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

Research Article

Chemical and Bioactivity Evaluation of Eryngium 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 Radu1,2

¹National Institute for Research-Development of Biological Sciences, Bucharest 6, 060031, Romania
²Faculty of Applied Chemistry and Materials Science, Politehnica University of Bucharest, Bucharest, Romania

Correspondence should be addressed to Gabriel Lucian Radu; glradu2006@gmail.com

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

Academic Editor: Min-Hui Li

Copyright © 2019 Gabriela Paun et al. This 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.

Abstract.

This study evaluated the biological activities of Eryngium planum and of Cnicus benedictus extracts enriched in polyphenols obtained by nanofiltration. The HPLC-MS analysis showed that E. planum contains mainly flavonoids, 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 first 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= 0.082 mg/mL), with IC₅₀ having a significantly 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 μg/mL for lipoxygenase and 24.6 μg/mL for hyaluronidase. Both polyphenolic-rich extracts had a higher inhibitory effect on α-amylase and α-glucosidase than that of the acarbose. The synergistic effect of ursolic acid, rutin, chlorogenic acid, rosmarinic acid, genistin, and daidzein identified in polyphenolic-rich extracts could be mainly responsible for the pharmacological potentials of the studied extracts used in managing inflammation and diabetes.

1. Introduction

Recently, the research is more and more focused on the polyphenol’s effect on the human health and on the natural antioxidants sources with low or no side effects to be used in medicine and in the food industry [1]. Data from literature firmly support the role of phenolic compounds in the prevention of degenerative diseases, such as neurodegenerative diseases and diabetes mellitus [2–4]. Several studies showed that the α-amylases and α-glucosidases have significant activities in the metabolism of polysaccharides. Dietary interventions based on plant natural inhibitors could successfully replace commercially drugs, such as acarbose, in the management of hyperglycaemia. The benefit stays in that there are no adverse side effects [5].

The frequent use of nonsteroidal anti-inflammatory drugs (NSAIDs) with various side effects determined lately a new direction in research toward the natural sources, effective against inflammatory disorders and with few side effects.

Lipoxygenases, which catalyse the addition of oxygen to arachidonic acid and other polyunsaturated fatty acids, and hyaluronidase, which hydrolyses hyaluronic acid, are involved in the inflammatory process [6, 7].

Development and use of efficient technologies for the bioactive compound’s extraction and concentration represent a current priority. Nowadays, ultrasonic-assisted extraction (UAE) and membrane technology have become the most important and efficient technique for extraction [7, 8], separation, and concentration of the phenolic compounds from various plant sources [8–13].

Eryngium planum L. (Apiaceae family) is a medicinal plant used in European and Asian traditional medicine, especially for various inflammatory disorders treatment. The Eryngium species aerial part bioactivity is mainly generated by polyphenols and saponins [14–16].

Cnicus benedictus L. (blessed thistle) belonging to the family Asteraceae is mainly distributed from Asia Minor through southern Europe. This medicinal plant contains alkaloids, cnicin, benedictin, mucilage, polyacetylene, triterpenoide, lignans, flavonoids, tannin, phytosterines, and volatile oils [17–19]. This is a plant with antidepressive, anti-inflammatory, antiseptic, cardiac, and antimicrobial properties and cnicin is the compound responsible for blessed thistle’s medicinal properties [20].

There are only several studies on the chemical composition of these herbs, yet very few phytopharmacological studies have been exhaustively investigated up till now. Taking into account the considerations mentioned above, the present study was undertaken to investigate the anti-inflammatory and the antidiabetic activities of Eryngium planum and Cnicus benedictus polyphenolic-rich extracts. In addition, the antioxidant activity and cytotoxicity of polyphenolic-rich extracts of selected herbs were also investigated.

However, up to date, these characteristics of E. planum and C. benedictus polyphenolic-rich extracts have not yet been studied; therefore, no reference is available regarding the UAE and the membrane concentration of polyphenolic compounds from these herbs.

2. Materials and Methods

2.1. Plant Material

Eryngium planum herb was collected from Valcea district (Romania) and Cnicus benedictus herb was collected from Razhdak district (Bulgaria). The voucher specimen number 657490 for Eryngium planum and voucher specimen number 665998 for Cnicus benedictus were preserved in the Botanic Garden of Cluj-Napoca, Romania.

2.2. Extraction and Processing

Dried aerial parts were grounded (10 g), mixed with the 50% (v/v) ethanol in water as solvent, and shaken for 5 minutes for the extraction process. Next, the extraction was performed by ultrasonic-assisted extraction (UAE) using a sonication water bath (Elma Transsonic T 460, Germany) at 35 kHz for 90 min. After these steps, extracts were filtered through the filter paper under vacuum and microfiltered through 0.45 μm pore size microfiltration membrane (Millipore). Nanofiltration was applied to concentrate biologically valuable components and obtain polyphenolic-rich extracts. The nanofiltration was performed with a tangential filtration KOCH Membrane laboratory unit equipped with an NF90 (Dow Filmtec, Sterlitech Company, USA), with a molecular weight cut-off of 200–300 Da, made of polyamide material.

