PART 1 Antioxidant And Anticoagulant Effects Of Phenylpropanoid Glycosides Isolated From Broomrapes (Orobanche Caryophyllacea, Phelipanche Arenaria, And P. Ramosa)
Mar 06, 2022
Bartosz Skalski a, Sylwia Pawelec b, Dariusz Jedrejek b, Agata Rolnik a, Rostyslav Pietukhov a,
Renata Piwowarczyk c, Anna Stochmal b, Beata Olas a,*
a University of Ło´d´z, Department of General Biochemistry, Faculty of Biology and Environmental Protection, 90-236 Ł´od´z, Poland
b Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, 24-100 Puławy, Poland
Center for Research and Conservation of Biodiversity, Department of Environmental Biology, Institute of Biology, Jan Kochanowski University, 25-406 Kielce, Poland
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 demonstrated antioxidant potential in human plasma treated with H2O2/Fe; however, the three tested PPGs 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.
For more information please contact: Joanna.jia@wecistanche.com
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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 one of the biggest damage to bio-macromolecules: proteins, lipids, and DNA. Oxidative stress 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 de- relatives 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 acetonide, 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), moreover, it equals or even exceeds the Cistanche spp. in terms of the content of active substances [8].
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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 Caryophyllaceae – OC, Pheli- panache Arenaria – PA, and P. ramosa – PR) rich in various phenyl-
prostanoids, 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-picrylhybrazil (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).
2. Materials and methods
2.1. Chemicals
2,2-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2′-azinobis-3-eth- ylbenzthiazoline-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 phenyl-
propanoic compounds tested in this work, including 2′-O-acetylacteoside (97%), 2′-O-acetylpoliumoside (98%), 3-O-methylpoliumoside(96%), acetonide (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. Ramos (L.) Pomel was identified by prof. Renata Piwowarczyk (Jan Kochanowski Uni- versity, Kielce, Poland) and collected from a natural source in Poland.
Voucher specimens (O. Caryophyllaceae – Chomento´wek (50.3349◦N, 20.4000◦E), xerothermic grassland, parasitize Galium boreale, May 2014; P. Arenaria – Zwierzyniec (50.3652◦N, 22.5801◦E), psammoma- lous 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 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).
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 per-
sulfate); the solutions of OC, PA, and PR extracts, at four concentration levels in the range of 100—400 μg/mL, and Trolox solutions, at six concentration levels 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: [(Absecon-
trol–Abssample)/Abscontrol] ×100.
The Trolox Equivalents (TE) of broomrapes’ extracts were calculated
using formula TE = msample/mstandard, 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: [(Absecontroll–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 (non-smoking 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].
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 else were [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
Letters indicate the results of Tukey’s test (p < 0.05), values with the same
letter within a row is not significantly different.
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 Malinow ska 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.
