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Echinacea complex – chemical view and anti-asthmatic profile
Author’s Accepted Manuscript Echinacea complex – chemical view and antiasthmatic profile Martina Šutovská, Peter Capek, Ivana Kazimierová, Lenka Pappová, Marta Jošková, Mária Matulová, Soňa Fraňová, Izabela Pawlaczyk, Roman Gancarz www.elsevier.com
PII: DOI: Reference:
S0378-8741(15)30132-X http://dx.doi.org/10.1016/j.jep.2015.09.007 JEP9730
To appear in: Journal of Ethnopharmacology Received date: 19 May 2015 Revised date: 4 September 2015 Accepted date: 6 September 2015 Cite this article as: Martina Šutovská, Peter Capek, Ivana Kazimierová, Lenka Pappová, Marta Jošková, Mária Matulová, Soňa Fraňová, Izabela Pawlaczyk and Roman Gancarz, Echinacea complex – chemical view and anti-asthmatic profile, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.09.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Echinacea complex – chemical view and anti-asthmatic profile
Martina Šutovskáa, Peter Capekb*, Ivana Kazimierováa, Lenka Pappováa, Marta Joškováa, Mária Matulováb , Soňa Fraňováa, Izabela Pawlaczykc, Roman Gancarzc
a
Department of Pharmacology, Jessenius Faculty of Medicine Comenius University,
Martin's Biomedical Center (BioMed) Malá Hora 11161 4C, Martin, Slovakia b
Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská
cesta 9, Bratislava, Slovakia c
Division of Organic and Pharmaceutical Technology, University of Technology, Wrocław,
Poland
ABSTRACT
Ethnopharmacological relevance Echinacea purpurea (L.) Moench is one of the mostly used herbs in the traditional medicine for the treatment of respiratory diseases. Modern interest in Echinacea is directed to its immunomodulatory activity. Recent studies have shown that secretion of asthmarelated cytokines in the bronchial epithelial cells can be reversed by Echinacea preparations. Aim of the study To examine the pharmacodynamics profile of Echinacea active principles, a complex has been isolated from its flowers by alkaline extraction and has been tested using an animal model of allergic asthma. Material and methods
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The structural features of Echinacea purpurea complex was determined using chemical and spectroscopic methods. Allergic inflammation of the airways was induced by repetitive exposure of guinea pigs to ovalbumin. Echinacea complex was then administered 14 days in 50 mg/kg b.w. daily dose perorally. Bronchodilatory effect was verified as decrease in the specific airway resistance (sRaw) in vivo and by reduced contraction amplitude (mN) of tracheal and pulmonary smooth muscle to cumulative concentrations of acetylcholine and histamine in vitro. The impact on mucociliary clearance evaluated measurement of ciliary beat frequency (CBF) in vitro using LabVIEW™ Software. Anti-inflammatory effect of Echinacea complex was verified by changes in exhaled NO levels and by Bio-Plex® assay of Th2 cytokine concentrations (IL-4, IL-5, IL-13 and TNF-alpha) in serum and bronchoalveolar lavage fluid (BALF). Results Chemical and spectroscopic studies confirmed the presence of carbohydrates, phenolic compounds and proteins, as well as the dominance of rhamnogalacturonan and arabinogalactan moieties in Echinacea complex. The significant decrease in sRaw values and suppressed histamine and acetylcholine-induced contractile amplitude of isolated airways smooth muscle that were similar to effects of control drug salbutamol confirmed Echinacea complex bronchodilatory activity. The anti-inflammatory effect was comparable with that of control agent budesonide and was verified as significantly reduced exhaled NO levels and concentration of Th2 cytokines in serum and BALF. The values of CBF were changed only insignificantly on long-term administration of Echinacea complex suggested its minimal negative impact on mucociliary clearance. Conclusion Pharmacodynamic studies have confirmed significant bronchodilatory and antiinflammatory effects of Echinacea complex that was similar to effects of classic synthetic 2
drugs. Thus, results provide a scientific basis for the application of this herb in traditional medicine.as a supplementary treatment of allergic disorders of the airways, such as asthma.
Keywords: Echinacea purpurea, Polysaccharide-phenolic-protein complex, NMR, Antiasthmatic effect, Bronchodilatory effect, Anti-inflammatory effect ____________________ *Corresponding author. Tel.:+421 2 59410209; fax: +421 2 59410222 E-mail address: [email protected] (P. Capek)
1. Introduction
Echinacea purpurea (L.) Moench (Purple coneflower or coneflower) is a perennial plant of Asteraceae family. Nowadays, it is regarded as the medicinal and as well ornamental plant. Several Echinacea species have been used in the traditional medicine to treat infections, cold, cough, bronchitis, inflammations, etc. (Barrett, 2003; Goel et al. 2004; Wagner et al. 1999). Echinacea is one of the most used plants in herbal medicine and in dietary supplements (Percival, 2000). Modern interest in Echinacea is directed to its immunomodulatory activity, in particular in the prevention and treatment of the common cold, cough, bronchitis, and other respiratory infections. Phytoconstituents of Echinacea species are still the subject of chemical and pharmacological research in order to identify their active ingredients, however, the description of all constituents is still not completed. A great deal of clinical trials was performed to verify the efficacy of Echinacea constituents isolated by different procedures/solvents from various Echinacea species to describe their active principles. Four main groups of constituents are considered to be active in Echinacea species - alkylamides, glycoproteins, phenylpropanoids and polysaccharides. It is believed that these compounds are responsible for immune3
stimulating, anti-inflammatory and anticoagulant activities (Pawlaczyk et al. 2009; Spelma et al. 2009). Concerning polymeric compounds, the modulation of immune system was reported by Echinacea polysaccharides, i.e. enhancing production of TNF-α, IL-6 , IL-10, and IL-1-β, adjuvant effects on human T-cell cytokine responses, increasing production of reactive oxygen intermediates, etc. (Roesler et al. 1991; Steinmuller et al. 1993; Currier and Miller, 2000; Currier et al. 2002; Fonseca et al. 2014). Furthermore, it was found that active polysaccharides did not stimulate B cells. Allergic asthma is a chronic obstructive disease of the lower airways, characterized by airway inflammation, reversible airflow obstruction, mucus hypersecretion, and airway hyperreactivity (Nakagome and Nagata, 2011). Many herbs have shown interesting results in various target specific biological activities such as bronchodilation, mast cell stabilization, immunomodulatory, antiinflammatory and inhibition of mediators such as leukotrienes and cytokines, in the treatment of asthma (Mali and Dhake, 2011). Recent studies have shown, that expression of asthma-related cytokine genes and product secretions in the bronchial epithelial cells can be reversed by Echinacea preparations (Barrett, 2003). From the literature it is evident, that phytoconstituents of E. purpurea are still subject of chemical and pharmacological attention. This fact inspired us to evaluate pharmacodynamic profile of E. purpurea complex using an animal model of allergic asthma. This model can mimic the pathological symptoms common in humans suffered from allergic bronchial asthma, for example airway hyperreactivity and inflammatory changes in small diameter bronchioles (Franova et al. 2013).
2. Material and methods
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2.1. Plant material and chemicals
Air-dried flowers of medicinal plant E. purpurea (L.) Moench were purchased from a local market in Wroclaw, Poland. The identity of the plant was certified by Prof. K. D. Kromer and J. Kochanowska from Botanical Garden of Wrocław University, Poland and a voucher specimen (No. 011493) has been deposited in the Botanical Garden of Wrocław University, Poland. Citric acid (AC) p.a., histamine, acetylcholine, methacholine, salbutamol, aluminium hydroxide, budesonide and chicken ovalbumine purchased from Sigma Aldrich (Lambda Life, Slovakia). Budesonide was prepared as suspension in 1 % TWEEN 80 (in 0.9 % saline) according to manufacturer’s instruction. All other above-mentioned drugs were dissolved in 0.9 % saline.
2.2. Isolation of Echinacea complex
The isolation of E. purpurea complex was made according to already described procedure (Pawlaczyk et al., 2009). Shortly, flowering parts were minced and suspended in 0.1 M sodium hydroxide at room temperature for 24 h and refluxed for 6 h. The rest of plant was removed by centrifugation. The supernatant was neutralized by 1 M HCl, concentrated to a lower volume and gradually extracted with hexane (1:1, v/v), diethyl ether (1:1, v/v), chloroform (1:1, v/v), and chloroform and ethanol mixture (3:2, v/v). Organic extracts were discarded while the water fraction was evaporated to a paste and treated with methanol at room temperature. The soluble methanolic part was filtered off and the dark residue was solubilized in deionized water, dialyzed and freeze-dried to give a dark brown Echinacea complex.