2.3. Total Phenolic and Flavonoid Content

The total phenolic content was determined using Folin-Ciocalteu assay [21]. The total phenolic content was calculated on the basis of a calibration curve of gallic acid (y=0.0027x+0.017, R² = 0.9965) and expressed as mg gallic acid equivalents per litter of extracts (mg GAE/L).

The total flavonoid content was measured using the AlCl₃ colorimetric method as described by Lin [22]. Results were expressed as quercetin (QE) mg/mL using the equation based on the calibration curve: y = 0.004x + 0.0535; R² = 0.9934.

2.4. HPLC-MS Analysis

The analysis of polyphenols was performed using a validated HPLC-MS method [23]. All solvents and reference compounds (p-coumaric acid, caffeic acid, ferulic acid, gallic acid, chlorogenic acid, rosmarinic acid, ellagic acid, rutin, luteolin, quercetin, quercetin 3-β-D-glucoside, apigenin, kaempferol, daidzein, genistein, and genistin) were purchased from Sigma (Sigma Aldrich, Germany), Fluka (Switzerland), and Roth (Carl Roth GmbH, Germany).

The liquid chromatography analysis was performed using a HPLC SHIMADZU system coupled to a MS detector, LCMS-2010 detector (liquid chromatography mass spectrometer), and equipped with an electrospray ionization (ESI) interface. For analysis, a C18 Kromasil 3.5, 2.1x100mm column was used. The mobile phase was composed of solvent A (formic acid in water, pH=3.0) and solvent B (formic acid in acetonitrile, pH=3.0). The polyphenolic compound’s separation was performed using binary gradient elution: 0 min 5% solvent B; 0.01-20 min 5-30% solvent B; 20-40 min 30% solvent B; 40.01-50 min 30-50% solvent B; and 50.01-52 min 50-5% solvent B. The flow rate was 0-5 min 0.1 mL/min; 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. The quantitative analysis of the components was achieved with reference to standards.

For ursolic acid HPLC-MS determination, a C18 Nucleosil 100-3.5, 100x2.1 mm, KROMASIL column was used. Mobile phase was formic acid in water (solvent A) and methanol (solvent B). The flow rate for solvent A was 0.042 mL/min and for solvent B was 0.258 mL/min. The injection volume was 20 μL and the temperature of the column was kept constant at 20°C. ESI source and negative ionization mode were used.

2.5. Antioxidant Activity

2.5.1. DPPH Radical Scavenging Activity

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the polyphenolic-rich extracts was performed according to the method described by Bondet with some modification [24]. 100 μl sample extract or standard (ascorbic acid) with varying concentrations was mixed with 2900 mL of DPPH∙ methanolic solution and the absorbance was measured at 519 nm after 30 min of reaction.

2.5.2. Reducing Power

The reduction of Fe³⁺ into the Fe²⁺ potential of the extracts was determined according to the method of Baba and Malik [25]. In brief, 100 μL of extract, 2.5 mL phosphate buffer (0.2M, pH 6.6), and 2.5 mL potassium ferricyanide (1%) were mixed and incubated at 50°C for 20 min. Then, 2.5 mL trichloroacetic acid (10%) was added, the mixture was centrifuged, and the upper layer (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL of 0.1% ferric chloride; then the absorbance was measured at 700 nm; ascorbic acid was used as standard.

2.6. In Vitro Anti-Inflammatory Assay

2.6.1. Lipoxygenase (LOX) Inhibition

The LOX inhibition was performed by the spectrophotometric assay [26]. The increase in absorbance at 234 nm was due to the formation of the product 13-hydroperoxyocta-decadienoic acid from the reaction of oxygen and linoleic acid catalysed by lipoxygenase. For the blank solution, 100 μL LOX solution 2200 U/mL was mixed with 100 μL linoleic acid and 3050 μL boric acid buffer 0.2 M (pH 9.0). For samples, 50 μL of borate buffer was replaced with 50 μL of the extract. The mixture was incubated for 15 min; then 100 μL linoleic acid was added to the mixture and the absorbance was read at 234 nm for 2 minutes. The % inhibition for different concentrations of the extracts was calculated as

where and are the increase of absorbance for the sample extracts and the buffer instead of inhibitor (sample). Ibuprofen is used as standard.

2.6.2. Hyaluronidase (HYA) Inhibition

The hyaluronidase inhibition was determined by the method described by Sahasrabudhe and Dedhar with slight modifications [27]. Briefly, 100 μl of hyaluronidase (type-1-S from bovine, 400 units/mL) in acetate buffer (0.1 M, pH 3.5) and 50 μL of plant extract was incubated for 20 min at 37°C; then 100 μL of CaCl₂ solution 12.5 mM was added and the mixture was incubated also at 37°C for 20 minutes. Then, the reaction was initiated by adding 250 μL of sodium hyaluronate (1.2 mg/mL) and incubated at 37°C for 40 min; 100 μL of sodium hydroxide solution 0.4 M and 100 μL of potassium borate (0.4 M) were added to the reaction mixture and incubated for 3 min in a boiling water bath. After cooling, 3000 μL of 10% -dimethylaminobenzaldehyde reagent was added and after 20 minutes the increase in absorbance was measured at 585 nm. The percentage of enzyme inhibition was calculated as

where and are the absorbance of the sample extracts and absorbance of control (buffer instead of inhibitor). Ibuprofen is used as standard.

