienoic acid from the where �ctr is the absorbance of the control (without extract/ reaction of oxygen and linoleic acid catalysed by lipoxyge- acarbose) and �s is the absorbance of the sample extract. nase. For the blank solution, 100 �L LOX solution 2200 U/mL was mixed with 100 �Llinoleicacidand3050�L boric acid . .. �-Glucosidase Inhibition Assay. �-Glucosidase inhibi- bufer 0.2 M (pH 9.0). For samples, 50 �Lofboratebuferwas tion was evaluated using the procedures described in the replaced with 50 �L of the extract. Te mixture was incubated literature [30]. Sample solution (50 �L) was preincubated for 15 min; then 100 �Llinoleicacidwasaddedtothemixture with 125 �Lof�-glucosidase from Saccharomyces cerevisiae and the absorbance was read at 234 nm for 2 minutes. Te (0.5 U/mL in phosphate bufer solution, pH 6.9) and 700 �Lof ∘ % inhibition for diferent concentrations of the extracts was phosphate bufer at 37 C for 15 minutes. Ten, 125 �Lof5mM calculated as p-nitrophenyl glucopyranoside (pNPG) was added to quench ∘ the reaction and the mixture was kept at 37 Cfor30minutes. Δ��/Δ� � ( ) =(1− ) × 100 Te reaction was stopped by adding 1000 �L of 0.2M Na2CO3 % Δ� /Δ� (1) ��� and the absorbance was recorded at 405 nm. Control sample was prepared to contain bufer instead of enzyme. Acarbose where As and Actr are the increase of absorbance for the sample extracts and the bufer instead of inhibitor (sample). wasusedaspositivecontrol. Ibuprofenisusedasstandard. � −� ��ℎ������� ( ) =( ����� ������ ) × 100 % � −� (4) ... Hyaluronidase (HYA) Inhibition. Te hyaluronidase ����� ������� inhibition was determined by the method described by where Ablank stands for the absorbance of the mixture Sahasrabudhe and Dedhar with slight modifcations [27]. without extract samples; Asample stands for the absorbance � Briefy, 100 l of hyaluronidase (type-1-S from bovine, 400 of polyphenolic-rich extract samples; Acontrol stands for the � units/mL) in acetate bufer (0.1 M, pH 3.5) and 50 Lof absorbance of the mixture without a-glucosidase. ∘ � plant extract was incubated for 20 min at 37 C; then 100 L IC50 value (concentration of phenolic-rich extracts of CaCl2 solution 12.5 mM was added and the mixture was ∘ required to inhibit 50% of the enzyme activity) was calculated incubated also at 37 C for 20 minutes. Ten, the reaction graphically by dose-dependent inhibition. was initiated by adding 250 �Lofsodiumhyaluronate(1.2 ∘ mg/mL) and incubated at 37 Cfor40min;100�Lofsodium . . Cytotoxicity. Te cell line of mouse fbroblast (NCTC hydroxide solution 0.4 M and 100 �L of potassium borate (0.4 clone 929 from the European Collection of cell Culture, M) were added to the reaction mixture and incubated for 3 Sigma-Aldrich, USA) was used to determine the cell viability min in a boiling water bath. Afer cooling, 3000 �Lof10%�- by the MTT test. Fibroblast cultures (NCTC) were grown in dimethylaminobenzaldehyde reagent was added and afer 20 MEM containing 10% fetal bovine serum (FBS) and PSN. Te minutes the increase in absorbance was measured at 585 nm. 4 Te percentage of enzyme inhibition was calculated as NCTC cell line was inoculated at 4 x 10 cells/mL density. Te cells cultured in standard conditions adhered to plastic Δ�� afer 24 hours of incubation; then the medium of culture � (%) =(1− ) × 100 (2) Δ���� changed with medium containing various concentrations of plant extracts (100, 250, and 500 �g/mL). Te plates with 96 where As and Actr are the absorbance of the sample extracts wells were incubated for 24 and 72 hours at 37C in 5% CO2 air and absorbance of control (bufer instead of inhibitor). atmosphere. Te culture control was untreated cells cultivated Ibuprofenisusedasstandard. in MEM and 10% FBS, and the positive control was H2O2 (2�L/mL). Afer incubation with MTT solution (3 hours, at ∘ . . In Vitro Antidiabetic Assays 37 C), the plates were put on a shaker for 15 min and the absorbance was read at 570 nm on a microplate Mithras LB . .. Inhibition of �-Amylase Activity. Te �-amylase inhib- 940 (Berthold Technologies). itory activity was evaluated according to Ranilla et al. with For the morphology, the culture of mouse fbroblast slight modifcation [28], as a previous reported paper [29]. cells (NCTC), fxed in methanol and Giemsa stained, was In brief, the plant extract and �-amylase from hog pancreas observed afer 72 hours afer the addition of the extracts and ∘ solution (0.5 mg/mL) were incubated at 37 Cfor20min. acquired with Zeiss AxioStar Plus microscope (Carl Zeiss, Afer that, a 1% starch solution was added and incubated at Germany). thesametemperaturefor30min.Tereactionwasstopped with dinitrosalicylic acid colour reagent. Te mixture was . . Statistical Analysis. All values are expressed as the mean incubated in a boiling water bath for 5 min, cooled to ± standard deviation and analyzed using Microsof Excel. room temperature, and diluted with distilled water and the Values were considered signifcant when p value is lower than absorbance was recorded at 540 nm. Acarbose was used as 0.05. positive control. Te rate of enzyme inhibition was calculated according to the following equation: 3. Results and Discussion

