Antioxidant And Anticoagulant Effects Of Phenylpropanoid Glycosides
Mar 18, 2022
for more information:ali.ma@wecistanche.com
Bartosz Skalski, et al
Abstract
Holoparasitic plants of the Orobanchaceae, including Cistanche, Orobanche, and Phelipanche spp, are known for their richness of phenylpropanoid glycosides (PPGs). Many PPG compounds have been found to possess a wide spectrum of activities, such as antimicrobial, anti-inflammatory, antioxidant, and memory-enhancing. To better explore the bioactivity potential of European broomrapes (O. Caryophyllaceae – OC, P. Arenaria – PA, P. ramosa – PR) and ten single isolated phenylpropanoid constituents, we investigated their antiradical action, protective effect against oxidation in plasma in vitro system, and influence on coagulation parameters. The tested extracts showed a scavenging activity of 50–70% of Trolox’s power. The OC extract, rich in acteoside, had over 20% better antiradical potential than PR extract which was the only one containing PPGs lacking a B-ring catechol moiety in the acyl unit. Moreover, it was found that only eight tested PPGs (phenylpropanoid glycosides) demonstrated antioxidant potential in human plasma treated with H2O2/Fe; however, the three tested PPGs (phenylpropanoid glycosides) possessed anticoagulant potential in addition to antioxidant properties. It appears that the structure of PPGs, especially the presence of acyl and catechol moieties, is mainly related to their antioxidant properties. The anticoagulant potential of these compounds is also related to their chemical structure. Selected PPGs exhibit the potential for treating cardiovascular diseases associated with oxidative stress.
Keywords: Broomrape, Phenylpropanoid glycosides, Oxidative stress, Plasma Hemostasis
Cistanche has Phenylpropanoid glycosides
1. Introduction
Oxidative stress is widely known for its negative impact on the health of living organisms, including accelerated aging and some cancers. The occurrence of oxidative stress is associated with a disturbed balance between the oxidative and antioxidative mechanisms (including enzymatic (catalase, glutathione peroxidase) and non-enzymatic (glutathione) defense) in the cells of the body [1]. The overproduction of reactive oxygen species (ROS), including oxidizing radicals and closed-shell species, is one of the main mechanisms behind the formation of oxidative stress. However, the biological effect caused by ROS depends largely on the concentration, time of exposure, and location. Under normal conditions (low concentration), oxygen/nitrogen radicals can play the role of secondary messengers, but at the higher level, they may start to react with biological structures, like cell membranes [2]. Among all ROS species, a hydroxyl radical (HO. ) is causing of Oxidative stresses is known to play an important role in a range of diseases, including cardiovascular ones. Disorders of the blood system have been correlated and/or preceded by changes in various parameters of hemostasis and plasma biomarkers [1,3].
On the other hand, many natural substances, such as polyphenols and polyunsaturated fatty acids, have been identified as potent antioxidants capable of preventing the formation and/or diminishing reactive oxygen species. Compounds with such properties are found in many food products and pharmaceutical preparations of plant origin. A diet enriched with fresh vegetables and fruits, and antioxidative therapies based on natural antioxidants, are therefore widely recommended as they can reduce the level of oxidative stress and prevent various pathophysiological processes [4,5]. Plant polyphenols are a diverse group of secondary metabolites, among which phenolic acids occupy an important place, as they are widely distributed and exhibit a variety of biological effects, such as antimicrobial, antioxidant, and anti-inflammatory. Phenylpropanoid glycosides (PPGs) are ester derivatives of hydroxycinnamic acid and they are the main/only class of secondary metabolites present in holoparasitic Orobanchaceae plants, including Cistanche, Orobanche, and Phelipanche spp. Several species of this family are serious pests of crops that farmers want to get rid of in the fields (example of Phelipanche ramosa), few are used in pharmacology, while most are of little importance to humans. Herba Cistanche is extensively used in Asian traditional medicine in the treatment of kidney deficiency and as an immunity- and memory-enhancing, anti-aging, and antifatigue agent [6]. Phytochemical analyses of various research groups have demonstrated that phenylpropanoid glycosides, such as acteoside, echinacoside, and podium side, are one of the main active ingredients of Herba Cistanche [7]. A recent study of several broomrape species found in Poland by Jedrejek et al. [8] has shown that this plant material has a similar qualitative composition (domination of PPGs) (phenylpropanoid glycosides), moreover, it equals or even exceeds the Cistanche spp. in terms of the content of active substances [8]
The present study was aimed at evaluating the antiradical and antioxidant potential, as well as the influence on hemostasis parameters of the three broomrape extracts (Orobanche caryophyllacea – OC, Phelipanche Arenaria – PA, and P. ramosa – PR) rich in various phenylpropanoids, as well as their single PPG constituents. The antiradical capacity was measured using 2,2′ -azinobis-3-ethylbenzthiazoline-6-sulphonic acid/Trolox Equivalent (ABTS/TE) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) tests. The oxidative stress in the plasma test system was induced using a hydroxyl radical (H2O2/Fe), then lipid peroxidation (thiobarbituric acid-reactive species (TBARS) assay), and the level of protein carbonyl and thiol groups were measured. Among the determined parameters of hemostasis were: activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT).