2.7. In Vitro Antidiabetic Assays

2.7.1. Inhibition of α-Amylase Activity

The α-amylase inhibitory activity was evaluated according to Ranilla et al. with slight modification [28], as a previous reported paper [29]. In brief, the plant extract and α-amylase from hog pancreas solution (0.5 mg/mL) were incubated at 37°C for 20 min. After that, a 1% starch solution was added and incubated at the same temperature for 30 min. The reaction was stopped with dinitrosalicylic acid colour reagent. The mixture was incubated in a boiling water bath for 5 min, cooled to room temperature, and diluted with distilled water and the absorbance was recorded at 540 nm. Acarbose was used as positive control. The rate of enzyme inhibition was calculated according to the following equation:

where is the absorbance of the control (without extract/acarbose) and is the absorbance of the sample extract.

2.7.2. α-Glucosidase Inhibition Assay

α-Glucosidase inhibition was evaluated using the procedures described in the literature [30]. Sample solution (50 μL) was preincubated with 125 μL of α-glucosidase from Saccharomyces cerevisiae (0.5 U/mL in phosphate buffer solution, pH 6.9) and 700 μL of phosphate buffer at 37°C for 15 minutes. Then, 125 μL of 5 mM p-nitrophenyl glucopyranoside (pNPG) was added to quench the reaction and the mixture was kept at 37°C for 30 minutes. The reaction was stopped by adding 1000 μL of 0.2M Na₂CO₃ and the absorbance was recorded at 405 nm. Control sample was prepared to contain buffer instead of enzyme. Acarbose was used as positive control.

where stands for the absorbance of the mixture without extract samples; stands for the absorbance of polyphenolic-rich extract samples; stands for the absorbance of the mixture without a-glucosidase.

IC₅₀ value (concentration of phenolic-rich extracts required to inhibit 50% of the enzyme activity) was calculated graphically by dose-dependent inhibition.

2.8. Cytotoxicity

The cell line of mouse fibroblast (NCTC clone 929 from the European Collection of cell Culture, Sigma-Aldrich, USA) was used to determine the cell viability by the MTT test. Fibroblast cultures (NCTC) were grown in MEM containing 10% fetal bovine serum (FBS) and PSN. The NCTC cell line was inoculated at 4 x 10⁴ cells/mL density. The cells cultured in standard conditions adhered to plastic after 24 hours of incubation; then the medium of culture changed with medium containing various concentrations of plant extracts (100, 250, and 500 μg/mL). The plates with 96 wells were incubated for 24 and 72 hours at 37C in 5% CO₂ air atmosphere. The culture control was untreated cells cultivated in MEM and 10% FBS, and the positive control was H₂O₂ (2μL/mL). After incubation with MTT solution (3 hours, at 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 940 (Berthold Technologies).

For the morphology, the culture of mouse fibroblast cells (NCTC), fixed in methanol and Giemsa stained, was observed after 72 hours after the addition of the extracts and acquired with Zeiss AxioStar Plus microscope (Carl Zeiss, Germany).

2.9. Statistical Analysis

All values are expressed as the mean ± standard deviation and analyzed using Microsoft Excel. Values were considered significant when p value is lower than 0.05.

3. Results and Discussion

3.1. Phytochemical Composition and Antioxidant Activity

Polyphenols (phenolic acids and flavonoids) are compounds exhibiting strong antioxidant activity and are responsible for these plants’ medicinal properties. The nanofiltration process (NF) was used for the polyphenol’s concentration to obtain the polyphenolic-rich extracts in the present study. The extracted fractions were evaluated by means of total phenolic compound (TPC), total flavonoid content (TFC), and antioxidant capacities (DPPH and reducing power assays) (Table 1).

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

Medicinal herb
Fraction
TPC,
TFC,
DPPH,
Reducing power,

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, microfiltration process; NF, nanofiltration process.

Antioxidant activity is expressed as half inhibitory concentration (EC₅₀); values are expressed as mean ± standard deviation of three measurements.

The results indicated a good concentration performance of the membrane processes, resulting in a significant increase of the polyphenols contents in the nanofiltrate fractions. C. benedictus nanofiltrate retentate was found to have the highest TPC level (2.659 ± 0.128 mg GAE/mL), of which over 17.5% (0.478 ± 0.045 mg QE/mL) can be attributed to the total flavonoids.

The polyphenolic compounds present in the MF and NF retentate fractions of E. planum and C. benedictus extracts were determined by HPLC-MS and the data presented in Table 2 can be correlated with Figures 1 and 2.

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

Class
Compound 

Eryngium planum
Cnicus benedictus

µg/mL
µg/mL

MF.
Conc.
MF.
Conc.