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2.3. General methods
Concentration of solutions was performed under reduced pressure at bath temperature not exceeding 40 C. The content of carbohydrate, phenolic and protein was estimated by the phenol–sulfuric acid, Folin–Ciocalteu and Lowry assays, respectively (Dubois et al. 1956; Lowry et al. 1951; Singleton et al. 1999) and the uronic acid content was determined by m-hydroxybiphenyl reagent (Blumenkrantz and Asboe-Hansen, 1973). Sample was hydrolysed with 2 M TFA for 1 h at 120 ºC and the quantitative determination of the neutral sugars was carried out in the form of their alditol acetates (Englyst and Cummings,1984), by gaschromatography on a Trace GC Ultra coupled with ITQ 900 (Thermo Scientific, USA) equipped with a Restek RT- 2330-NB column (0.32 mm × 105 m), the temperature program of 80 ºC (12 min)–160 ◦C (8 ºC /min)–250 ºC (4 ºC /min, 25 min at 250 ºC)–265 ◦C (20 ºC /min, 10 min at 265 ºC) and the flow rate of helium was 1 mL/min. Molecular mass determination of a sample was performed with HPLC Shimadzu apparatus (Vienna, Austria) equipped with a differential refractometer RID-6A and a UV– vis detector SPD-10AV using the column HEMA-BIO 1000 (8 mm×250 mm) of particle size 10 m (Tessek, Prague, Czech Republic). As a mobile phase 0.02 M phosphate buffer pH 7.2 containing 0.1 M NaCl was used at a flow rate 0.8 mL/min. A set of dextran standards was used for of the column (Gearing Scientific, Polymer Lab., Hertfordshire, UK). The colorimetric assays were measured using UVVIS 1800 spectrophotometer (Shimadzu, Japan). Fourier-transform infrared (FT-IR) were obtained on a NICOLET Magna 750 spectrometer with DTGS detector and OMNIC 3.2 software, where 128 scans were recorded with 4 cm−1 resolution. NMR spectra of conjugate were recorded in D2O at 60 ºC on Varian 400 NMR spectrometer on direct 5 mm PFG AutoX probe. Sample was
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twice freeze-dried from D2O before measurements. For 1H and 13C NMR spectra, chemical shifts were referenced to internal standard – acetone ( 2.22 and 31.07, respectively). For the assignment of signals one-dimensional (1H NMR) and twodimensional Heteronuclear Single Quantum Correlation experiment (HSQC) were used.
2.3. Animals
All experiments were approved by Institutional Ethics Committee of the Jessenius Faculty of Medicine, Comenius University in Martin, Slovakia, registered in Institutional Review Board/Institutional Ethic Board Office (IRB 00005636), complied with Slovakian and European Community regulations for the use of laboratory animals and follow the criteria of experimental animal’s well fare (decision No. 1249/2013). Adult male Trik strain guinea pigs (200-350 g) were obtained from approved breeding facility situated in Department of Experimental Pharmacology, Slovak Academy of Sciences (Dobra Voda, Slovakia) and were housed in approved animal holding facility for one-week adapting period and subsequent several days adaptation to experimental conditions. The animals were divided into the five groups, each consisting of 10 animals: Negative controls – (1) Healthy group and (2) Group of ovalbumin-sensitized animals (OVA+) received saline (sodium chloride 0.9 %) per orally (p.o.); (3) Positive controls – sensitized guinea pigs received salbutamol (10 mg/kg b.w.) intraperitoneally (i.p.) once daily long-term (Sal LT) or (4) received budesonide (3 mg/mL) by inhalation for 5 min to 14 days, respectively (Bud LT); (5) Experimental group – sensitized animals underwent long-term therapy with Echinacea complex (50 mg/kg b.w.) per orally (p.o.), long-term (Ep LT). The doses of control drugs and tested complex from medicinal plant E. purpurea were selected according to literature data
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and results of our previous experiments (Whelan et al., 1993; Prisenžňáková et al., 2005; Šutovská et al., 2014) and following the manufacturer’s instruction.
2.4. Antigen-induced airway hyperresponsiveness
Sensitization of animals by repetitive doses of antigen ovalbumin, which causes airway reactivity changes on immunological basis, was performed during 21 days (Franova et al. 2013). Briefly, Al (OH)3 adsorbed ovalbumin was administered intraperitoneally and subcutaneously (1st day of sensitization) and intraperitoneally (3rd day). Further, 1-2 min lasted inhalations of allergen was performed at 9th, 12th, 15th, 18th and 20th days using bodyplethysmograph for small laboratory animals (HSE type 855, Hugo Sachs Elektronik, Germany). Allergic inflammation was confirmed as skin reaction increase in exhaled nitric oxide (eNO) and in basal specific airway resistance (sRaw) values. Twenty four hours after the last administration of allergen animals have been treated by saline, control drugs and Ep isolate for a period of 14 days. Budesonide and salbutamol were used as the anti-inflammatory and bronchodilatory controls, respectively. After these treatments, animals have been used for evaluation of the Echinacea effect on defence reflexes of airways and its ability to suppress allergic inflammation process by different in vivo and in vitro methods.
2.5. The evaluation of airway smooth muscle reactivity in vivo
In vivo airway smooth muscle (ASM) reactivity was evaluated using a bodyplethysmograph consisting of head and body chambers. The values of sRaw calculated by Pennock et al. (1979) and their changes were regarded as indicator of in vivo
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airways reactivity. The sRaw is proportional to phase difference between nasal and thoracic respiratory airflow. The changes in sRaw were measured under the basal conditions and then during 1 min consecutively after the short (30 s) exposure to contractile mediators: citric acid (0.3 M), histamine and methacholine (both 10-6 M). Between the bronchoprovoking agent exposure and measurement of sRaw was an interval 1 min, during which fresh air was insufflate into the nasal chamber. The effect of Echinacea was compared to salbutamol and saline.
2.5.1. The evaluation of airway smooth muscle reactivity in vitro
The changes of the ASM reactivity on cumulative doses of contractive mediators (acetylcholine and histamine) were tested by organ tissue bath method (Sutovska et al. 2012). Briefly, guinea pigs were killed by transversal interruption of neck spinal cord. Consequently the respiratory organs were removed. Four strips (two of tracheal and two of pulmonary smooth muscle) obtained from each animal were placed into organ bath chambers filled with Krebs-Henseleit´s buffer saturated by pneumoxide (95 % O2 + 5 % CO2), held at the temperature 36 ± 0.5 ºC, maintained at pH 7.5 ± 0.1. Single strips were fixed onto the sliding arm and the other end was bound with a thin thread to a hook of a transducer (Experimetria Ltd., Hungary). The tension was used to monitor the intensity of contractile responses. The amplitude of isometric contraction (mN) of tracheal and pulmonary smooth muscle to cumulative doses of contractile mediator acetylcholine and histamine at concentrations 10-8–10-3 M was used for evaluation of ASM reactivity.
2.6. Measurement of eNO in vivo
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The changes in exhaled nitrogen oxide (eNO) values were used as rough indicator of airway inflammation and anti-inflammatory effect (Sutovska et al. 2013). Animals were placed into the offline chamber sampling connected with NIOX Flex Offline Start Kit 041210-F (Aerocrine AB, Sweden), breathed NO free air for 5 min. Subsequently the exhaled gas (flow rate 5 mL/s) was analysed during 7 s. NIOX uses the high sensitivity and high specificity of chemiluminescence gas analyser, together with integrated software, to accurately measure NO molecules at very low concentrations (particle per billion, ppb).
2.7. The evaluation of ciliary beat frequency (CBF) in vitro
The experiments were carried out under the standard laboratory conditions (temperature 21–24 °C and humidity at 55 ± 10 %). The temperature of the microscopic glass slide and the saline used as a nutritive medium for cilia, were kept in the range of 37–38 °C. Following transversal interruption of the male guinea pigs neck spinal cord, transverse access to the trachea was made approximately in the middle of its normal length. Ciliated samples were obtained by cytology brush, which was dipped into the saline and then gently rotated on the mucosal surface of the animal trachea. Tracheal brushings were immediately placed into saline solution and were processed to microscopic preparation. Microscopic preparations were examined 3 minutes after brushing the tracheal cilia using phase contrast inverted biological microscope (Kvant model IM1C, Slovakia). Beating ciliated cells were recorded using a digital high speed video camera (Basler A504kc; Basler AG, Germany) on frame rate from 256 to 512 fps (frames per second). There were approximately 10-12 video records of the same microscopic preparation performed at 1 minute intervals and duration of each record was approximately 5-10
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seconds. Video records were analysed using LabVIEW™ Software to generate a ciliary region of interest (ROI), intensity variation in selected ROI and intensity variance curve. Curve was then analysed according to Hargas et al. (2011) method using fast Fourier transform algorithm (FFT). Fourier spectrum of each intensity variance curve was then equal to frequency spectrum of cilia beating in each ROI. The median of frequency (Hz) for each ROI and their arithmetic means referred to definite CBF value for each microscopic preparation.