Cistanche has Phenylpropanoid glycosides
2. Materials and methods
2.1. Chemicals
2,2-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2′ -azinobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), potassium persulfate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), dimethylsulfoxide (DMSO), thiobarbituric acid (TBA), formic acid (LC-MS grade), and H2O2 were purchased from Sigma-Aldrich (St. Louis, MO., USA). Methanol (HPLC gradient grade) and acetonitrile (LC-MS grade) were acquired from Merck (Darmstadt, Germany). Ten phenylpropanoid compounds tested in this work, including 2′ -O-acetylacteoside (97%), 2′ -O-acetylpoliumoside (98%), 3-O-methylpoliumoside (96%), acteoside (99%), arena inside (97%), crenatoside (98%), teniposide (99%), poliumoside (99%), tubuloside A (96%), and wiedemannioside D (96%) were previously isolated by us from below-given plant material [8]. The purity of compounds was assessed using a UHPLC-PDA-MS analysis. Ultrapure water was prepared in-house using a Milli-Q water purification system (Millipore Co.). Other reagents were of analytical grade and were provided by domestic commercial suppliers.
2.2. Plant material
Flowering plants of three broomrape species, including Orobanche Caryophyllaceae Sm., Phelipanche Arenaria Pomel, and P. ramosa (L.) Pomel was identified by prof. Renata Piwowarczyk (Jan Kochanowski University, Kielce, Poland) and collected from a natural source in Poland. Voucher specimens (O. Caryophyllaceae – Chomentowek ´ (50.3349◦N, 20.4000◦E), xerothermic grassland, parasitize Galium boreale, May 2014; P. Arenaria – Zwierzyniec (50.3652◦N, 22.5801◦E), psammophilous grassland and fallow, parasitize Artemisia campestris, June 2014; P. ramosa – Szewce (50.3553◦N, 22.3038◦E), field, parasitize Solanum Lycopersicum, September 2014) are deposited at the Herbarium of the Jan Kochanowski University in Kielce (KTC). The plant material was lyophilized and finely ground before extraction.
2.3. Preparation of broomrapes' extracts
Powdered plant material (O. Caryophyllaceae (OC) – 2 g, P. Arenaria (PA) – 3 g and P. ramosa (PR) – 3 g) was extracted with 80% MeOH at 40 ◦C and 1500 psi (solvent pressure) using an ASE 200 accelerated solvent extractor (Dionex, Sunnyvale, CA, USA). The extracts were evaporated and freeze-dried (Gamma 2–16 LSC freeze dryer, Christ, Germany). The extraction efficiency for OC, PA, and PR was 55%, 37%, and 43% by weight of the plant material, respectively. Due to the high content of carbohydrates (data not shown), the raw extracts were further purified by solid-phase extraction (SPE) on Oasis HLB micro-column (500 mg; Waters, Milford, MA, USA). The sugars were removed with 1% MeOH, then compounds of interest were eluted with 80% MeOH. After removing the solvent, the OC, PA, and PR extracts were lyophilized (Gamma 2–16 LSC freeze dryer), and the yields of SPE purification were 53% (OC), 67% (PA), and 51% (PR).