Flavonols
Rutin
183.63
290.52
1.87
3.56

Kaempferol
3.55
4.20
0.62
0.73

Isoquercetin
23.40
36.11
7.54
9.75

Isorhamnetin
0.17
0.47
-
-

Quercetol
2.06
3.03
-
-

Flavone
Luteolin
0.72
0.93
0.50
0.62

Apigenin
-
-
0.20
0.56

Phenolic acids
Caffeic acid
4.51
5.68
4.07
5.58

Rosmarinic acid
15.53
18.95
7.62
10.17

Ferulic acid
6.39
7.07
-
-

Chlorogenic acid
40.33
63.02
315.11
454.29

p-Coumaric acid
4.29
4.66
7.52
8.35

Sinapic acid
0.67
9.93
109.74
249.85

Ellagitannins
Ellagic acid
1.29
1.32
1.19
1.54

Isoflavones
Genistein
1.42
1.49
2.20
2.47

Genistin
2.09
2.80
1.80
2.61

Daidzein
0.17
0.35
2.31
4.68

“-”, under limit of detection.

Figure 1: HPLC-MS chromatograms of the Eryngium planum polyphenolic-rich extract (1, chlorogenic acid, 2, p-coumaric acid, 3, rutin, 4, quercetin 3-β-D-glucoside, 5, ferulic acid, 6, rosmarinic acid, 7, daidzein, 8, luteolin, 9, quercetol, 10, genistein, 11, kaempferol, and 12, isorhamnetin).

Figure 2: HPLC-MS chromatograms of the Cnicus benedictus polyphenolic-rich extract (1, chlorogenic acid, 2, caffeic acid, 3, p-coumaric acid, 4, sinapic acid, 5, ellagic acid, 6, rosmarinic acid, 7, luteolin, 8, quercetol, 9, genistein, and 10, kaempferol).

The HPLC-MS results showed that the extracts from E. planum contain mainly flavonoids, especially rutin and isoquercitrin, while in C. benedictus extracts show the high concentration of the phenolic acids, especially the chlorogenic acid and sinapic acid, in agreement with data reported in the literature [14, 31].

Our study’s significant contribution consists of the identification and quantifying of new polyphenols for the studied herbal extracts. However, this is the first time that genistin and isorhamnetin are reported in E. planum and C. benedictus extracts.

In the studied extracts, ursolic acid, a triterpene responsible for many pharmacological effects, was identified and quantified also. Ursolic acid was concentrated from 76.77 μg/mL to 158.04 μg/mL in E. planum extracts and from 46.84 μg/mL to 146.50 μg/mL in C. benedictus extracts.

Antioxidant activity has been studied also, due to the fact that oxidative stress is considered to be the key factor in the anti-inflammatory process and in the pathogenesis of diabetic complications. Screening the antioxidant activity by DPPH and reducing power assays showed that for both plants extracts the nanofiltration retentates are more effective.

Table 1 shows that C. benedictus nanofiltrate retentate had the high scavenging activity (IC₅₀ = 0.0081 mg/mL) comparable to that of ascorbic acid (IC₅₀ = 0.0012 mg/mL) and a higher reducing power (IC₅₀ = 0.082 mg/mL), with IC₅₀ value significantly lower than that of the ascorbic acid (0.125 mg/mL), used as control. E. planum nanofiltrate retentate had a similar reducing power to control but a lower scavenging activity comparable to that of ascorbic acid. A significant correlation (p< 0.05) was found between antioxidant activity and polyphenol and flavone contents (Table 1S from supplementary material), indicating a significant contribution of these compounds to the antioxidant activity for these polyphenolic-rich extracts.

The bioactive compounds from herbal extracts concentrated by ultrafiltration (UF) and nanofiltration (NF) have been recently investigated as an alternative solution to the conventional methodologies. Paun and collaborators tested the ultrafiltration membrane for the concentration of antioxidants from Phyllitis scolopendrium L. extract [32]; Tylkowsky et al. investigated the NF process using with membranes with MWCO in the range of 200–500 Da for the treatment of ethanolic extracts from Sideritis spp. L. in order to concentrate polyphenols [33]; Conidi and collaborators concentrated clarified artichoke wastewater using a NF membrane (NF 270, polyamide) with MWCO of 200–300 Da [34]. All these researches proved the efficiency of both ultrafiltration and nanofiltration procedures to recover and concentrate polyphenols from different herbal extracts.

3.2. In Vitro Anti-Inflammatory Activity

Several enzymes have been stated as mediators of inflammation and appear to be involved in inflammatory disorders. Among them, lipoxygenases and hyaluronidases were used as a criterion of anti-inflammatory potential [35]. In vitro anti-inflammatory activity of the E. planum and C. benedictus polyphenolic-rich extracts against lipoxygenase (LOX) and hyaluronidase (HYA) (Table 3) has been tested.

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

Extract fractions

IC₅₀, µg/ml

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
125.9±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.

All studied extracts had inhibitory effects on the tested enzymes. Among the fractions, the E. planum polyphenolic-rich extract exhibited the highest inhibitory activity with IC₅₀ value of 31.3±2.1 μg/ml for LOX and 24.6±1.5 μg/ml for HYA.