2.8. The assessment of cytokine level in vitro
The blood from guinea pigs heart was collected immediately after transversal spinal cord interruption. To measure inflammatory mediators in the airways, bronchoalveolar lavage was performed with warm saline (37 °C) in volume calculated according body weight of animal (10 mL/kg). Saline was injected and withdrawal via the cannula placed into the right bronchus. Serum and supernatant from biological fluids were obtained by centrifugation – blood at the centrifugal force 2054 g for 5 min and bronchoalveolar lavage fluid (BALF) at 377 g for 2 min. The apparatus Bio-Plex® 200 System and TH1/TH2 panel Human Cytokine (Bio-Rad, USA) were used for cytokine levels assessment. The assay was designed on magnetic beads according to a capture sandwich immunoassay format. The capture antibody– coupled beads were first incubated with antigen standards, samples, or controls followed by incubation with biotinylated detection antibodies. After washing away the unbound biotinylated antibodies, the beads were incubated with a reporter streptavidinphycoerythrin (S-P) conjugate. Following the removal of S-P excess, the beads were passed through the Bio-Plex 200 suspension array reader equipped with two lasers, one
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(532 nm excitation) for analytes quantification and second (635 nm excitation) for cytokine identification in small volume samples, which measures the fluorescence of the beads and of the bound S-P. All washes were performed using a Bio-Plex Pro wash station. High-speed digital processor manages data output and Bio-Plex Manager™ 6.0 software presents results as concentration in pg/mL.
2.9. Statistics
All obtained data were evaluated with one-way analysis of variance (ANOVA) with the post hoc Bonferroni test using GraphPad Prism 6 software. Data are presented as mean ± standard error of the mean (SEM). The results with p<0.05 was considered as statistically significant. Significances of p<0.05, p<0.01 and p<0.001 are shown by one, two and three symbols (● vs unsensitized healthy animals, * vs negative control OVA+, or + vs positive control drugs).
3. Results and discussion
3.1. Chemical and spectroscopic characteristics of Echinacea complex
The hot alkaline extraction of E. purpurea flowers followed by neutralization and multi-step extractions with organic solvents, dialysis and freeze-drying afforded a dark brown Echinacea complex in 1.8% yield on dry plant. HPLC and GPC analyses showed one single peak of molecule mass at around 10 kDa indicating thus its molecular homogeneity (Pawlaczyk et al. 2009). Compositional analyses of Echinacea complex revealed carbohydrates (26.3%), phenolics (17.5% or 1.03 mM of GAE/1 g), protein (14%) and uronic acids (11.2 %). Analysis of the carbohydrate part showed the presence
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of uronic acids (30%), galactose (Gal, 22%), arabinose (Ara, 17%) and rhamnose residues (Rha, 13%), while other sugars were found in lower contents, i.e. xylose (Xyl, 9%), glucose (Glc, 6%), mannose (Man, 2%) and fucose (Fuc, 1%). The FTIR analysis confirmed the presence of three structural components in Echinacea conjugate, i.e. carbohydrates and phenolics as the dominant constituents, while proteins were found in a small amount (Pawlaczyk et al. 2009). In the 1H NMR spectrum of Echinacea complex (Fig. 1), three regions of signals could be distinguished. Signals in the region at δ 9–6 reveal the presence of aromatic rings in phenolic compounds; carbohydrate signals were present mainly in the region at δ 5.5–3, while those due CH2, CH3 signals of deoxy sugars, proteins, phenolics and acetyl groups were located in the region at δ 3–0.6. Broad signals reflect high molecular mass of the conjugate. Dominant sugar components, particularly galacturonic acid (GalA), Gal, Ara and Rha, have been identified by sugar analysis. Consequently, they afforded dominant signals in NMR spectra. Characteristic 1H/13C chemical shifts of signals found in HSQC spectrum (Fig. 2) were used for identification of sugars, their linkage types as well as types of carbohydrate polymers present in Echinacea complex. In the α-anomeric region the most important signals in form of singlets were due to Araf at δ 5.21/110.25 and 5.04/108.62. All other signals were of low intensity and very broad. The most intensive overlapped β-anomeric H1/C1 cross peaks at characteristic chemical shifts at δ 4.48/104.34 and 4.44/104.10 have been identified as 1,3,6-β-Gal and 1,6-linked βGal. The presence of 6-linked βGal units was evident from H6/C6 cross peaks with characteristic chemical shifts at δ 4.01, 3.88/70.61. These data are in a very good agreement with data published for arabinogalactan type II isolated from C. arabica (Capek et al. 2010) with a highly branched 1,3-β-galactan backbone with short 1,6-linked βGal side chains, which were also
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branched at O3 by βGal and αAraf. The 1,3-linked βGal signals were not found. Characteristic chemical shifts of Araf units confirmed two types of linkages: one as βGal O3-linked (δ 5.21/110.25) and the other as Araf, branched at O5 (δ 5.04/108.62). A downfield shift of αAraf C5 signal due to 1,5-linkage was confirmed by H5/C5 cross peaks at δ 3.885, 3.795/67.84. These facts indicate that one component in the Echinacea complex is the highly branched arabinogalactan. A doublet H6 signal at δ 1.214 (6Hz, d) with C6 chemical shift δ 17.56 gave a clear evidence about Rhap presence. It can form a branching element in arabinogalactans (Nunes et al. 2008), but due to a high content of GalA in EP rhamnose should be also a component of rhamnogalacturonan which is usually a homopolymer formed by 1,4-linked αGalA. It was found that HSQC contains also signals of two O-methyl groups at H/C δ 3.45/58.60 and 3.45/60.78. For their location identification the comparison of cross peaks chemical shifts in the HSQC spectrum of Echinacea complex with published NMR data of not substituted, acetylated and O-methylated 1,4-linked galacturonans was used for analysis. Chemical shift of OMe group at C6 in GalA ester was identified at δ 3.80/54.1 and H5/C5 at δ 5.13/72.0 (Popov et al. 2011). Based on OMe chemical shifts and missing signal at H5/C5 at δ 5.13/72.0 a conclusion was made that GalA in Echinacea complex is not C6 esterified by OMe. Further analysis has shown that OMe H/C chemical shifts values are in agreement with data published for substituted 1,6-linked β-galactan at C3 by OMe isolated from Salvia officinalis L. (Capek, 2008). Thus the location of OMe on βGal units is highly probable. The presence of acetyl groups in NMR spectra was reveal by CH3 signals of OAc cross peaks at H/C 2.020/23.527, 2.060/21.28 and 2.158/21.28 and downfield shifted signals due to acetylation (broad signals at δ 4.675/72.87 and 4.60/72.32). Cross peak at δ 3.933/43.40 was characteristic chemical shifts of CH2-N group. Together with cross peaks due to CH2 at δ 2.009/28.49, 2.226/34.28 and CH3
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groups at δ 0.857/15.84, 0.867/18.69 and 0.846/22.78 they are the most important signals due to proteic part of the complex. All other signals are too broad and of low intensity to be detected at given experimental conditions in HSQC spectrum. NMR data suggest the presence of complex mixture of arabinogalactan type II and rhamnogalacturonans polymers, O-methylated and acetylated, as the main carbohydrate components. Chemical shifts of Araf units do not indicate the presence of arabinans, but galactans can’t be excluded. Polyphenolics show large signals in 1H NMR spectrum which not allow their better identification.