Δ�� .. Phytochemical Composition and Antioxidant Activity. � (%) =(1− ) × 100 (3) Δ���� Polyphenols (phenolic acids and favonoids) are compounds 4 BioMed Research International

Table 1: Te active biological compounds content and antioxidant activity of extract fractions.

TPC, TFC, DPPH, Reducing power, Medicinalherb Fraction mgGAE/mL mgQE/mL EC50,mg/mL EC50,mg/L E. planum MF 0.749±0.062 0.176±0.014 0.316±0.034 0.209±0.020 NF retentate 1.882±0.104 0.378±0.035 0.197±0.031 0.128±0.012 C. benedictus MF 1.847±0.134 0.223±0.020 0.711±0.042 0.140±0.012 NF retentate 2.659±0.128 0.478±0.045 0.0081±0.0005 0.082±0.006 Ascorbic acid 0.0012±0.0001 0.125±0.010 MF, microfltration process; NF, nanofltration process. Antioxidant activity is expressed as half inhibitory concentration (EC50); values are expressed as mean ± standard deviation of three measurements.

Table 2: HPLC-MS polyphenolic profle of extracts fractions.

Eryngium planum Cnicus benedictus Class Compound � � [M/z]- g/mL g/mL MF. Conc. MF. Conc. Rutin [609] 183.63 290.52 1.87 3.56 Kaempferol [285] 3.55 4.20 0.62 0.73 Flavonols Isoquercetin [463] 23.40 36.11 7.54 9.75 Isorhamnetin [477] 0.17 0.47 - - Quercetol [301] 2.06 3.03 - - [285] 0.72 0.93 0.50 0.62 Flavone Luteolin Apigenin [269] - - 0.20 0.56 Cafeic acid [19] 4.51 5.68 4.07 5.58 Rosmarinic acid [359] 15.53 18.95 7.62 10.17 [193] Phenolic acids Ferulic acid 6.39 7.07 - - Chlorogenic acid [353] 40.33 63.02 315.11 454.29 p-Coumaric acid [163] 4.29 4.66 7.52 8.35 Sinapic acid [223] 0.67 9.93 109.74 249.85 Ellagitannins Ellagic acid [301] 1.29 1.32 1.19 1.54 Genistein [269] 1.42 1.49 2.20 2.47 Isofavones Genistin [433] 2.09 2.80 1.80 2.61 Daidzein [253] 0.17 0.35 2.31 4.68 “-”, under limit of detection. exhibiting strong antioxidant activity and are responsible Te HPLC-MS results showed that the extracts from for these ’ medicinal properties. Te nanofltration E. planum contain mainly favonoids, especially rutin and process (NF) was used for the polyphenol’s concentration to isoquercitrin, while in C. benedictus extracts show the high obtain the polyphenolic-rich extracts in the present study. concentration of the phenolic acids, especially the chloro- Te extracted fractions were evaluated by means of total phe- genic acid and sinapic acid, in agreement with data reported nolic compound (TPC), total favonoid content (TFC), and in the literature [14, 31]. antioxidant capacities (DPPH and reducing power assays) Our study’s signifcant contribution consists of the iden- (Table 1). tifcation and quantifying of new polyphenols for the studied Te results indicated a good concentration performance herbal extracts. However, this is the frst time that genistin of the membrane processes, resulting in a signifcant increase and isorhamnetin are reported in E. planum and C. benedictus of the polyphenols contents in the nanofltrate fractions. extracts. C. benedictus nanofltrate retentate was found to have the In the studied extracts, ursolic acid, a triterpene respon- highest TPC level (2.659 ± 0.128 mg GAE/mL), of which over sible for many pharmacological efects, was identifed and 17.5%(0.478 ± 0.045 mg QE/mL) can be attributed to the total quantifed also. Ursolic acid was concentrated from 76.77 favonoids. �g/mL to 158.04 �g/mL in E. planum extracts and from 46.84 Te polyphenolic compounds present in the MF and NF �g/mL to 146.50 �g/mL in C. benedictus extracts. retentate fractions of E. planum and C. benedictus extracts Antioxidant activity has been studied also, due to the were determined by HPLC-MS and the data presented in fact that oxidative stress is considered to be the key factor Table 2 can be correlated with Figures 1 and 2. in the anti-infammatory process and in the pathogenesis of BioMed Research International 5