The phenolic acids, flavonoids, and isoflavones from plants were reported to have anti-inflammatory activity [36–39]. Therefore, the anti-inflammatory properties of E. planum and C. benedictus polyphenolic-rich extracts may be assigned to the synergic effect of ursolic acid and polyphenols such as rutin, chlorogenic acid, rosmarinic acid, genistin, and daidzein previously investigated for anti-inflammatory effects [36–39].

Ursolic acid present in high quantities in the concentrated extracts is of particular interest due to antioxidant, antimicrobial, anti-inflammatory, and hypoglycemic activities [40].

3.3. α-Amylase and α-Glucosidase Inhibition

Due to their inhibition capacity against α-amylase and α-glucosidase, these medicinal plant extracts provide a therapeutic approach, aiming at postprandial hyperglycemia reducing and controlling on diabetes disease [41]. Acarbose is an inhibitor of carbohydrates digesting enzymes used to decrease the glucose absorbance, but it has some gastrointestinal side effects [42]. A wide range of medicinal plants could be related to the inhibition of carbohydrate enzymes; therefore lately the studies on new α-amylase and α-glucosidase natural inhibitors are extended. [43].

The present study investigating enzyme’s inhibitory potential of the selected herbs is the first report on the α-amylase and α-glucosidase inhibitory effects of E. planum and C. benedictus polyphenolic-rich extracts. The results about the interaction of the E. planum and C. benedictus polyphenolic-rich extracts with α-amylase and α-glucosidase are presented in Table 4.

Table 4: IC₅₀ values (μg/ml) of α-amylase and α-glucosidase inhibition activity of polyphenolic-rich extracts.

Extract fractions

α-amylase
α-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

All of the tested extracts inhibited α-amylase and α-glucosidase in a dose-dependent manner. As it can be observed in Table 4, both of the polyphenolic-rich extracts had higher inhibitory effect on α-amylase (IC₅₀ = 8.27 μg/mL, resp., IC₅₀ = 1.2 μg/mL) than the acarbose (IC₅₀ = 17.68 μg/mL).

In this study on the α-glucosidase inhibition, our data revealed that all the herbal extracts have stronger activity than acarbose (used as a standard inhibitor). The best α-glucosidase inhibition was determined in E. planum polyphenolic-rich extract (NF retentate) with IC₅₀ of 5.94 μg/mL, where the α-glucosidase content was almost 46 times lower than its value in case of using acarbose (272.58 μg/mL).

The data revealed that both polyphenolic-rich extracts (NF retentate) had the highest inhibitory activity, correlating with their total phenol and flavonoid high contents.

The presented results may provide fundamental data for further studies in designing new plant-based drugs or nutraceuticals. The phenolic compounds (phenolic acids and flavonoids) found in these extracts exert their protective properties against diabetes and these compounds were mentioned previously as inhibitors of α-amylase and α-glucosidase [44]. According to the previous findings, ursolic acid, a triterpene, has a significant inhibitory activity to α-amylase and α-glucosidase and therefore might play an important role in the treatment of diabetes mellitus and its complications [45]. The high contents of ursolic acid, rutin, and chlorogenic acid from E. planum and C. benedictus polyphenolic-rich extracts might be considered responsible for their α-amylase and α-glucosidase inhibition capacity.

For efficient therapeutic use in the treatment of hyperglycaemia, these natural inhibitors are preferred to have mild inhibitory activity against α-amylase and strong α-glucosidase inhibitory activity, because inhibition of both enzymes could have side gastrointestinal effect [46]. In support of this idea, E. planum polyphenolic-rich extract can be considered more relevant in the management of diabetes mellitus.

On the other hand, previously studies established that chronic inflammation is linked with type II diabetes and therefore targeting inflammation may ameliorate diabetes. Results presented in the paper showed that E. planum polyphenolic-rich extract has the highest anti-inflammatory activity, correlating with the highest antidiabetic activity based on the enzyme’s strong inhibition.

3.4. Cytotoxicity

For the further therapeutic application of the medicinal plant extracts in discussion, the effect of E. planum and C. benedictus polyphenolic-rich extracts on the NCTC clone 929 stabilized cell line was tested. The toxicity of each extract was determined to carry out both quantitative and qualitative methods.

The E. planum polyphenolic-rich extract tested on NCTC line produced proliferative effect after 24 h, noncytotoxic effect after 48 h and 72 h within the concentration range of 100-500 μg/mL, and a slightly cytotoxic effect at 750μg/mL after 72 h (Figure 3). Cytotoxicity analysis for the C. benedictus polyphenolic-rich extract showed a noncytotoxic effect after 24 h and 48 h, but after 72 h this extract was slightly cytotoxic starting at 500 μg/mL and moderately cytotoxic at 750 μg/mL (Figure 4).

Figure 3: Cell viability of NCTC clone 929-line cells with E. planum polyphenolic-rich extract.

Figure 4: Cell viability of NCTC clone 929-line cells with C. benedictus polyphenolic-rich extract.

These data determined the optimal range of noncytotoxic concentrations for each of the plant extracts: up to 500 μg/mL for E. planum and 250 μg/mL for C. benedictus.