3.2. The influence of Echinacea and control drugs on defence reflexes of the airways
In this study the attention was focused on the pharmacodynamic profile of E. purpurea complex using animal model of allergic airway inflammation induced by exposure of guinea pigs to ovalbumin (OVA+). Repetitive exposure of animals to allergen leads to complex of changes that almost mimic the asthma phenotype in human, e.g. airway hyperreactivity (AHR) and allergic inflammation of the airways characterized by typical histological features and changes in cytokines and mediators production (Franova et al. 2013, Sutovska et al. 2015). Results of in vivo measurements showed the changes in basal specific airway resistance values and sRaw induced by citric acid (AC), histamine and methacholine (Fig. 3) used as mediators, each producing bronchoconstriction through different pathway involved in AHR. The significant increase in basal hyperreactivity and in the response on contracting mediators is the common feature in asthma and it was confirmed in sensitized, saline-treated animals (OVA+). Echinacea complex significantly decreased bronchoconstriction induced by AC, histamine or methacholine (AC and histamine,
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p<0.001; methacholine, p<0.01). Furthermore, Echinacea complex significantly reduced basal hyperreactivity (p<0.001) and its effect was similar to that of salbutamol, a standard bronchodilatory antiasthmatic drug. The measurement of sRaw is widely used by paediatric pulmonologists and important therapeutic decisions are based on this approach. According to Mahut et al. (2009) sRaw is very sensitive parameter appropriate to detect mild levels airway obstruction. The long-term treatment of sensitized animals by Echinacea complex significantly decreased sRaw induced by bronchoconstrictors used as well as basal sRaw. These values characterized basal hyperreactivity. These findings strongly support possible effectiveness of Echinacea complex in allergic asthma. In vitro studies were based on a contraction change of tracheal and pulmonary smooth muscles after treatment with acetylcholine and histamine, used as exogenous contractile agents in cumulative concentrations. In the Figs. 4 and 5 the highest amplitudes are observed in sensitized (OVA+) tissues, while the lowest ones are in tissues of animals’ long term treated by Echinacea complex. Moreover, amplitudes are even significantly lower than those due to salbutamol treated animals, especially at low concentration of histamine (10-6 M) added directly into the pulmonary muscle strips or at high concentration of histamine (10-4 and 10-3 M) in the case of tracheal smooth muscle. The inhibition of airway smooth muscles (ASM) reactivity by Echinacea complex evaluated in vitro was consistent with in vivo findings. Furthermore, in comparison to salbutamol effect, the long-term administration of Echinacea complex results in a greater reduction in histamine-induced contraction of pulmonary smooth muscle tissue. It is generally accepted, that asthma is a disease of small diameter intraparenchymal bronchi and thus a good response of the lung tissue to Echinacea complex be regarded as a very positive outcome.
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The evaluation of a ciliary beat frequency (CBF) of healthy and treated animals is shown in Fig. 6. Results showed that allergic inflammation induced by repetitive exposure to ovalbumin is associated with significant enhancement of CBF (p<0.05). Furthermore, long-term treatment of animals with the Echinacea complex did not change the frequency of cilia beating, that is, Echinacea complex has no effect on the natural cilia clearance. The control drug budesonide, tested under the same condition, changed CBF only insignificantly. The cilia of respiratory epithelium are a relatively vulnerable structures targeted by various stimuli, e.g. serotonin or TNF-α caused the increase in the frequency of cilia beating (Weiterer et al. 2014) and IL-13 inhibits not only CBF but also alter the morphology of the ciliated cells (Laoukili et al. 2001) . CBF is a key parameter of mucociliary clearance and ciliary transport efficiency is linearly dependent on the CBF (Braiman and Priel, 2001). Our study showed that both, budesonide and Echinacea do not affect the effectiveness of mucociliary clearance.
3.3. The influence of Echinacea and control drugs on allergic inflammation of airways
Two ways, i.e. indirect and direct were used to evaluate the influence of Echinacea on allergic inflammation of airways. The indirect method of exhaled nitrogen oxide (eNO) measurement is based on the fact, that the level of eNO is higher in animals with a developed inflammation of airways. As can be seen from the Fig. 7, the levels of eNO measured in animals’ long term treated by Echinacea complex was significantly lower than that of sensitized (diseased) animals. It was found that levels of eNO in animals treated by Echinacea complex were comparable with those of animals treated by classic anti-inflammatory drug budesonide.
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Indicative results of Echinacea complex on allergic inflammation of airways were confirmed by measurement of cytokine levels in BALF and plasma. TH1/TH2 panel Human Cytokine allows us to evaluate changes in cytokines that are responsible for key features of allergic asthma: IL-4, IL-5, IL-13 and TNF α. Results showed that the treatment of ovalbumine sensitized animals (OVA+) by Echinacea complex decreased cytokine levels in both, BALF and plasma (Fig. 8). Furthermore, the induced decrease by Echinacea complex was more significant than the budesonide, with the exception of plasma levels of IL-5 and IL-13. The reduction in the BALF cytokines appears to be the most important results with regard to allergic inflammation, as this shows that Echinacea complex is able to very effectively reduce the local, epithelium-mediated pulmonary inflammation. Exhaled NO levels were measured to estimate the degree of inflammation in response to repeated exposure to ovalbumin, as well as anti-inflammatory action of Echinacea complex. As reported earlier, significantly increased eNO was regularly found in the developed allergic inflammation of the airways and eNO is normally reduced by corticosteroids administration (Kocmalova et al., 2015; Sutovska et al., 2013). The presented study confirmed increase in eNO of ovalbumin – sensitized untreated animals and showed a significant reduction in the levels of eNO in animals sensitized with ovalbumin and treated long-term with Echinacea complex. Several studies have shown an enhanced in exhaled NO levels due to upregulated inducible NO-synthase (iNOS) in human subjects suffering from untreated asthma and have found correlations between eNO and airway eosinophilia (Redington, 2006). Eosinophils, T cells and mast cells have been shown not only as a source of iNOS, but also the mediators and cytokines, e.g., IL-4, IL-5, IL-13 and TNF-α by stimulated increased of iNOS expression in airway epithelial
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cells (Benson et al., 2011; Paoliello-Paschoalato et al., 2005) and played a key role in asthma (Feske et al., 2012). The significant anti-inflammatory activity of Echinacea complex, determined with eNO was supported also by the results of the Bio-Plex® assay, which showed a significant decrease in levels of key cytokines mediating asthma in plasma as well as in BALF. The similar changes in cytokine production were reported by Altamirano-Dimas et al. (2009) and Sharma et al. (2009). Authors studied profile of produced cytokines and chemokines and changes induced by Echinacea preparations on human bronchial rhinovirustransfected cell lines. Reduced BALF levels of IL-4, IL-5, IL-13 and TNF-α evidenced the Echinacea complex suppressive effect on secretory functions of respiratory epithelium, which represents the most important tissue-organizing inflammatory response in asthma (Holgate, 2011).
4. Conclusion
It can be concluded that long-term administration of Echinacea complex, composed of carbohydrate, phenolic and protein components, showed significant bronchodilator and anti-inflammatory effects and maintains unchanged frequency of cilia beating in guinea pigs with experimentally induced allergic airway inflammation. Using animal model of allergic asthma, Echinacea showed potency to suppress both, airway hyperreactivity and airway inflammation. Bronchodilation was similar to the effect of salbutamol, a classic antiasthmatic bronchodilator and suppression of allergic inflammation was comparable with that of budesonide, an anti-inflammatory corticosteroid agent. Preliminary studies confirmed that Echinacea complex is beneficial to patients suffering from allergic
19
disorders of the airways, such as asthma and thus provide a scientific basis for the application of this herb in traditional medicine.
Acknowledgements
Studies were supported by the project BioMed co-financed from EC sources and by the grants VEGA No. 1/0165/14 and 2/0018/15, MZ No. 2012/35-UKMA-12 and APVV project No. 0305-12.