2.4. Phytochemical characteristics of broomrapes' extracts
Qualitative and quantitative analyses of broomrape extracts were performed using an ACQUITY UPLC system (Waters) connected to a photodiode array detector (PDA) and a tandem quadrupole mass spectrometer (TQD-MS/MS). Freeze-dried OC, PA, and PR extracts were dissolved in 50% methanol at a concentration of 0.50 mg/mL and then chromatographed on BEH C18 column (100 × 2.1 mm, 1.7 µm, Waters). Chromatographic conditions were as follows: oven temperature – 25 ◦C, linear-gradient 10→25% of mobile phase B (0.1% formic acid in acetonitrile) in mobile phase A (0.1% formic acid in H2O) over 12 min, flow rate – 0.4 mL/min, injection volume – 2 μL, UV range – 190–490 nm (3.6 nm resolution). The MS analysis was performed in negative ion mode with electrospray ionization (ESI), using the following settings: scan range 100–1200 m/z; capillary voltage 2.8 kV; cone voltage 35 V; source temperature 150 ◦C; desolvation temperature 450 ◦C; desolvation gas flow 900 L/h, and cone gas flow 100 L/h. Data acquisition and processing were performed using Waters MassLynx 4.1 software.
Phenylpropanoid glycoside (PPG) peaks were identified by comparison of the obtained LC-MS data with those of previously isolated compounds [8]. Quantitation of PPGs (phenylpropanoid glycosides) in broomrape extracts was based on the UPLC-UV method with detection at 330 nm, and an external standard calibration using acteoside (Sigma-Aldrich, ≥ 99%, HPLC) as group standard. A linear calibration curve was prepared in six concentrations within the range of 1–200 μg/mL and showed good linearity (R2 ≥ 0.999). Quantitative results represent the mean ± SD value of three injections and were expressed as milligrams acteoside equivalents (eq) per gram of extract (mg acteoside eq/g).
Cistanche has Phenylpropanoid glycosides
2.5. Antiradical activity in vitro
2.5.1. ABTS radical scavenging assay
The ABTS antiradical test was carried out using the method described by Kontek et al. [9], with slight modifications as follows: 20% MeOH was used to prepare reagents (7 mM ABTS and 4.9 mM potassium persulfate); the solutions of OC, PA, and PR extracts, at four concentration level in the range of 100− 400 μg/mL, and Trolox solutions, at six concentration level in the range of 10− 250 μg/mL, were prepared with 50% MeOH. The proportion of sample to ABTS+ working solution was 1:25 (v/v). The absorbance at 734 nm was measured after 30 min incubation in the dark using a UV–vis spectrophotometer (Evolution 260 Bio, Thermo Fisher Scientific Inc., Waltham, MA, USA).
The absorbance inhibition (%) was calculated as follows: [(Abscontrol–Abssample)/Abscontrol] ×100.
The Trolox Equivalents (TE) of broomrapes’ extracts were calculated using formula TE = sample/standard, where m is the slope of the straight line curves (absorbance inhibition vs. concentration). The TE value of the sample describes its normalized activity against Trolox (TEstandard = 1.0). The IC50 values for OC, PA, and PR extracts and Trolox were reached experimentally, then were calculated from their straight-line curves (absorbance inhibition vs. concentration) and are expressed in μg/mL.
The assay was performed in triplicate, and the results are presented as means ± standard deviations (SD).
2.5.2. DPPH radical scavenging assay
The DPPH antiradical test was carried out using the method described by Jedrejek et al. [8] and Brand-Williams et al. [10], with slight modifications as follows: the solutions of OC, PA, and PR extracts, at four concentration levels in the range of 50− 250 μg/mL, and Trolox solutions, at six concentration level in the range of 10− 250 μg/mL, were prepared with 50% MeOH. The proportion of sample to DPPH was 1:19 (v/v). The absorbance at 517 nm was measured after 30 min incubation in the dark using a UV–vis spectrophotometer (Evolution 260 Bio).
The absorbance inhibition (%) was calculated as follows: [(Abscontrol–Abssample)/Abscontrol] ×100.
The Trolox Equivalent (TE) and IC50 values of test samples were calculated in the same manner as in the ABTS test (Section 2.5.1). The assay was performed in triplicate, and the results are presented as means ± SD.
2.6. Stock solutions of tested plant compounds and extracts for experiments with human plasma
Stock solutions of the tested compounds and plant extracts were prepared in 50% DMSO. The final concentration of DMSO in the tested samples was lower than 0.05% and its effects were determined in all experiments.