There are no references for the cytotoxic activity on fibroblasts for Eryngium planum and for Cnicus benedictus, but other Eryngium and Cnicus species were tested on various cancer cell lines [47–49].

The cytotoxic effect of the analyzed extracts against the cell lines is presented in the photomicrographs (Figure 5). In both of the cases implying untreated cells (Figure 5(a), left) and the cells treated with 100 μg/mL and 500 mg/L concentrations of E. planum polyphenolic-rich extract, no morphology or cell density changes could be noticed (Figure 5(a)). Cells were homogeneously distributed on the plate and exhibited the typical spindle-shape morphology of normal fibroblasts.

(a) Effects of E. planum polyphenolic-rich extract on NCTC cell morphology, after 72 h

(b) Effects of C. benedictus polyphenolic-rich extract on NCTC cell morphology, after 72 h

Figure 5

Withal, untreated cells (Figure 5(b), left) and cells treated with 100 μg/mL of C. benedictus polyphenolic-rich extract did not exhibit morphological and cell density changes (Figure 5(b), middle), whereas for the cells treated with the same extract at 500 mg/L concentrations an alteration in cell morphology and decreased cell density were noticed, indicating a moderate cytotoxic effect (Figure 5(b), right).

4. Conclusions

In conclusion, this is the first report about the determination of ursolic acid, genistin, and isorhamnetin in E. planum and C. benedictus and also the first study to evaluate the E. planum and C. benedictus polyphenolic-rich extracts potentially used in therapy for hyperglycaemia.

The study results demonstrated that Eryngium planum and Cnicus benedictus extracts obtained by ultrasound-assisted extraction and concentrated by nanofiltration lead to obtaining an enriched fraction of bioactive polyphenolic compounds, consequently preserving a high antioxidant activity.

Both polyphenolic-rich extracts exhibited inhibition of the enzymatic activity relevant to type II diabetes management and anti-inflammatory correlation with a high content of ursolic acid and polyphenolic compounds. Moreover, phenolic acids and flavonoids were found to be mainly responsible for these bioactivities of the extracts according to Pearson’s correlation coefficients. These findings provide new support for the traditional anti-inflammatory and antidiabetic application of E. planum and C. benedictus and indicate that both polyphenolic-rich extracts could be utilized as potential antidiabetic and anti-inflammatory agents.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

This work was funded by Nucleus Program, with the support of Romanian Ministry of Research and Innovation, within the Contract no. 25N/2019 (PN 19270101/2019).

Supplementary Materials

The correlation between bioactive compounds, antioxidant activity, and enzymes inhibitory effects of extracts is presented in Table 1S. (Supplementary Materials)

References

F. Shahidi, “Nutraceuticals, functional foods and dietary supplements in health and disease,” Journal of Food and Drug Analysis, vol. 20, no. 1, pp. 226–230, 2012.

A. Scalbert, C. Manach, C. Morand, C. Rémésy, and L. Jiménez, “Dietary polyphenols and the prevention of diseases,” Critical Reviews in Food Science and Nutrition, vol. 45, no. 4, pp. 287–306, 2005.

N. Pantidos, A. Boath, V. Lund, S. Conner, and G. J. McDougall, “Phenolic-rich extracts from the edible seaweed, ascophyllum nodosum, inhibit α-amylase and α-glucosidase: Potential anti-hyperglycemic effects,” Journal of Functional Foods, vol. 10, pp. 201–209, 2014.

S. Lalegani, H. Ahmadi Gavlighi, M. H. Azizi, and R. Amini Sarteshnizi, “Inhibitory activity of phenolic-rich pistachio green hull extract-enriched pasta on key type 2 diabetes relevant enzymes and glycemic index,” Food Research International, vol. 105, pp. 94–101, 2018.

F. J. Alarcon-Aguilara, R. Roman-Ramos, S. Perez-Gutierrez, A. Aguilar-Contreras, C. C. Contreras-Weber, and J. L. Flores-Saenz, “Study of the anti-hyperglycemic effect of plants used as antidiabetics,” Journal of Ethnopharmacology, vol. 61, no. 2, pp. 101–110, 1998.

R. Mashima and T. Okuyama, “The role of lipoxygenases in pathophysiology; new insights and future perspectives,” Redox Biology, vol. 6, pp. 297–310, 2015.

K. S. Girish, K. Kemparaju, S. Nagaraju, and B. S. Vishwanath, “Hyaluronidase inhibitors: a biological and therapeutic perspective,” Current Medicinal Chemistry, vol. 16, no. 18, pp. 2261–2288, 2009.

M. Ramić, S. Vidović, Z. Zeković, J. Vladić, A. Cvejin, and B. Pavlić, “Modeling and optimization of ultrasound-assisted extraction of polyphenolic compounds from Aronia melanocarpa by-products from filter-tea factory,” Ultrasonics Sonochemistry, vol. 23, pp. 360–368, 2015.

X. Luo, J. Cui, H. Zhang et al., “Ultrasound assisted extraction of polyphenolic compounds from red sorghum (Sorghum bicolor L.) bran and their biological activities and polyphenolic compositions,” Industrial Crops and Products, vol. 112, pp. 296–304, 2018.