Table 3: Lipoxygenase and hyaluronidase inhibitory activity of E. planum and C. benedictus extract fractions.

IC50, �g/ml Extract fractions LOX HYA E. planum extract MF 104.8±9.6 37.9±2.1 NF retentate 31.3±2.1 24.6±1.5 C. benedictus extract MF 115.1±7. 2 1 2 5 . 9 ±8.4 NF retentate 52.7±3.4 93.6±7. 0 Ibuprofen 69.7±0.005 14.3±0.8 Data are expressed as mean ± SD of triplicate determinations.

(×1,000,000) a similar reducing power to control but a lower scavenging 2.25 10 activity comparable to that of ascorbic acid. A signifcant 2.00 3 7 9 correlation (p< 0.05) was found between antioxidant activity 1.75 1 6 and polyphenol and favone contents (Table 1S from sup- 1.50 12 plementary material), indicating a signifcant contribution 1.25 2 5 of these compounds to the antioxidant activity for these 1.00 polyphenolic-rich extracts. 0.75 0.50 Te bioactive compounds from herbal extracts con- centrated by ultrafltration (UF) and nanofltration (NF) 0.25 4 8 11 0.00 have been recently investigated as an alternative solution to the conventional methodologies. Paun and collaborators 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 tested the ultrafltration membrane for the concentration Figure 1: HPLC-MS chromatograms of the Eryngium planum of antioxidants from Phyllitis scolopendrium L. extract [32]; polyphenolic-rich extract (1, chlorogenic acid, 2, p-coumaric acid, � Tylkowsky et al. investigated the NF process using with 3, rutin, 4, quercetin 3- -D-glucoside, 5, ferulic acid, 6, rosmarinic membranes with MWCO in the range of 200–500 Da for acid,7,daidzein,8,luteolin,9,quercetol,10,genistein,11,kaempferol, the treatment of ethanolic extracts from Sideritis spp. L. in and 12, isorhamnetin). order to concentrate polyphenols [33]; Conidi and collabora- tors concentrated clarifed artichoke wastewater using a NF (×1,000,000) membrane (NF 270, polyamide) with MWCO of 200–300 2.75 2.50 Da [34]. All these researches proved the efciency of both 2.25 ultrafltration and nanofltration procedures to recover and 2.00 concentrate polyphenols from diferent herbal extracts. 1.75 1 5 78 3 1.50 9 .. In Vitro Anti-Inflammatory Activity. Several enzymes 1.25 10 have been stated as mediators of infammation and appear 1.00 to be involved in infammatory disorders. Among them, 0.75 6 0.50 lipoxygenases and hyaluronidases were used as a criterion of 0.25 4 anti-infammatory potential [35]. In vitro anti-infammatory 0.00 2 activity of the E. planum and C. benedictus polyphenolic- rich extracts against lipoxygenase (LOX) and hyaluronidase 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 (HYA) (Table 3) has been tested. Figure 2: HPLC-MS chromatograms of the Cnicus benedictus All studied extracts had inhibitory efects on the tested polyphenolic-rich extract (1, chlorogenic acid, 2, cafeic acid, 3, p- enzymes. Among the fractions, the E. planum polyphenolic- coumaric acid, 4, sinapic acid, 5, ellagic acid, 6, rosmarinic acid, 7, rich extract exhibited the highest inhibitory activity with IC50 luteolin, 8, quercetol, 9, genistein, and 10, kaempferol). value of 31.3±2.1 �g/ml for LOX and 24.6±1.5 �g/ml for HYA. Te phenolic acids, favonoids, and isofavones from plants were reported to have anti-infammatory activity diabetic complications. Screening the antioxidant activity by [36–39]. Terefore, the anti-infammatory properties of E. DPPH and reducing power assays showed that for both plants planum and C. benedictus polyphenolic-rich extracts may be extracts the nanofltration retentates are more efective. assigned to the synergic efect of ursolic acid and polyphenols Table 1 shows that C. benedictus nanofltrate retentate such as rutin, chlorogenic acid, rosmarinic acid, genistin, had the high scavenging activity (IC50 = 0.0081 mg/mL) and daidzein previously investigated for anti-infammatory comparable to that of ascorbic acid (IC50 = 0.0012 mg/mL) efects [36–39]. and a higher reducing power (IC50 = 0.082 mg/mL), with IC50 Ursolic acid present in high quantities in the concentrated value signifcantly lower than that of the ascorbic acid (0.125 extracts is of particular interest due to antioxidant, antimicro- mg/mL), used as control. E. planum nanofltrate retentate had bial, anti-infammatory, and hypoglycemic activities [40]. 6 BioMed Research International