2.7. Human plasma isolation
Human blood, or plasma, was obtained from six regular donors (nonsmoking men and women) to a blood bank (Lodz, Poland) and a medical center (Lodz, Poland). Blood was collected as a CPD solution (citrate/ phosphate/dextrose; 9:1; v/v blood/CPD) or CPDA solution (citrate/ phosphate/dextrose/adenine; 8.5:1; v/v; blood/CPDA). The donors had not taken any medication or addictive substances (including tobacco, alcohol, and antioxidant supplementation) for at least two weeks before a donation. Our analysis of the blood samples was performed under the guidelines of the Helsinki Declaration for Human Research and approved by the Committee on the Ethics of Research in Human Experimentation at the University of Lodz. Plasma was prepared by centrifugation of fresh human blood at 4500x g for 25 min at room temperature. The protein concentration was calculated by measuring the absorbance of the tested samples at 280 nm, according to the procedure of Whitaker and Granum [11].
Cistanche has Phenylpropanoid glycosides
2.8. Markers of oxidative stress in human plasma
2.8.1. Lipid peroxidation measurement
Plasma lipid peroxidation was quantified by measuring the concentration of thiobarbituric acid reactive substances (TBARS). TBARS concentration was calculated using the molar extinction coefficient (ε = 156,000 M− 1 cm− 1 ). The method is described more thoroughly elsewhere [12,13].
2.8.2. Carbonyl group measurement
The level of carbonyl groups was calculated using the molar extinction coefficient (ε = 22,000 M− 1 cm− 1 ) and was expressed as nmol carbonyl groups/mg of plasma protein, according to Bartosz [13] and Levine et al. [14].
2.8.3. Thiol group determination
The thiol group content in plasma proteins was measured spectrophotometrically using a SPECTROstar Nano Microplate Reader (BMG LABTECH, Germany) by absorbance at 412 nm with 5,5′ -dithiol-bis-(2- nitrobenzoic acid). The method is described in more detail elsewhere [15–17].
2.9. Parameters of hemostasis
2.9.1. The measurement of prothrombin time (PT)
PT was determined coagulometrically using an Optic Coagulation Analyser (model K-3002, Kselmed, Grudziadz, Poland) according to Malinowska et al. [18].
2.9.2. The measurement of thrombin time (TT)
The TT was determined coagulometrically using an Optic Coagulation Analyser (model K-3002, Kselmed, Grudziadz, Poland), according to the method described by Malinowska et al. [18].
2.9.3. The measurement of activated partial thromboplastin time (APTT)
The APTT was determined coagulometrically using a K-3002 Optic Coagulation Analyser (Kselmed, Grudziadz, Poland) according to Malinowska et al. [18].
2.10. Data analysis
The Q-Dixon test was performed to eliminate uncertain data. The data were tested for normal distribution with the Shapiro-Wilk test and equality of variance with Levene’s test. Statistically significant differences were identified using ANOVA, followed by Tukey’s multiple comparisons test or the Kruskal-Wallis test. Comparisons were considered significant at p < 0.05. The values are presented as means ± SD/SEM.
3. Results and discussion
Ten previously isolated by us phenylpropanoid glycosides [8], including 2′ -O-acetylacteoside, 2′ -O-acetylpoliumoside, 3-O-methylpoliumoside, acteoside, arena inside, crenatoside, pheliposide, podium side, tubuloside A, and wiedemannioside D, together with three broomrape extracts (Orobanche Caryophyllaceae (OC), Phelipanche arenaria (PA), and P. ramosa (PR)) were currently studied for alleviation oxidative stress and anticoagulant properties in a human plasma system. Chemical structures of the phenylpropanoids tested are presented in Fig. 1, and, as can be seen, they are all built according to a similar pattern, with the same/similar sub-units: hydroxytyrosol, monosaccharides (glucose, rhamnose, and/or xylose), and hydroxycinnamic acid. Most of the examined PPG compounds are substituted with caffeic acid, but this may be replaced with either coumaric or ferulic acid.