References Altamirano-Dimas, M., Sharma, M., Hudson, JB., 2009. Echinacea and anti-inflammatory cytokine responses: Results of a gene and protein array analysis. Pharm. Biol. 47, 500– 508. Barrett, B., 2003. Medicinal properties of Echinacea: A critical review. Phytomed. 10 (1), 66– 86. Benson, R.C., Hardy, K.A., Morris, C.R., 2011. Arginase and arginine dysregulation in asthma. J. Allergy (Cairo), 736319. doi: 10.1155/2011/736319. Blumenkrantz, N., Asboe-Hansen, O., 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54, 484–489. Braiman, A., Priel, Z., 2001. Intracellular stores maintain stable cytosolic Ca2+ gradients in epithelial cells by active Ca2+ redistribution. Cell Calcium, 30, 361–371. Capek, P., Matulova, M., Navarini, L., Suggi Liverani, F., 2010. Structural features of an arabinogalactan-protein isolated from instant coffee powder of Coffea arabica beans. 20
Carbohydr. Polym. 80, 180–185. Capek, P., 2008. An arabinogalactan containing 3-O-methyl-D-galactose residues isolated from the aerial parts of Salvia officinalis L. Carbohydr. Res. 343, 1390–1393. Currier, N.L., Lejtenyi, D., Miller, S.C., 2002. The effect with time of administration in vivo, of the polysaccharide, arabinogalactan, on the immune and hemopoietic cell lineages in murine spleen and bone marrow. Phytomed. 10, 145–153. Currier, N.L., Miller, S.C., 2000. Natural killer cells from aging mice treated with extracts from Echinacea purpurea are quantitatively and functionally rejuvenated. Exp. Gerontol. 35, 627–639. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Biochem. 28 (3), 350– 355. Englyst, H.N., Cummings, J.H., 1984. Simplified method for the measurement of total nonstarch polysaccharides by gas-liquid chromatography of constituent sugars as alditol acetates. Analyst, 109, 937-942. Feske, S., Skolnik, E.Y., Prakriya, M., 2012. Ion channels and transporters in lymphocyte function and imunity. Nat. Rev. Immunol. 12, 532-547. Fonseca, F.N., Papanicolaou, G., Lin, H., Lau, C.B.S., Kennelly, E.J., Cassileth, B.R., Cunningham-Rundles, S., 2014. Echinacea purpurea (L.) Moench modulates human Tcell cytokine response. Int. Immunopharm. 19, 94–102. Franova, S., Joskova, M., Sadlonova, V., Pavelcikova, D., Mesarosova, L., Novakova, E.,
21
Sutovska, M., 2013. Experimental model of allergic asthma. Ad. Exp. Med. Biol. 756, 4955. Goel, V., Lovlin, R., Barton, R., Lyon, M.R., Bauer, R., Lee, T.D.,Basu, T.K., 2004. Efficacy of a standardized echinacea preparation (Echinilin) for the treatment of the common cold: a randomized, double-blind, placebo-controlled trial. J. Clin. Pharm. Ther. 29, 75–83. Hargaš, L., Koniar, D., Štofan, S., 2011. Sophisticated Biomedical Tissue Measurement Using Image Analysis and Virtual Instrumentation, in LabVIEW – Practical Applications and Solutions, in: Folea, S. (Editor), InTECH, pp. 155-180. Holgate, S.T., 2011. The sentinel role of the airway epithelium in asthma pathogenesis. Immunol. Rev. 242, 205-219. Kocmalova, M., Oravec, M., Adamkov, M., Sadlonova, V., Kazimierova, I., Medvedova, I., Joskova, M., Franova, S., Sutovska, M., 2015. Potassium ion channels and allergic asthma. Adv. Exp. Med. Biol. 838, 35-45. Laoukili, J., Perret, E., Willems, T., Minty, A., Parthoens, E., Houcine, O., Coste, A., Jorissen, M., Marano, F., Caput, D., Tournier, F., 2001. IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells. J. Clin. Invest. 108, 1817–1824. Lowry, O., Rosebrough, N., Farr, L., Randall, R., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Mahut, B., Trinquart, L., Bokov, P., Le Bourgeois, M., Waernessyckle, S., Peiffer, C., Delclaux, C., 2009. Relationships between Specific Airway Resistance and Forced 22
Expiratory Flows in Asthmatic Children. Plos One, 4 (4), e5270. Doi: 10.1371/journal.pone.0005270. Mali, R.G., Dhake, A.S., 2011. A review on herbal antiasthmatics. Orient. Pharm. Exp. Med. 11, 77-90. Nakagome, K., Nagata, M., 2011. Pathogenesis of airway inflammation in bronchial asthma. Auris Naxus Larynx, 38, 555-563. Nunes, F.M., Reis, F.M., Silva, V.A., Domingues, A.M.S., Coimbra, M.A., 2008. Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee arabinogalactans. Phytochem. 69, 1573–1585. Paoliello-Paschoalato, A.B., Oliveira, S.H., Cunha, F.Q., 2005. Interleukin 4 induces the expression of inducible nitric oxide synthase in eosinophils. Cytokine, 30, 116-124. Pawlaczyk, I., Czerchawski, L., Pilecki, W., Lamer-Zarawska, E., Gancarz, R., 2009. Polyphenolic-polysaccharide compounds from selected medicinal plants of Asteraceae and Rosaceae families: chemical characterization and blood anticoagulant activity. Carbohydr. Polym. 77, 568–575. Pennock, B.E., Cox, C.P., Rogers, R.M., Cain, W.A.,Wells, J.H., 1979. A non-invasive technique for measurement of changes in specific airway resistance. J. Appl. Physiol. 46, 399-406. Percival, S.S., 2000. Use of echinacea in medicine. Biochem. Pharm. 60, 155-158. Popov, S.V., Ovodova, R.G., Golovchenko, V.V., Popova, G.Y., Viatyasev, F.V., Shashkov, A.S., Ovodov,Y.S., 2011. Chemical composition and anti-inflammatory activity of a pectic polysaccharide isolated from sweet pepper using a simulated gastric medium. Food Chem. 124, 309–315. 23
Prisenžňáková, L., Nosáľová, G., Hromádková, Z., Ebringerová, A., 2011. The pharmacological activity of wheat bran polysaccharides. Fitoterapia. 81, 1037-1044. Redington, AE., 2006. Modulation of nitric oxide pathways: therapeutic potential in asthma and chronic obstructive pulmonary disease. Eur. J. Pharm. 533, 263-276. Roesler, J., Steinmuller, C., Kiderlen, A., Emmendorffer, A., Wagner, H., LohmannMatthes, M.L., 1991. Application of purified polysaccharides from cell cultures of the plant Echinacea purpurea to mice mediates protection against systemic infections with Listeria monocytogenes and Candida albicans. Int. J. Immunopharmacol. 13, 27–37. Sharma, M., Schoop, R., Hudson, J.B., 2009. Echinacea as an antiinflammatory agent: the influence of physiologically relevant parameters. Phytother. Res. 23, 863- 867. Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M., 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods Enzymol. 299, 152–178. Spelman, K., Wetschler, M.H., Cech, N.B., 2009. Comparison of alkylamide yield in ethanolic extracts prepared from fresh versus dry Echinacea purpurea utilizing HPLCESI-MS. J. Pharm. Biomed. Anal. 49, 1141–1149. Steinmuller, C., Roesler, J., Grottrup, E., Franke, G., Wagner, H., Lohmann-Matthes, M.L., 1993. Polysaccharides isolated from plant cell cultures of Echinacea purpurea enhance the resistance of immunosuppressed mice against systemic infections with Candida albicans and Listeria monocytogenes. Int. J. Immunopharmacol. 15, 605–14. Sutovska, M., Kocmalova, M., Joskova, M., Adamkov, M., Franova, S., 2015. The Effect of Long-term Administered CRAC Channels Blocker on the Functions of Respiratory 24
Epithelium in Guinea Pig Allergic Asthma Model. Gen. Physiol. Biophys. 34(2), 167-76. Sutovska, M., Kocmalova, M., Adamkov, M., Vybohova, D., Mikolka, P., Mokra, D., Hatok, J., Antosova, M., Franova, S. (2013). The long-term administration of Orai1 antagonist possesses antitussive, bronchodilatory and anti-inflammatory effects in experimental asthma model. Gen. Physiol. Biophys. 32, 251-259. Šutovská, M., Capek, P., Fraňová, S., Pawlaczyk, I., Gancarz, R., 2012. Antitussive and bronchodilatory effects of Lythrum salicaria polysaccharide-polyphenolic conjugate. Int. J. Biol. Macromol. 51, 794-799. Šutovská, M., Capek, P., Kočmalová, M., Pawlaczyk, I., Zaczyńska, E., Czarny, A., Uhliariková, I., Gancarz, R., Fraňová, S., 2014. Characterization and pharmacodynamic properties of Arnica montana complex. Int. J. Biol. Macromol. 69, 214-221. Wagner, H., Kraus, S., Jurcic, K., 1999. Search for immunostimulating agents fromplants and other natural sources, in: Wagner, H. (Ed.), Immunomodulatory Agents from Plants. Birkhäuser, Verlag, Basel-Boston-Berlin, pp. 1–40. Weiterer, S., Schulte, D., Müller, S., Kohlen, T., Uhle, F., Weigand, M.A., Henrich, M. 2014. Tumor Necrosis Factor Alpha Induces a Serotonin Dependent Early Increase in Ciliary Beat Frequency and Epithelial Transport Velocity in Murine Tracheae. Plos One, 9, e91705. doi: 10.1371/journal.pone.009170. Whelan, C.J., Johnson, M., Vardey, C.J., 1993. Comparison of the anti-inflammatory properties of formoterol, salbutamol and salmeterol in guinea-pig skin and lung. Br. J. Pharmacol. 110, 613-618. 25
Figure legends
Fig. 1. 1H NMR spectrum of Echinacea complex.