Table 4: IC 50 values (�g/ml) of �-amylase and �-glucosidase inhibition activity of polyphenolic-rich extracts.

Extract fractions �-amylase �-glucosidase E. planum extract MF 10.72 ± 0.8 24.99 ± 1.8 NF retentate 8.27 ± 0.5 5.94 ± 0.35 C. benedictus extract MF 5.41 ± 0.26 84.83 ± 3.2 NF retentate 1.2 ± 0.01 48.54 ± 2.6 Acarbose 17.68 ± 1.2 272.58 ± 5.4

.. �-Amylase and �-Glucosidase Inhibition. Due to their 140 � � inhibition capacity against -amylase and -glucosidase, 120 these medicinal plant extracts provide a therapeutic ap- proach, aiming at postprandial hyperglycemia reducing and 100 controlling on diabetes disease [41]. Acarbose is an inhibitor 80 of carbohydrates digesting enzymes used to decrease the glucose absorbance, but it has some gastrointestinal side 60 efects [42]. A wide range of medicinal plants could be related % cells viability 40 to the inhibition of carbohydrate enzymes; therefore lately the studies on new �-amylase and �-glucosidase natural 20 inhibitors are extended. [43]. 0 Te present study investigating enzyme’s inhibitory Mc 100 250 500 750 M+ potential of the selected herbs is the frst report on the �- Extract concentration (mg/mL) � amylase and -glucosidase inhibitory efects of E. planum 24 h and C. benedictus polyphenolic-rich extracts. Te results 48 h about the interaction of the E. planum and C. benedictus 72 h polyphenolic-rich extracts with �-amylase and �-glucosidase are presented in Table 4. Figure 3: Cell viability of NCTC clone 929-line cells with E. planum polyphenolic-rich extract. All of the tested extracts inhibited �-amylase and �- glucosidase in a dose-dependent manner. As it can be observed in Table 4, both of the polyphenolic-rich extracts For efcient therapeutic use in the treatment of hyper- had higher inhibitory efect on �-amylase (IC50 =8.27�g/mL, � glycaemia, these natural inhibitors are preferred to have resp., IC50 =1.2 g/mL) than the acarbose (IC50 =17.68 � � �g/mL). mild inhibitory activity against -amylase and strong - � glucosidase inhibitory activity, because inhibition of both In this study on the -glucosidase inhibition, our data enzymes could have side gastrointestinal efect [46]. In revealed that all the herbal extracts have stronger activity than � support of this idea, E. planum polyphenolic-rich extract can acarbose (used as a standard inhibitor). Te best -glucosi- be considered more relevant in the management of diabetes dase inhibition was determined in E. planum polyphenolic- � mellitus. rich extract (NF retentate) with IC50 of 5.94 g/mL, where On the other hand, previously studies established that the �-glucosidase content was almost 46 times lower than its � chronic infammation is linked with type II diabetes and value in case of using acarbose (272.58 g/mL). therefore targeting infammation may ameliorate diabetes. Te data revealed that both polyphenolic-rich extracts Results presented in the paper showed that E. planum (NF retentate) had the highest inhibitory activity, correlating polyphenolic-rich extract has the highest anti-infammatory with their total phenol and favonoid high contents. activity, correlating with the highest antidiabetic activity Te presented results may provide fundamental data based on the enzyme’s strong inhibition. for further studies in designing new plant-based drugs or nutraceuticals. Te phenolic compounds (phenolic acids .. Cytotoxicity. For the further therapeutic application of and favonoids) found in these extracts exert their protec- the medicinal plant extracts in discussion, the efect of E. tive properties against diabetes and these compounds were planum and C. benedictus polyphenolic-rich extracts on the mentioned previously as inhibitors of �-amylase and �- NCTC clone 929 stabilized cell line was tested. Te toxicity glucosidase [44]. According to the previous fndings, ursolic of each extract was determined to carry out both quantitative acid, a triterpene, has a signifcant inhibitory activity to and qualitative methods. �-amylase and �-glucosidase and therefore might play an Te E. planum polyphenolic-rich extract tested on NCTC important role in the treatment of diabetes mellitus and its line produced proliferative efect afer 24 h, noncytotoxic complications [45]. Te high contents of ursolic acid, rutin, efect afer 48 h and 72 h within the concentration range of and chlorogenic acid from E. planum and C. benedictus 100-500 �g/mL, and a slightly cytotoxic efect at 750�g/mL polyphenolic-rich extracts might be considered responsible afer 72 h (Figure 3). Cytotoxicity analysis for the C. benedic- for their �-amylase and �-glucosidase inhibition capacity. tus polyphenolic-rich extract showed a noncytotoxic efect BioMed Research International 7