Apart from individual PPG compounds, three broomrape extracts – OC, PA, and PR, which are mixtures of several PPGs (phenylpropanoid glycosides) and served previously as starting material for compound isolation, were also included in the biological study. Another reason for selecting three different species was the large difference in the phytochemical profile among them, as can be seen in Fig. 2. A more detailed comparison of the OC, PA, and PR extracts, including the quantitative data, is presented in Table 1. Acteoside was the main constituent of the O. Caryophyllaceae extract (690 mg/g), teniposide, and arena inside dominated in P. Arenaria (together 550 mg/g), while poliumoside and its acetylated derivative were the most important metabolites in P. ramosa extract (together 640 mg/g). The examined extracts also differed in the overall content of phenylpropanoids, the highest amount was found in OC (810 mg/g), a little lower in PR (795 mg/g), and lower in PA (685 mg/g). Moreover, it is worthy to note that the presence of PPGs with other than caffeoyl moieties, such as coumaroyl or feruloyl, was detected only in P. ramosa extract, where these compounds constituted about one-sixth of the total PPGs (about 120 mg/g) (Table 1).
Previous studies of the antiradical activity of phenylpropanoid glycosides by Heilmann et al. [19] and Jedrejek et al. [8], including about 30 different PPGs (phenylpropanoid glycosides) such as acteoside, isoacteoside, and crenatoside, have revealed its strong relation to the structure of acyl moieties (phenolic acid and tyrosol). In general, the modification or substitution of catechol moiety of acyl unit resulted in a significant decrease in the scavenging activity against reactive oxygen species (ROS) and DPPH radical. In the current study, the antiradical in vitro potential of three broomrape extracts (OC, PA, and PR) was examined with ABTS and DPPH assays, and results were compared both with each other as well as with the activity of individual phenylpropanoid components measured in our previous study [8]. The results were expressed as Trolox Equivalents (TE) and IC50 values (Table 2). In general, all three extracts were good scavengers of both ABTS and DPPH radicals but also differences were observed among the samples tested (estimated TE was in the range of 0.5–0.7; 1.0 was the equivalent of Trolox). Antiradical scavenging activity of samples was in the following order: Trolox > OC > PA > PR. The extract of Orobanche Caryophyllaceae (IC50 = 155–275 µg/mL) had over 20% greater activity than Phelipanche ramosa extract (IC50 = 200–320 µg/mL).
The reported highest radical scavenging activity of the OC extract can be explained by the highest PPGs (phenylpropanoid glycosides) content in this sample, as well as to the input of acteoside, its dominant ingredient, which according to the previous research [8,19] is one of the strongest free radical scavengers among the metabolites from this group (TEDPPH = 0.87; [4]). However, considering the mutual relationship of antiradical activity and the content of phenylpropanoids in the OC, PA, and PR extracts, no simple correlation was found between these two factors (r < 0.5), indicating a significant input of qualitative profile. This is mainly related to the P. ramosa extract, which despite the high level of PPGs (phenylpropanoid glycosides) (0.8 g/g) was characterized by the lowest biological activity among the tested samples (TE ~ 0.5). The PR extract, as mentioned above, was the only sample to possess the phenylpropanoids with coumaric or ferulic acids, substances lacking a B-ring catechol moiety, which have been reported to have decreased anti-oxidative potential. Four PPG compounds having modified caffeic acid, including 3-O-methylpoliumoside, ramose A, and wiedemannioside D, tested by us previously had the TEDPPH of around 0.3 [8]. Thus, current results are in accordance with and confirm the findings of the previous antiradical in vitro experiments on phenylpropanoids.
As Chen et al. [20] described, it is associated with greater H-donating ability or stabilization of the radical by various functional groups of a mixture of compounds. Several structural elements have been identified as enhancing the direct antioxidant activity of polyphenols, especially those associated with the number and position of hydroxyl groups. It is believed that free radical scavenging activity increases with the increasing number of –OH groups. However, the position of these groups, in a molecule, has an even greater impact on the exerted activity. Relatively stable potent compounds are those possessing 3,4-dihydroxy moiety in their structures, as well as those possessing more than two hydroxyl groups [21]. The chemical structure of the antioxidant substance allows an understanding of the antioxidant reaction mechanism. Lopez-Munguía et al. [22] based on density functional theory (DFT) calculations determined that the PPGs (phenylpropanoid glycosides) antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). However, Li et al. [23] attempt to explore the mechanisms of phenolic phenylpropanoid antioxidants, concluded, that PPGs (acteoside, forsythoside B, and poliumoside) may be involved in multiple pathways to exert the antioxidant action, enhanced the role of sugar-residues.