U. Balyan and B. Sarkar, “Integrated membrane process for purification and concentration of aqueous Syzygium cumini (L.) seed extract,” Food and Bioproducts Processing, vol. 98, pp. 29–43, 2016.

C. Conidi, A. Cassano, F. Caiazzo, and E. Drioli, “Separation and purification of phenolic compounds from pomegranate juice by ultrafiltration and nanofiltration membranes,” Journal of Food Engineering, vol. 195, pp. 1–13, 2017.

B. Tylkowski, M. Nowak, I. Tsibranska et al., “Concentration and fractionation of polyphenols by membrane operations,” Current Pharmaceutical Design, vol. 23, no. 2, pp. 231–241, 2017.

G. Paun, E. Neagu, V. Moroeanu et al., “Anti-inflammatory and antioxidant activities of the Impatiens noli-tangere and Stachys officinalis polyphenolic-rich extracts,” Revista Brasileira de Farmacognosia, vol. 28, no. 1, pp. 57–64, 2018.

S. Conea, L. Vlase, and I. Chirila, “Comparative study on the polyphenols and pectin of three eryngium species and their antimicrobial activity,” Cellulose Chemistry and Technology, vol. 50, no. 3-4, pp. 473–481, 2016.

M. Kikowska, J. Budzianowski, A. Krawczyk, and B. Thiem, “Accumulation of rosmarinic, chlorogenic and caffeic acids in in vitro cultures of Eryngium planum L.,” Acta Physiologiae Plantarum, vol. 36, no. 6, pp. 2425–2433, 2014.

M. Kowalczyk, M. Masullo, B. Thiem, S. Piacente, A. Stochmal, and W. Oleszek, “Three new triterpene saponins from roots of Eryngium planum,” Natural Product Research (Formerly Natural Product Letters), vol. 28, no. 9, pp. 653–660, 2014.

A. Ulubelen and T. Berkan, “Triterpenic and steroidal compounds of Cnicus benedictus,” Planta Medica, vol. 31, no. 4, pp. 375–377, 1977.

V. Tešević, S. Milosavljević, V. Vajs et al., “Quantitative analysis of sesquiterpene lactone cnicin in seven Centaurea species wild-growing in Serbia and Montenegro using 1H-NMR spectroscopy,” Journal of the Serbian Chemical Society, vol. 72, no. 12, pp. 1275–1280, 2007.

M. Ghiasy-Oskoee, M. AghaAlikhani, F. Sefidkon, A. Mokhtassi-Bidgoli, and M. Ayyari, “Blessed thistle agronomic and phytochemical response to nitrogen and plant density,” Industrial Crops and Products, vol. 122, pp. 566–573, 2018.

Z. Can, N. Baltaş, S. Keskin, O. Yıldız, and S. Kolaylı, “Properties of antioxidant and anti-inflammatory activity and phenolic profiles of Şevketi Bostan (Cnicus benedictus L.) cultivated in Aegean Region from Turkey,” Turkish Journal of Agriculture - Food Science and Technology, vol. 5, no. 4, pp. 308–314, 2017.

V. L. Singleton and J. A. Rossi, “Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents,” American Journal of Enology and Viticulture, vol. 16, no. 3, pp. 144–158, 1965.

J.-Y. Lin and C.-Y. Tang, “Determination of total phenolic and flavonoid contents in selected fruits and vegetables, as well as their stimulatory effects on mouse splenocyte proliferation,” Food Chemistry, vol. 101, no. 1, pp. 140–147, 2006.

C. Albu, S. A. V. Eremia, R. Penu et al., “Characterization of the phenolics and free radical scavenging of romanian red wine,” Analytical Letters, vol. 50, no. 4, pp. 591–606, 2017.

V. Bondet, W. Brand-Williams, and C. Berset, “Kinetics and mechanisms of antioxidant activity using the DPPH• free radical method,” LWT- Food Science and Technology, vol. 30, no. 6, pp. 609–615, 1997.

S. A. Baba and S. A. Malik, “Evaluation of antioxidant and antibacterial activity of methanolic extracts of Gentiana kurroo royle,” Saudi Journal of Biological Sciences, vol. 21, no. 5, pp. 493–498, 2014.

H. Wangensteen, A. B. Samuelsen, and K. E. Malterud, “Antioxidant activity in extracts from coriander,” Food Chemistry, vol. 88, no. 2, pp. 293–297, 2004.

A. Sahasrabudhe and M. Deodhar, “Anti-hyaluronidase, anti-elastase activity of Garcinia indica,” International Journal of Botany, vol. 6, no. 3, pp. 299–303, 2010.

L. G. Ranilla, Y.-I. Kwon, E. Apostolidis, and K. Shetty, “Phenolic compounds, antioxidant activity and in vitro inhibitory potential against key enzymes relevant for hyperglycemia and hypertension of commonly used medicinal plants, herbs and spices in Latin America,” Bioresource Technology, vol. 101, no. 12, pp. 4676–4689, 2010.