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60 % cells viability 40

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0 Mc 100 250 500 750 M+ Extract concentration (g/mL) 24 h 48 h 72 h Figure 4: Cell viability of NCTC clone 929-line cells with C. benedictus polyphenolic-rich extract.

Untreated cells 100 g/mL E. planum 500 g/mL E. planum (a) Efects of E. planum polyphenolic-rich extract on NCTC cell morphology, afer 72 h

Untreated cells 100 g/mL C. benedictus 500 g/mL C. benedictus (b) Efects of C. benedictus polyphenolic-rich extract on NCTC cell morphology, afer 72 h

Figure 5 afer 24 h and 48 h, but afer 72 h this extract was slightly Te cytotoxic efect of the analyzed extracts against the cytotoxic starting at 500 �g/mL and moderately cytotoxic at cell lines is presented in the photomicrographs (Figure 5). In 750 �g/mL (Figure 4). both of the cases implying untreated cells (Figure 5(a), lef) Tese data determined the optimal range of noncytotoxic and the cells treated with 100 �g/mL and 500 mg/L concentra- concentrations for each of the plant extracts: up to 500 �g/mL tions of E. planum polyphenolic-rich extract, no morphology for E. planum and 250 �g/mL for C. benedictus. or cell density changes could be noticed (Figure 5(a)). Cells Tere are no references for the cytotoxic activity on were homogeneously distributed on the plate and exhibited fbroblasts for Eryngium planum and for Cnicus benedictus, the typical spindle-shape morphology of normal fbroblasts. but other Eryngium and Cnicus species were tested on various Withal, untreated cells (Figure 5(b), lef) and cells treated cancer cell lines [47–49]. with 100 �g/mL of C. benedictus polyphenolic-rich extract 8 BioMed Research International did not exhibit morphological and cell density changes Reviews in Food Science and Nutrition,vol.45,no.4,pp.287– (Figure 5(b), middle), whereas for the cells treated with the 306, 2005. same extract at 500 mg/L concentrations an alteration in [3] N. Pantidos, A. Boath, V.Lund, S. Conner, and G. J. McDougall, cell morphology and decreased cell density were noticed, “Phenolic-rich extracts from the edible seaweed, ascophyllum indicating a moderate cytotoxic efect (Figure 5(b), right). nodosum, inhibit �-amylase and �-glucosidase: Potential anti- hyperglycemic efects,” Journal of Functional Foods,vol.10,pp. 4. Conclusions 201–209, 2014. [4]S.Lalegani,H.AhmadiGavlighi,M.H.Azizi,andR.Amini In conclusion, this is the frst report about the determination Sarteshnizi, “Inhibitory activity of phenolic-rich pistachio green of ursolic acid, genistin, and isorhamnetin in E. planum and hull extract-enriched pasta on key type 2 diabetes relevant C. benedictus and also the frst study to evaluate the E. planum enzymes and glycemic index,” Food Research International,vol. and C. benedictus polyphenolic-rich extracts potentially used 105, pp. 94–101, 2018. in therapy for hyperglycaemia. [5] F. J. Alarcon-Aguilara, R. Roman-Ramos, S. Perez-Gutierrez, Te study results demonstrated that Eryngium planum A. Aguilar-Contreras, C. C. Contreras-Weber, and J. L. Flores- and Cnicus benedictus extracts obtained by ultrasound- Saenz, “Study of the anti-hyperglycemic efect of plants used as assisted extraction and concentrated by nanofltration lead antidiabetics,” Journal of Ethnopharmacology,vol.61,no.2,pp. 101–110, 1998. to obtaining an enriched fraction of bioactive polyphenolic compounds, consequently preserving a high antioxidant [6] R. Mashima and T. Okuyama, “Te role of lipoxygenases in pathophysiology; new insights and future perspectives,” Redox activity. Biology,vol.6,pp.297–310,2015. Both polyphenolic-rich extracts exhibited inhibition of the enzymatic activity relevant