Studies have shown that plant-derived antioxidants are effective modulators of hemostasis in cardiovascular diseases [24–26]. Various plants used in traditional medicine contain significant levels of PPGs [27,28]. In addition, PPGs (phenylpropanoid glycosides) are known to have a range of biological activities, including anti-inflammatory, anti-nephritic, and anti-hepatoxic properties [29–33].
In their recent study, Jedrejek et al. [8] described the isolation of PPGs (phenylpropanoid glycosides) from three Polish broomrapes and assessed their antioxidant activity by the DPPH test. Based thereupon, the present study evaluates whether the ten selected PPGs isolated from these plants could reduce oxidative stress in human plasma treated with a strong biological oxidant, i.e. the hydroxyl radical donor H2O2/Fe, and modulate coagulation properties of plasma in vitro. The antioxidant properties of ten isolated PPGs were determined according to selected parameters of oxidative stress: TBARS level as a marker of lipid peroxidation, together with carbonyl group and thiol group levels, as markers of oxidative protein damage.
Both plasma lipid peroxidation and protein carbonylation levels in plasma induced by H2O2/Fe were significantly reduced in the presence of eight tested compounds, viz. acteoside, crenatoside, 2′ -O-acetylacteoside, pheliposide, arena inside, tubuloside A, poliumoside and 3- O-methylpolimuoside, at all tested concentrations (1, 5 and 50 µg/ mL); however, neither effect was observed for two of the tested compounds, viz. 2′ -O-acetylpoliumoside and wiedemannioside D, or any of the tested extracts at any concentration (1, 5, and 50 µg/mL). In addition, none of the tested compounds or tested extracts were found to protect the plasma against H2O2/Fe – induced thiol group oxidation in proteins (Figs. 3–5). However, the tested extracts may be the source of compounds with various biological properties.
For the first time, the results of the present study indicate that eight of the tested PPGs (phenylpropanoid glycosides) demonstrate antioxidant potential in human plasma in the presence of exogenous reactive oxygen species by inhibiting lipid peroxidation and protein carbonylation in plasma treated with H2O2/Fe. In addition, 2′ -O-acetylpoliumoside and wiedemannioside D did not present any such effect. In general, our findings are consistent with previous in vitro experiments on PPGs. Heilmann et al. [19] and Jedrejek et al. [8] report a correlation between the chemical structure of PPGs and their activities. Antioxidant properties of PPGs (phenylpropanoid glycosides) appear to be primarily related to the structure of their acyl moieties, i.e. the phenolic acid and phenylpropanoid unit, including the presence and/or modification of catechol moiety. For example, wiedemannioside D was found to lose its antioxidative potential towards plasma treated with H2O2/Fe following the replacement of its caffeoyl moiety with a feruloyl moiety.
Changes in the coagulation process often result from oxidative stress; these changes can modulate the functions of the cardiovascular system and can lead to the development of cardiovascular diseases [1]. Out of the ten plant compounds and three plant extracts tested in the present study, tubuloside, podium side, and 3-O-methylpoliumoside and all tested extracts demonstrated to significantly prolong thrombin time at all tested concentrations, viz. 1, 5, and 50 µg/mL (Fig. 6B). However, none of these extracts, nor any of the tested compounds, changed PT or APTT (Fig. 6A and C).
Fig. 6. Effects of tested compounds (acteoside, crenatoside, 2′-O-acetylacteoside, pheliposide, arenarioside, tubuloside A, poliumoside, 3-O-methylpoliumoside, 2′-O-cetylpolimuoside, wiedemannioside D) and plant extracts (P. arenaria extract - PA, P. ramosa extract – PR, and O. caryophyllacea extract - OC) (1–50 µg/mL) on selected haemostatic parameters of plasma: PT (A), TTn (B) and APTT (C). Data represent means ± SEM of six independent experiments.n* p < 0.05 (vs. control), n.s. – p > 0.05 (vs. control).
Table 3 compares the effects of the PPGs (5 µg/mL) on biomarkers of oxidative stress in plasma treated with H2O2/Fe and their influence on coagulation. Eight of the tested PPGs (phenylpropanoid glycosides) demonstrated antioxidant potential only in the treated human plasma; however, three tested PPGs were found to possess both antioxidant properties and anticoagulant potential. Interestingly, the results of the DPPH test did not coincide with those obtained in the biological model using human plasma treated with H2O2/Fe: the antioxidant potential of the tested extracts may be blocked by certain compounds present in the plasma.