G. Paun, E. Neagu, C. Albu, V. Moroeanu, and G.-L. Radu, “Antioxidant activity and inhibitory effect of polyphenolic-rich extract from betonica officinalis and Impatiens noli-tangere herbs on key enzyme linked to type 2 diabetes,” Journal of the Taiwan Institute of Chemical Engineers, vol. 60, pp. 1–7, 2016.

P. Mccue, Y.-I. Kwon, and K. Shetty, “Anti-amylase, anti-glucosidase and anti-angiotensin I-converting enzyme potential of selected foods,” Journal of Food Biochemistry, vol. 29, no. 3, pp. 278–294, 2005.

R. Jaiswal, J. Kiprotich, and N. Kuhnert, “Determination of the hydroxycinnamate profile of 12 members of the Asteraceae family,” Phytochemistry, vol. 72, no. 8, pp. 781–790, 2011.

G. Paun, E. Neagu, C. Albu, and G. L. Radu, “Application of the polyphenylene ether-ether-sulfone ultrafiltration membrane for concentration of antioxidants from the Phyllitis scolopendrium L. extract,” New Journal of Chemistry, vol. 39, no. 2, pp. 1154–1160, 2015.

B. Tylkowski, I. Tsibranska, R. Kochanov, G. Peev, and M. Giamberini, “Concentration of biologically active compounds extracted from Sideritis ssp. L. by nanofiltration,” Food and Bioproducts Processing, vol. 89, no. 4, pp. 307–314, 2011.

C. Conidi, A. D. Rodriguez-Lopez, E. M. Garcia-Castello, and A. Cassano, “Purification of artichoke polyphenols by using membrane filtration and polymeric resins,” Separation and Purification Technology, vol. 144, pp. 153–161, 2015.

Y. Bellik, L. Boukraâ, H. A. Alzahrani et al., “Molecular mechanism underlying anti-inflammatory and anti-Allergic activities of phytochemicals: an update,” Molecules, vol. 18, no. 1, pp. 322–353, 2013.

N. Chockalingam and S. Muruhan, “Anti-inflammatory properties of rosmarinic acid - a review,” International Journal of Research in Pharmaceutical Sciences, vol. 8, no. 4, pp. 656–662, 2017.

M. Naveed, V. Hejazi, M. Abbas et al., “Chlorogenic acid (CGA): a pharmacological review and call for further research,” Biomedicine & Pharmacotherapy, vol. 97, pp. 67–74, 2018.

M.-H. Pan, C.-S. Lai, and C.-T. Ho, “Anti-inflammatory activity of natural dietary flavonoids,” Food & Function, vol. 1, no. 1, pp. 15–31, 2010.

C. Danciu, Ș. Avram, I. Z. Pavel et al., “Main isoflavones found in dietary sources as natural anti-inflammatory agents,” Current Drug Targets, vol. 19, no. 7, pp. 841–853, 2018.

D. Kashyap, H. S. Tuli, and A. K. Sharma, “Ursolic acid (UA): A metabolite with promising therapeutic potential,” Life Sciences, vol. 146, pp. 201–213, 2016.

A. O. Ademiluyi and G. Oboh, “Soybean phenolic-rich extracts inhibit key-enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin I converting enzyme) in vitro,” Experimental and Toxicologic Pathology, vol. 65, no. 3, pp. 305–309, 2013.

M. F. Di Carli, J. Janisse, G. Grunberger, and J. Ager, “Role of chronic hyperglycemia in the pathogenesis of coronary microvascular dysfunction in diabetes,” Journal of the American College of Cardiology, vol. 41, no. 8, pp. 1387–1393, 2003.

A. B. Shori, “Screening of antidiabetic and antioxidant activities of medicinal plants,” Journal of Integrative Medicine, vol. 13, no. 5, pp. 297–305, 2015.

A. Kam, K. M. Li, V. Razmovski-Naumovski et al., “A comparative study on the inhibitory effects of different parts and chemical constituents of pomegranate on α-amylase and α-glucosidase,” Phytotherapy Research, vol. 27, no. 11, pp. 1614–1620, 2013.

B.-W. Zhang, Y. Xing, C. Wen et al., “Pentacyclic triterpenes as α-glucosidase and α-amylase inhibitors: structure-activity relationships and the synergism with acarbose,” Bioorganic & Medicinal Chemistry Letters, vol. 27, no. 22, pp. 5065–5070, 2017.

E. Apostolidis, Y. I. Kwon, and K. Shetty, “Potential of cranberry-based herbal synergies for diabetes and hypertension management,” Asia Pacific Journal of Clinical Nutrition, vol. 15, no. 3, pp. 433–441, 2006.

K. Cianfaglione, E. E. Blomme, L. Quassinti et al., “Cytotoxic essential oils from eryngium campestre and eryngium amethystinum (Apiaceae) growing in central Italy,” Chemistry & Biodiversity, vol. 14, no. 7, Article ID e1700096, 2017.

M. Kikowska, M. Dworacka, I. Kędziora, and B. Thiem, “Eryngium creticum – Ethnopharmacology, phytochemistry and pharmacological activity. A review,” Revista Brasileira de Farmacognosia, vol. 26, no. 3, pp. 392–399, 2016.

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.