to type II diabetes manage- [7]K.S.Girish,K.Kemparaju,S.Nagaraju,andB.S.Vishwanath, “Hyaluronidase inhibitors: a biological and therapeutic per- ment and anti-infammatory correlation with a high content spective,” Current Medicinal Chemistry,vol.16,no.18,pp.2261– of ursolic acid and polyphenolic compounds. Moreover, 2288, 2009. phenolic acids and favonoids were found to be mainly [8] M. Rami´c, S. Vidovi´c,Z. Zekovi´c, J. Vladi´c, A. Cvejin, and responsible for these bioactivities of the extracts according B. Pavli´c, “Modeling and optimization of ultrasound-assisted to Pearson’s correlation coefcients. Tese fndings pro- extraction of polyphenolic compounds from Aronia melanocar- vide new support for the traditional anti-infammatory and pa by-products from flter-tea factory,” Ultrasonics Sonochem- antidiabetic application of E. planum and C. benedictus and istry,vol.23,pp.360–368,2015. indicate that both polyphenolic-rich extracts could be utilized [9]X.Luo,J.Cui,H.Zhangetal.,“Ultrasoundassistedextraction as potential antidiabetic and anti-infammatory agents. of polyphenolic compounds from red sorghum (Sorghum bicolor L.) bran and their biological activities and polyphenolic Data Availability compositions,” Industrial Crops and Products,vol.112,pp.296– 304, 2018. Te data used to support the fndings of this study are [10] U. Balyan and B. Sarkar, “Integrated membrane process for available from the corresponding author upon request. purifcation and concentration of aqueous Syzygium cumini (L.) seed extract,” Food and Bioproducts Processing,vol.98,pp. Conflicts of Interest 29–43, 2016. [11] C.Conidi,A.Cassano,F.Caiazzo,andE.Drioli,“Separationand Te authors declare that there are no conficts of interest purifcation of phenolic compounds from pomegranate juice by regarding the publication of this paper. ultrafltration and nanofltration membranes,” Journal of Food Engineering,vol.195,pp.1–13,2017. Acknowledgments [12] B. Tylkowski, M. Nowak, I. Tsibranska et al., “Concentration and fractionation of polyphenols by membrane operations,” Tis work was funded by Nucleus Program, with the support Current Pharmaceutical Design,vol.23,no.2,pp.231–241,2017. of Romanian Ministry of Research and Innovation, within the [13] G. Paun, E. Neagu, V. Moroeanu et al., “Anti-infammatory Contract no. 25N/2019 (PN 19270101/2019). and antioxidant activities of the Impatiens noli-tangere and Stachys ofcinalis polyphenolic-rich extracts,” Revista Brasileira Supplementary Materials de Farmacognosia,vol.28,no.1,pp.57–64,2018. [14] S. Conea, L. Vlase, and I. Chirila, “Comparative study on the Te correlation between bioactive compounds, antioxidant polyphenols and pectin of three eryngium species and their activity, and enzymes inhibitory efects of extracts is pre- antimicrobial activity,” Cellulose Chemistry and Technology,vol. sented in Table 1S. (Supplementary Materials) 50,no.3-4,pp.473–481,2016. [15] M. Kikowska, J. Budzianowski, A. Krawczyk, and B. Tiem, References “Accumulation of rosmarinic, chlorogenic and cafeic acids in in vitro cultures of Eryngium planum L.,” Acta Physiologiae [1] F.Shahidi, “Nutraceuticals, functional foods and dietary supple- Plantarum,vol.36,no.6,pp.2425–2433,2014. ments in health and disease,” Journal of Food and Drug Analysis, [16] M. Kowalczyk, M. Masullo, B. Tiem, S. Piacente, A. Stochmal, vol. 20, no. 1, pp. 226–230, 2012. and W. Oleszek, “Tree new triterpene saponins from roots of [2] A. Scalbert, C. Manach, C. Morand, C. R´em´esy, and L. Jim´enez, Eryngium planum,” Natural Product Research (Formerly Natural “Dietary polyphenols and the prevention of diseases,” Critical Product Letters),vol.28,no.9,pp.653–660,2014. BioMed Research International 9