Fig. 2. HSQC spectrum of Echinacea complex.
Fig. 3. The changes in specific airways resistance (sRaw) under the basal conditions and changes in this parameter induced by short-time inhalation of citric acid (AC), histamine (His) and methacholine (Met) measured in unsensitized (healthy) and sensitized vehicletreated animals (OVA+), and in groups of sensitized guinea pigs received long-term positive control drug salbutamol (Sal LT) or E. purpurea complex (Ep LT). ●p < 0.05 and
●●●
p<
0.001 vs healthy; ** p < 0.01 and *** p < 0.001 vs OVA+.
Fig. 4. The contractile response of tracheal smooth muscle (mN) on cumulative concentrations of acetylcholine and histamine (10-8–10-3 M) added to isolated strips to bath chamber. The tracheal reactivity of animals treated long-term by E. purpurea complex (Ep LT) was compared to contractile response of control sensitized groups treated by vehicle (OVA+) and salbutamol (Sal LT). *p < 0.05 and **p < 0.01 vs OVA+; +p < 0.05 vs salbutamol.
Fig. 5. The changes in pulmonary smooth muscle (mN) contractile response on cumulative concentrations of acetylcholine and histamine (10-8–10-3 M) registered in animals treated long-term by Echinacea complex (Ep), vehicle (OVA+) and salbutamol (Sal). *p < 0.05; **p < 0.01 and ***p < 0.001 vs OVA+; +p < 0.05 vs salbutamol.
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Fig. 6. The changes in ciliary beat frequency (CBF) evaluated in unsensitized (healthy) animals and following sensitized groups: Echinacea complex (Ep LT), negative control (OVA+) and positive budenoside control (Bud LT) as the response on long-term administration of Echinacea complex, vehicle and budesonide. ●p < 0.05 vs healthy animals.
Fig. 7. The variations in values of exhaled NO (eNO) measured in unsensitized (healthy) guinea pigs, sensitized negative control group treated by vehicle (OVA+), experimental group of animals treated long-term by Echinacea complex (Ep LT) and positive control group received long-term budesonide (Bud LT). ●●p < 0.01 vs healthy; *p < 0.01 vs OVA+.
Fig. 8. The comparison of changes in levels of cytokines IL-4, IL-5, IL-13 and TNF-α measured in BALF (part A) and plasma (part B) obtained from unsensitised (healthy) guinea pigs (healthy), ovalbumin-sensitized negative control (OVA+), animals treated long-term by Echinacea complex (Ep LT) and budesonide (Bud LT).●p<0.05, ●●p<0.01 and ●●●p<0.001 vs healthy animals; *p<0.05, **p<0.01 and ***p<0.001 vs OVA+; ++p<0.01 and +++p<0.001 vs budesonide.
27
Abbreviations
Ep, Echinacea complex; sRaw, specific airway resistance; CBF, ciliary beat frequency; NO, nitrogen oxide; BALF, bronchoalveolar lavage fluid; Th2 cytokine, T helper cell cytokine; IL-1-, IL-4, IL-5, IL-6, IL-10, IL-13, interleukins 1, 4, 5, 6, 10, 13; TNF-, tumor necrosis factor alpha; NMR, nuclear magnetic resonaces; AC, Citric acid; HCl, hydrochloric acid; TFA, trifluoroacetic acid; HPLC, high-performance liquid chromatography; UV-vis, ultraviolet–visible; NaCl, sodium chloride; DTGS detector, deuterated-triglycine sulfate detector; D2O, deuterium oxide; HSQC, heteronuclear single quantum correlation experiment; OVA+, ovalbumin-sensitized animals; i.p., intraperitoneally; Sal LT, salbutamol long term; Bud LT, budenoside long term; Ep LT, Echinacea complex long term; Al(OH)3, aluminium hydroxide; eNO, exhaled nitric oxide; ASM, airway smooth muscle; ppb, particle per billion; ROI, region of interest; FFT, Fourier transform algorithm; GalA, galacturonic acid; OMe, O-methyl; βGal, β-linked galactose; Araf, arabinofuranose; AHR, airway hyperreactivity; iNOS, inducible NO-synthase; Gal, galactose; Ara, arabinose; Rha, rhamnose; Xyl, xylose; Glc, glucose; Man, mannose; Fuc, fucose; GalA, galacturonic acid;
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Influence on airway defense mechanisms Ovalbumin sensitized
Echinacea flowers PPP complex 0.1 M NaOH
PPP: polysaccharide-polyphenolic-protein
in vivo/in vitro tests
Influence on allergic inflammation
Graphical abstract
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Fig. 1. Sutovska et al., J Ethnopharm
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Fig. 2. Sutovska et al., J Ethnopharm
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Fig. 8.Sutovska et al., J Ethnopharm
PII: DOI: Reference:
S0378-8741(15)30132-X http://dx.doi.org/10.1016/j.jep.2015.09.007 JEP9730
To appear in: Journal of Ethnopharmacology Received date: 19 May 2015 Revised date: 4 September 2015 Accepted date: 6 September 2015 Cite this article as: Martina Šutovská, Peter Capek, Ivana Kazimierová, Lenka Pappová, Marta Jošková, Mária Matulová, Soňa Fraňová, Izabela Pawlaczyk and Roman Gancarz, Echinacea complex – chemical view and anti-asthmatic profile, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.09.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Echinacea complex – chemical view and anti-asthmatic profile
Martina Šutovskáa, Peter Capekb*, Ivana Kazimierováa, Lenka Pappováa, Marta Joškováa, Mária Matulováb , Soňa Fraňováa, Izabela Pawlaczykc, Roman Gancarzc
a
Department of Pharmacology, Jessenius Faculty of Medicine Comenius University,
Martin's Biomedical Center (BioMed) Malá Hora 11161 4C, Martin, Slovakia b
Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská
cesta 9, Bratislava, Slovakia c
Division of Organic and Pharmaceutical Technology, University of Technology, Wrocław,
Poland
ABSTRACT
Ethnopharmacological relevance Echinacea purpurea (L.) Moench is one of the mostly used herbs in the traditional medicine for the treatment of respiratory diseases. Modern interest in Echinacea is directed to its immunomodulatory activity. Recent studies have shown that secretion of asthmarelated cytokines in the bronchial epithelial cells can be reversed by Echinacea preparations. Aim of the study To examine the pharmacodynamics profile of Echinacea active principles, a complex has been isolated from its flowers by alkaline extraction and has been tested using an animal model of allergic asthma. Material and methods
1
The structural features of Echinacea purpurea complex was determined using chemical and spectroscopic methods. Allergic inflammation of the airways was induced by repetitive exposure of guinea pigs to ovalbumin. Echinacea complex was then administered 14 days in 50 mg/kg b.w. daily dose perorally. Bronchodilatory effect was verified as decrease in the specific airway resistance (sRaw) in vivo and by reduced contraction amplitude (mN) of tracheal and pulmonary smooth muscle to cumulative concentrations of acetylcholine and histamine in vitro. The impact on mucociliary clearance evaluated measurement of ciliary beat frequency (CBF) in vitro using LabVIEW™ Software. Anti-inflammatory effect of Echinacea complex was verified by changes in exhaled NO levels and by Bio-Plex® assay of Th2 cytokine concentrations (IL-4, IL-5, IL-13 and TNF-alpha) in serum and bronchoalveolar lavage fluid (BALF). Results Chemical and spectroscopic studies confirmed the presence of carbohydrates, phenolic compounds and proteins, as well as the dominance of rhamnogalacturonan and arabinogalactan moieties in Echinacea complex. The significant decrease in sRaw values and suppressed histamine and acetylcholine-induced contractile amplitude of isolated airways smooth muscle that were similar to effects of control drug salbutamol confirmed Echinacea complex bronchodilatory activity. The anti-inflammatory effect was comparable with that of control agent budesonide and was verified as significantly reduced exhaled NO levels and concentration of Th2 cytokines in serum and BALF. The values of CBF were changed only insignificantly on long-term administration of Echinacea complex suggested its minimal negative impact on mucociliary clearance. Conclusion Pharmacodynamic studies have confirmed significant bronchodilatory and antiinflammatory effects of Echinacea complex that was similar to effects of classic synthetic 2
drugs. Thus, results provide a scientific basis for the application of this herb in traditional medicine.as a supplementary treatment of allergic disorders of the airways, such as asthma.