In conclusion, our present findings shed new light on the antioxidant potential and anticoagulant properties of PPGs (phenylpropanoid glycosides). It appears that the structure of PPGs (phenylpropanoid glycosides), especially the presence of acyl and catechol moieties, is mainly related to their antioxidant and anticoagulant properties. Selected PPGs may well have the potential for the treatment of cardiovascular diseases associated with oxidative stress. However, further experiments are needed to determine the concentrations of these compounds needed for in vivo models.
Conflict of interest statement
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
[1] A. Daiber, S. Chlopicki, Revisiting pharmacology of oxidative stress and endothelial dysfunction in cardiovascular disease: evidence for redox-based therapies, Free Radic. Biol. Med. 157 (2020) 15–37.
[2] J.-J. Sauvain, A. Setyan, P. Wild, P. Tacchini, G. Lagger, F. Storti, S. Deslarzes, M. J. Guillemin, M. Rossi, M. Riediker, Biomarkers of oxidative stress and its association with the urinary reducing capacity in bus maintenance workers, J. Occupat. Med. Toxicol. 6 (2011) 18–23.
[3] P. Nowak, B. Olas, B. Wachowicz, Oxidative stress and hemostasis, Post. Biochem. 56 (2010) 239–247.
[4] M. Martinez-Huelamo, J. Rodriguez-Morato, A. Boronat, R. de la Torre, Modulation of Nrf2 by olive oil and wine polyphenols and neuroprotection, Antioxidants 6 (2017) 73.
[5] S. Amor, P. Chalons, V. Aires, D. Delmas, Polyphenol extracts from red wine and grapevine: potential effects on cancers, Diseases 6 (2018) 1–12.
[6] Z. Li, H. Lin, L. Gu, J. Gao, C.M. Tzeng, Herba Cistanche (Rou Cong-Rong): one of the best pharmaceutical gifts of traditional Chinese medicine, Front. Pharmacol. 7 (2016) 41–49.
[7] Y. Jiang, P.-F. Tu, Review: analysis of chemical constituents in Cistanche species, J. Chromatogr. 1216 (2009) 1970–1979.
[8] D. Jedrejek, S. Pawelec, R. Piwowarczyk, L. Precio, A. Stochmal, Identification and occurrence of phenylethanoid and iridoid glycosides in six Polish broomrapes (Orobanche spp. and Phelipanche spp., Orobanchaceae), Phytochemistry 170 (2020), 112189.
[9] B. Kontek, D. Jedrejek, W. Oleszek, B. Olas, Antiradical and antioxidant activity in vitro of hops-derived extracts rich in bitter acids and xanthohumol, Ind. Crops Prod. 161 (2021) 1–9.
[10] W. Brand-Williams, M.E. Cuvelier, C. Berset, Use of a free radical method to evaluate antioxidant activity, Lebensm. Wiss. Technol. 28 (1995) 25–30.
[11] J.R. Whitaker, P.E. Granum, An absolute method for protein determination based on the difference in absorbance at 235 and 280 nm, Anal. Biochem. 109 (1980) 156–159.
[12] B. Wachowicz, Adenine nucleotides in thrombocytes of birds, Cell Biochem. Funct. 2 (1984) 167–170.
[13] G. Bartosz, Druga twarz tlenu, Warsz. PWN 1 (2008) 7.
[14] R.L. Levine, D. Garland, C.N. Oliver, A. Amici, I. Climent, A.G. Lenz, B.W. Ahu, S. Shaltier, E.R. Stadtman, Determination of carbonyl content in oxidatively modified proteins, Methods Enzym. 186 (1990) 464–478.
[15] D.M. Joel, J. Gressel, L.J. (Eds.), Musselman, Parasitic Orobanchaceae: Parasitic Mechanisms and Control Strategies, Springer, Heidelberg, Germany, 2013, pp. 1–10.
[16] Y. Ando, M. Steiner, Sulphydryl and disulfide groups of platelet membranes: determination of disulfide groups, Biochim. Biophys. Acta 311 (1973) 26–37.
[17] Y. Ando, M. Steiner, Sulphydryl and disulfide groups of platelet membranes: determination of sulphydryl groups, Biochim. Biophys. Acta 311 (1973) 38–44.