[17] A. Ulubelen and T. Berkan, “Triterpenic and steroidal com- [32]G.Paun,E.Neagu,C.Albu,andG.L.Radu,“Application pounds of Cnicus benedictus,” Planta Medica,vol.31,no.4,pp. of the polyphenylene ether-ether-sulfone ultrafltration mem- 375–377, 1977. brane for concentration of antioxidants from the Phyllitis [18] V. Teˇsevi´c, S. Milosavljevi´c, V. Vajs et al., “Quantitative analysis scolopendrium L. extract,” New Journal of Chemistry,vol.39, of lactone cnicin in seven Centaurea species no. 2, pp. 1154–1160, 2015. wild-growing in Serbia and Montenegro using 1H-NMR spec- [33] B. Tylkowski, I. Tsibranska, R. Kochanov, G. Peev, and M. troscopy,” Journal of the Serbian Chemical Society,vol.72,no.12, Giamberini, “Concentration of biologically active compounds pp.1275–1280,2007. extracted from Sideritis ssp. L. by nanofltration,” Food and Bioproducts Processing, vol. 89, no. 4, pp. 307–314, 2011. [19] M. Ghiasy-Oskoee, M. AghaAlikhani, F. Sefdkon, A. Mokhtassi-Bidgoli, and M. Ayyari, “Blessed thistle agronomic [34]C.Conidi,A.D.Rodriguez-Lopez,E.M.Garcia-Castello,and and phytochemical response to nitrogen and plant density,” A. Cassano, “Purifcation of artichoke polyphenols by using Industrial Crops and Products,vol.122,pp.566–573,2018. membrane fltration and polymeric resins,” Separation and Purification Technology, vol. 144, pp. 153–161, 2015. [20] Z. Can, N. Baltas¸,S.Keskin,O.Yıldız,andS.Kolaylı,“Properties [35] Y. Bellik, L. Boukraˆa, H. A. Alzahrani et al., “Molecular mecha- of antioxidant and anti-infammatory activity and phenolic nism underlying anti-infammatory and anti-Allergic activities profles of S¸evketi Bostan (Cnicus benedictus L.) cultivated in of phytochemicals: an update,” Molecules,vol.18,no.1,pp.322– Aegean Region from Turkey,” Turkish Journal of Agriculture - 353, 2013. Food Science and Technology,vol.5,no.4,pp.308–314,2017. [36] N. Chockalingam and S. Muruhan, “Anti-infammatory prop- [21] V. L. Singleton and J. A. Rossi, “Colorimetry of total phenolics erties of rosmarinic acid - a review,” International Journal of with phosphomolybdic-phosphotungstic acid reagents,” Ameri- Research in Pharmaceutical Sciences,vol.8,no.4,pp.656–662, can Journal of Enology and Viticulture,vol.16,no.3,pp.144–158, 2017. 1965. [37] M. Naveed, V. Hejazi, M. Abbas et al., “Chlorogenic acid [22] J.-Y. Lin and C.-Y. Tang, “Determination of total phenolic and (CGA): a pharmacological review and call for further research,” favonoid contents in selected fruits and vegetables, as well as Biomedicine & Pharmacotherapy,vol.97,pp.67–74,2018. their stimulatory efects on mouse splenocyte proliferation,” [38] M.-H. Pan, C.-S. Lai, and C.-T. Ho, “Anti-infammatory activity Food Chemistry,vol.101,no.1,pp.140–147,2006. of natural dietary favonoids,” Food & Function,vol.1,no.1,pp. [23] C. Albu, S. A. V.Eremia, R. Penu et al., “Characterization of the 15–31, 2010.