Keywords: Echinacea purpurea, Polysaccharide-phenolic-protein complex, NMR, Antiasthmatic effect, Bronchodilatory effect, Anti-inflammatory effect ____________________ *Corresponding author. Tel.:+421 2 59410209; fax: +421 2 59410222 E-mail address: [email protected] (P. Capek)
1. Introduction
Echinacea purpurea (L.) Moench (Purple coneflower or coneflower) is a perennial plant of Asteraceae family. Nowadays, it is regarded as the medicinal and as well ornamental plant. Several Echinacea species have been used in the traditional medicine to treat infections, cold, cough, bronchitis, inflammations, etc. (Barrett, 2003; Goel et al. 2004; Wagner et al. 1999). Echinacea is one of the most used plants in herbal medicine and in dietary supplements (Percival, 2000). Modern interest in Echinacea is directed to its immunomodulatory activity, in particular in the prevention and treatment of the common cold, cough, bronchitis, and other respiratory infections. Phytoconstituents of Echinacea species are still the subject of chemical and pharmacological research in order to identify their active ingredients, however, the description of all constituents is still not completed. A great deal of clinical trials was performed to verify the efficacy of Echinacea constituents isolated by different procedures/solvents from various Echinacea species to describe their active principles. Four main groups of constituents are considered to be active in Echinacea species - alkylamides, glycoproteins, phenylpropanoids and polysaccharides. It is believed that these compounds are responsible for immune3
stimulating, anti-inflammatory and anticoagulant activities (Pawlaczyk et al. 2009; Spelma et al. 2009). Concerning polymeric compounds, the modulation of immune system was reported by Echinacea polysaccharides, i.e. enhancing production of TNF-α, IL-6 , IL-10, and IL-1-β, adjuvant effects on human T-cell cytokine responses, increasing production of reactive oxygen intermediates, etc. (Roesler et al. 1991; Steinmuller et al. 1993; Currier and Miller, 2000; Currier et al. 2002; Fonseca et al. 2014). Furthermore, it was found that active polysaccharides did not stimulate B cells. Allergic asthma is a chronic obstructive disease of the lower airways, characterized by airway inflammation, reversible airflow obstruction, mucus hypersecretion, and airway hyperreactivity (Nakagome and Nagata, 2011). Many herbs have shown interesting results in various target specific biological activities such as bronchodilation, mast cell stabilization, immunomodulatory, antiinflammatory and inhibition of mediators such as leukotrienes and cytokines, in the treatment of asthma (Mali and Dhake, 2011). Recent studies have shown, that expression of asthma-related cytokine genes and product secretions in the bronchial epithelial cells can be reversed by Echinacea preparations (Barrett, 2003). From the literature it is evident, that phytoconstituents of E. purpurea are still subject of chemical and pharmacological attention. This fact inspired us to evaluate pharmacodynamic profile of E. purpurea complex using an animal model of allergic asthma. This model can mimic the pathological symptoms common in humans suffered from allergic bronchial asthma, for example airway hyperreactivity and inflammatory changes in small diameter bronchioles (Franova et al. 2013).
2. Material and methods
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2.1. Plant material and chemicals
Air-dried flowers of medicinal plant E. purpurea (L.) Moench were purchased from a local market in Wroclaw, Poland. The identity of the plant was certified by Prof. K. D. Kromer and J. Kochanowska from Botanical Garden of Wrocław University, Poland and a voucher specimen (No. 011493) has been deposited in the Botanical Garden of Wrocław University, Poland. Citric acid (AC) p.a., histamine, acetylcholine, methacholine, salbutamol, aluminium hydroxide, budesonide and chicken ovalbumine purchased from Sigma Aldrich (Lambda Life, Slovakia). Budesonide was prepared as suspension in 1 % TWEEN 80 (in 0.9 % saline) according to manufacturer’s instruction. All other above-mentioned drugs were dissolved in 0.9 % saline.
2.2. Isolation of Echinacea complex
The isolation of E. purpurea complex was made according to already described procedure (Pawlaczyk et al., 2009). Shortly, flowering parts were minced and suspended in 0.1 M sodium hydroxide at room temperature for 24 h and refluxed for 6 h. The rest of plant was removed by centrifugation. The supernatant was neutralized by 1 M HCl, concentrated to a lower volume and gradually extracted with hexane (1:1, v/v), diethyl ether (1:1, v/v), chloroform (1:1, v/v), and chloroform and ethanol mixture (3:2, v/v). Organic extracts were discarded while the water fraction was evaporated to a paste and treated with methanol at room temperature. The soluble methanolic part was filtered off and the dark residue was solubilized in deionized water, dialyzed and freeze-dried to give a dark brown Echinacea complex.
5
2.3. General methods
Concentration of solutions was performed under reduced pressure at bath temperature not exceeding 40 C. The content of carbohydrate, phenolic and protein was estimated by the phenol–sulfuric acid, Folin–Ciocalteu and Lowry assays, respectively (Dubois et al. 1956; Lowry et al. 1951; Singleton et al. 1999) and the uronic acid content was determined by m-hydroxybiphenyl reagent (Blumenkrantz and Asboe-Hansen, 1973). Sample was hydrolysed with 2 M TFA for 1 h at 120 ºC and the quantitative determination of the neutral sugars was carried out in the form of their alditol acetates (Englyst and Cummings,1984), by gaschromatography on a Trace GC Ultra coupled with ITQ 900 (Thermo Scientific, USA) equipped with a Restek RT- 2330-NB column (0.32 mm × 105 m), the temperature program of 80 ºC (12 min)–160 ◦C (8 ºC /min)–250 ºC (4 ºC /min, 25 min at 250 ºC)–265 ◦C (20 ºC /min, 10 min at 265 ºC) and the flow rate of helium was 1 mL/min. Molecular mass determination of a sample was performed with HPLC Shimadzu apparatus (Vienna, Austria) equipped with a differential refractometer RID-6A and a UV– vis detector SPD-10AV using the column HEMA-BIO 1000 (8 mm×250 mm) of particle size 10 m (Tessek, Prague, Czech Republic). As a mobile phase 0.02 M phosphate buffer pH 7.2 containing 0.1 M NaCl was used at a flow rate 0.8 mL/min. A set of dextran standards was used for of the column (Gearing Scientific, Polymer Lab., Hertfordshire, UK). The colorimetric assays were measured using UVVIS 1800 spectrophotometer (Shimadzu, Japan). Fourier-transform infrared (FT-IR) were obtained on a NICOLET Magna 750 spectrometer with DTGS detector and OMNIC 3.2 software, where 128 scans were recorded with 4 cm−1 resolution. NMR spectra of conjugate were recorded in D2O at 60 ºC on Varian 400 NMR spectrometer on direct 5 mm PFG AutoX probe. Sample was
6
twice freeze-dried from D2O before measurements. For 1H and 13C NMR spectra, chemical shifts were referenced to internal standard – acetone ( 2.22 and 31.07, respectively). For the assignment of signals one-dimensional (1H NMR) and twodimensional Heteronuclear Single Quantum Correlation experiment (HSQC) were used.
2.3. Animals
All experiments were approved by Institutional Ethics Committee of the Jessenius Faculty of Medicine, Comenius University in Martin, Slovakia, registered in Institutional Review Board/Institutional Ethic Board Office (IRB 00005636), complied with Slovakian and European Community regulations for the use of laboratory animals and follow the criteria of experimental animal’s well fare (decision No. 1249/2013). Adult male Trik strain guinea pigs (200-350 g) were obtained from approved breeding facility situated in Department of Experimental Pharmacology, Slovak Academy of Sciences (Dobra Voda, Slovakia) and were housed in approved animal holding facility for one-week adapting period and subsequent several days adaptation to experimental conditions. The animals were divided into the five groups, each consisting of 10 animals: Negative controls – (1) Healthy group and (2) Group of ovalbumin-sensitized animals (OVA+) received saline (sodium chloride 0.9 %) per orally (p.o.); (3) Positive controls – sensitized guinea pigs received salbutamol (10 mg/kg b.w.) intraperitoneally (i.p.) once daily long-term (Sal LT) or (4) received budesonide (3 mg/mL) by inhalation for 5 min to 14 days, respectively (Bud LT); (5) Experimental group – sensitized animals underwent long-term therapy with Echinacea complex (50 mg/kg b.w.) per orally (p.o.), long-term (Ep LT). The doses of control drugs and tested complex from medicinal plant E. purpurea were selected according to literature data
7
and results of our previous experiments (Whelan et al., 1993; Prisenžňáková et al., 2005; Šutovská et al., 2014) and following the manufacturer’s instruction.