[18] J. Malinowska, J. Kołodziejczyk-Czepas, M. Moniuszko-Szajwaj, I. Kowalska, W. Oleszek, A. Stochmal, B. Olas, Phenolic fractions from Trifolium pallidum and Trifolium scabrum aerial parts in human plasma protect against changes induced by hyperhomocysteinemia, Food Chem. Toxicol. 50 (2012) 4023–4027.
[19] J. Heilmann, I. Calis, H. Kirmizibekmez, W. Schuhly, S. Harput, O. Stiher, Radical scavenger activity of phenylpropanoid glycosides in FMLP stimulated polymorphonuclear leukocytes: structure-activity relationships, Planta Med. 66 (2000) 746–748.
[20] H. Chen, Y. Zhou, Y. Shao, F. Chen, Free phenolic acids in Shanxi aged vinegar: changes during aging and synergistic antioxidant activities, Int. J. Food Prop. 19 (2015) 1183–1193.
[21] C.A. Rice-Evans, N.J. Miller, G. Paganga, Structure-antioxidant activity relationships of flavonoids and phenolic acids, Free Radic. Biol. Med. 20 (1996) 933–956.
[22] A. Lopez-Munguía, ´ Y. Hernandez-Romero, ´ J. Pedraza-Chaverri, A. MirandaMolina, I. Regla, A. Martínez, E. Castillo, Phenylpropanoid glycoside analogs: enzymatic synthesis, antioxidant activity and theoretical study of their free radical scavenger mechanism, PloS One 6 (2011) 20115.
[23] X. Li, Y. Xie, K. Li, A. Wu, H. Xie, Q. Guo, P. Xue, Y. Maleshibek, W. Zhao, J. Guo, D. Chen, Antioxidation and cytoprotection of acteoside and its derivatives: comparison and mechanistic chemistry, Molecules 23 (2018) 498.
[24] S.E. Kulling, H.M. Rawel, Chokeberry (Aronia melanocarpa) – a review on the characteristic components and potential health effects, Planta Med. 74 (2008) 1625–1634.
[25] B.J. Mc Even, The influence of diet and nutrients on platelet function, Semin. Thromb. Hemost. 40 (2008) 214–226.
[26] B. Olas, The multifunctionality of berries toward blood platelets and the role of berry phenolics in cardiovascular disorders, Platelets 5 (2016) 1–10.
[27] U.B. Ismailoglu, I. Saracoglu, U.S. Harput, I. Sahin-Eredemli, Effects of phenylpropanoid and iridoid glycosides on free radical-induced impairment of endothelium-dependent relaxation in rat aortic rings, J. Ethnopharmacol. 79 (2002) 193–197.
[28] Z.F. Bai, Y. Liu, X.Q. Wang, Investigation of ethnic medicinal plants Orobanche, Cistanche and Boschniakia, Zhongguo Zhong Yao Zhi 93 (2014) 4548–4552.
[29] K. Hayashi, T. Nagamatsu, M. Ito, H. Yagita, Y. Suzuki, Acteoside, a component of Stachys sieboldii MIQ, may be a promising antinephritic agent (3): effect of acetonide on the expression on intercellular adhesion molecule-1 in experimental nephritic glomeruli in rats and cultured endothelial cells, Jpn. J. Pharmacol. 70 (1996) 157–168.
[30] Q. Xiong, K. Hase, Y. Tezuka, T. Tani, T. Namba, S. Kadota, Hepatoprotective activity of phenylpropanoids from Cistanche deserticola, Planta Med. 64 (1998) 120–125.
[31] W.F. Chiou, L.C. Lin, C.F. Chen, Acteoside protects endothelial cells against free radical-induced oxidative stress, J. Pharm. Pharm. 56 (2004) 743–748.
[32] S. Sahpaz, N. Garbacki, M. Tits, F. Bailleul, Isolation and pharmacological activity of phenylpropanoid esters from Marrubium vulgare, J. Ethnopharmacol. 79 (2002) 389–392.
[33] L. Lie-Chwen, W. Yea-Hwey, H. Yu-Chang, Ch Shiou, L. Kuo-Tong, Ch Yueh-Ching, W. Wen-Yen, S. Yoh-Chiang, The inhibitory effect of phenylpropanoid glycosides and iridoid glucosides on free radical production and β2 integrin expression in human leucocytes, J. Pharm. Pharmacol. 58 (2006) 129–135.