phenolics and free radical scavenging of romanian red wine,” [39] C. Danciu, S, . Avram, I. Z. Pavel et al., “Main isofavones found Analytical Letters,vol.50,no.4,pp.591–606,2017. in dietary sources as natural anti-infammatory agents,” Current [24]V.Bondet,W.Brand-Williams,andC.Berset,“Kineticsand Drug Targets,vol.19,no.7,pp.841–853,2018. mechanisms of antioxidant activity using the DPPH∙ free [40] D. Kashyap, H. S. Tuli, and A. K. Sharma, “Ursolic acid (UA): A radical method,” LWT- Food Science and Technology,vol.30,no. metabolite with promising therapeutic potential,” Life Sciences, 6, pp. 609–615, 1997. vol.146,pp.201–213,2016. [25] S. A. Baba and S. A. Malik, “Evaluation of antioxidant and [41] A. O. Ademiluyi and G. Oboh, “Soybean phenolic-rich extracts antibacterial activity of methanolic extracts of Gentiana kurroo inhibit key-enzymes linked to type 2 diabetes (�-amylase and royle,” Saudi Journal of Biological Sciences,vol.21,no.5,pp.493– �-glucosidase) and hypertension (angiotensin I converting 498, 2014. enzyme) in vitro,” Experimental and Toxicologic Pathology,vol. [26] H. Wangensteen, A. B. Samuelsen, and K. E. Malterud, “Antiox- 65, no. 3, pp. 305–309, 2013. idant activity in extracts from coriander,” Food Chemistry,vol. [42] M. F. Di Carli, J. Janisse, G. Grunberger, and J. Ager, “Role 88, no. 2, pp. 293–297, 2004. of chronic hyperglycemia in the pathogenesis of coronary microvascular dysfunction in diabetes,” Journal of the American [27] A. Sahasrabudhe and M. Deodhar, “Anti-hyaluronidase, anti- College of Cardiology,vol.41,no.8,pp.1387–1393,2003. elastase activity of Garcinia indica,” International Journal of Botany,vol.6,no.3,pp.299–303,2010. [43] A. B. Shori, “Screening of antidiabetic and antioxidant activities of medicinal plants,” Journal of Integrative Medicine,vol.13,no. [28]L.G.Ranilla,Y.-I.Kwon,E.Apostolidis,andK.Shetty,“Phe- 5, pp. 297–305, 2015. nolic compounds, antioxidant activity and in vitro inhibitory [44] A. Kam, K. M. Li, V. Razmovski-Naumovski et al., “A com- potential against key enzymes relevant for hyperglycemia and parative study on the inhibitory efects of diferent parts and hypertension of commonly used medicinal plants, herbs and chemical constituents of pomegranate on �-amylase and �- spices in Latin America,” Bioresource Technology,vol.101,no. glucosidase,” Phytotherapy Research,vol.27,no.11,pp.1614– 12, pp. 4676–4689, 2010. 1620, 2013. [29] G. Paun, E. Neagu, C. Albu, V. Moroeanu, and G.-L. Radu, [45] B.-W. Zhang, Y. Xing, C. Wen et al., “Pentacyclic triterpenes “Antioxidant activity and inhibitory efect of polyphenolic-rich as �-glucosidase and �-amylase inhibitors: structure-activity extract from betonica ofcinalis and Impatiens noli-tangere relationships and the synergism with acarbose,” Bioorganic & herbs on key enzyme linked to type 2 diabetes,” Journal of the Medicinal Chemistry Letters,vol.27,no.22,pp.5065–5070,2017. Taiwan Institute of Chemical Engineers,vol.60,pp.1–7,2016. [46] E. Apostolidis, Y. I. Kwon, and K. Shetty, “Potential of cran- [30] P. Mccue, Y.-I. Kwon, and K. Shetty, “Anti-amylase, anti- berry-based herbal synergies for diabetes and hypertension glucosidase and anti-angiotensin I-converting enzyme poten- management,” Asia Pacific Journal of Clinical Nutrition,vol.15, tial of selected foods,” Journal of Food Biochemistry,vol.29,no. no. 3, pp. 433–441, 2006. 3, pp. 278–294, 2005. [47]K.Cianfaglione,E.E.Blomme,L.Quassintietal.,“Cyto- [31] R. Jaiswal, J. Kiprotich, and N. Kuhnert, “Determination of toxic essential oils from eryngium campestre and eryngium the hydroxycinnamate profle of 12 members of the Asteraceae amethystinum (Apiaceae) growing in central Italy,” Chemistry family,” Phytochemistry, vol. 72, no. 8, pp. 781–790, 2011. &Biodiversity, vol. 14, no. 7, Article ID e1700096, 2017. 10 BioMed Research International

[48] M. Kikowska, M. Dworacka, I. Kędziora, and B. Tiem, “Eryngium creticum – Ethnopharmacology, phytochemistry and pharmacological activity. A review,” Revista Brasileira de Farmacognosia,vol.26,no.3,pp.392–399,2016. [49] V. Steenkamp and M. C. Gouws, “Cytotoxicity of six South African medicinal plant extracts used in the treatment of cancer,” South African Journal of Botany,vol.72,no.4,pp.630– 633, 2006. M EDIATORSof INFLAMMATION

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