2.4. Antigen-induced airway hyperresponsiveness
Sensitization of animals by repetitive doses of antigen ovalbumin, which causes airway reactivity changes on immunological basis, was performed during 21 days (Franova et al. 2013). Briefly, Al (OH)3 adsorbed ovalbumin was administered intraperitoneally and subcutaneously (1st day of sensitization) and intraperitoneally (3rd day). Further, 1-2 min lasted inhalations of allergen was performed at 9th, 12th, 15th, 18th and 20th days using bodyplethysmograph for small laboratory animals (HSE type 855, Hugo Sachs Elektronik, Germany). Allergic inflammation was confirmed as skin reaction increase in exhaled nitric oxide (eNO) and in basal specific airway resistance (sRaw) values. Twenty four hours after the last administration of allergen animals have been treated by saline, control drugs and Ep isolate for a period of 14 days. Budesonide and salbutamol were used as the anti-inflammatory and bronchodilatory controls, respectively. After these treatments, animals have been used for evaluation of the Echinacea effect on defence reflexes of airways and its ability to suppress allergic inflammation process by different in vivo and in vitro methods.
2.5. The evaluation of airway smooth muscle reactivity in vivo
In vivo airway smooth muscle (ASM) reactivity was evaluated using a bodyplethysmograph consisting of head and body chambers. The values of sRaw calculated by Pennock et al. (1979) and their changes were regarded as indicator of in vivo
8
airways reactivity. The sRaw is proportional to phase difference between nasal and thoracic respiratory airflow. The changes in sRaw were measured under the basal conditions and then during 1 min consecutively after the short (30 s) exposure to contractile mediators: citric acid (0.3 M), histamine and methacholine (both 10-6 M). Between the bronchoprovoking agent exposure and measurement of sRaw was an interval 1 min, during which fresh air was insufflate into the nasal chamber. The effect of Echinacea was compared to salbutamol and saline.
2.5.1. The evaluation of airway smooth muscle reactivity in vitro
The changes of the ASM reactivity on cumulative doses of contractive mediators (acetylcholine and histamine) were tested by organ tissue bath method (Sutovska et al. 2012). Briefly, guinea pigs were killed by transversal interruption of neck spinal cord. Consequently the respiratory organs were removed. Four strips (two of tracheal and two of pulmonary smooth muscle) obtained from each animal were placed into organ bath chambers filled with Krebs-Henseleit´s buffer saturated by pneumoxide (95 % O2 + 5 % CO2), held at the temperature 36 ± 0.5 ºC, maintained at pH 7.5 ± 0.1. Single strips were fixed onto the sliding arm and the other end was bound with a thin thread to a hook of a transducer (Experimetria Ltd., Hungary). The tension was used to monitor the intensity of contractile responses. The amplitude of isometric contraction (mN) of tracheal and pulmonary smooth muscle to cumulative doses of contractile mediator acetylcholine and histamine at concentrations 10-8–10-3 M was used for evaluation of ASM reactivity.
2.6. Measurement of eNO in vivo
9
The changes in exhaled nitrogen oxide (eNO) values were used as rough indicator of airway inflammation and anti-inflammatory effect (Sutovska et al. 2013). Animals were placed into the offline chamber sampling connected with NIOX Flex Offline Start Kit 041210-F (Aerocrine AB, Sweden), breathed NO free air for 5 min. Subsequently the exhaled gas (flow rate 5 mL/s) was analysed during 7 s. NIOX uses the high sensitivity and high specificity of chemiluminescence gas analyser, together with integrated software, to accurately measure NO molecules at very low concentrations (particle per billion, ppb).
2.7. The evaluation of ciliary beat frequency (CBF) in vitro
The experiments were carried out under the standard laboratory conditions (temperature 21–24 °C and humidity at 55 ± 10 %). The temperature of the microscopic glass slide and the saline used as a nutritive medium for cilia, were kept in the range of 37–38 °C. Following transversal interruption of the male guinea pigs neck spinal cord, transverse access to the trachea was made approximately in the middle of its normal length. Ciliated samples were obtained by cytology brush, which was dipped into the saline and then gently rotated on the mucosal surface of the animal trachea. Tracheal brushings were immediately placed into saline solution and were processed to microscopic preparation. Microscopic preparations were examined 3 minutes after brushing the tracheal cilia using phase contrast inverted biological microscope (Kvant model IM1C, Slovakia). Beating ciliated cells were recorded using a digital high speed video camera (Basler A504kc; Basler AG, Germany) on frame rate from 256 to 512 fps (frames per second). There were approximately 10-12 video records of the same microscopic preparation performed at 1 minute intervals and duration of each record was approximately 5-10
10
seconds. Video records were analysed using LabVIEW™ Software to generate a ciliary region of interest (ROI), intensity variation in selected ROI and intensity variance curve. Curve was then analysed according to Hargas et al. (2011) method using fast Fourier transform algorithm (FFT). Fourier spectrum of each intensity variance curve was then equal to frequency spectrum of cilia beating in each ROI. The median of frequency (Hz) for each ROI and their arithmetic means referred to definite CBF value for each microscopic preparation.
2.8. The assessment of cytokine level in vitro
The blood from guinea pigs heart was collected immediately after transversal spinal cord interruption. To measure inflammatory mediators in the airways, bronchoalveolar lavage was performed with warm saline (37 °C) in volume calculated according body weight of animal (10 mL/kg). Saline was injected and withdrawal via the cannula placed into the right bronchus. Serum and supernatant from biological fluids were obtained by centrifugation – blood at the centrifugal force 2054 g for 5 min and bronchoalveolar lavage fluid (BALF) at 377 g for 2 min. The apparatus Bio-Plex® 200 System and TH1/TH2 panel Human Cytokine (Bio-Rad, USA) were used for cytokine levels assessment. The assay was designed on magnetic beads according to a capture sandwich immunoassay format. The capture antibody– coupled beads were first incubated with antigen standards, samples, or controls followed by incubation with biotinylated detection antibodies. After washing away the unbound biotinylated antibodies, the beads were incubated with a reporter streptavidinphycoerythrin (S-P) conjugate. Following the removal of S-P excess, the beads were passed through the Bio-Plex 200 suspension array reader equipped with two lasers, one
11
(532 nm excitation) for analytes quantification and second (635 nm excitation) for cytokine identification in small volume samples, which measures the fluorescence of the beads and of the bound S-P. All washes were performed using a Bio-Plex Pro wash station. High-speed digital processor manages data output and Bio-Plex Manager™ 6.0 software presents results as concentration in pg/mL.
2.9. Statistics
All obtained data were evaluated with one-way analysis of variance (ANOVA) with the post hoc Bonferroni test using GraphPad Prism 6 software. Data are presented as mean ± standard error of the mean (SEM). The results with p<0.05 was considered as statistically significant. Significances of p<0.05, p<0.01 and p<0.001 are shown by one, two and three symbols (● vs unsensitized healthy animals, * vs negative control OVA+, or + vs positive control drugs).
3. Results and discussion
3.1. Chemical and spectroscopic characteristics of Echinacea complex
The hot alkaline extraction of E. purpurea flowers followed by neutralization and multi-step extractions with organic solvents, dialysis and freeze-drying afforded a dark brown Echinacea complex in 1.8% yield on dry plant. HPLC and GPC analyses showed one single peak of molecule mass at around 10 kDa indicating thus its molecular homogeneity (Pawlaczyk et al. 2009). Compositional analyses of Echinacea complex revealed carbohydrates (26.3%), phenolics (17.5% or 1.03 mM of GAE/1 g), protein (14%) and uronic acids (11.2 %). Analysis of the carbohydrate part showed the presence
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of uronic acids (30%), galactose (Gal, 22%), arabinose (Ara, 17%) and rhamnose residues (Rha, 13%), while other sugars were found in lower contents, i.e. xylose (Xyl, 9%), glucose (Glc, 6%), mannose (Man, 2%) and fucose (Fuc, 1%). The FTIR analysis confirmed the presence of three structural components in Echinacea conjugate, i.e. carbohydrates and phenolics as the dominant constituents, while proteins were found in a small amount (Pawlaczyk et al. 2009). In the 1H NMR spectrum of Echinacea complex (Fig. 1), three regions of signals could be distinguished. Signals in the region at δ 9–6 reveal the presence of aromatic rings in phenolic compounds; carbohydrate signals were present mainly in the region at δ 5.5–3, while those due CH2, CH3 signals of deoxy sugars, proteins, phenolics and acetyl groups were located in the region at δ 3–0.6. Broad signals reflect high molecular mass of the conjugate. Dominant sugar components, particularly galacturonic acid (GalA), Gal, Ara and Rha, have been identified by sugar analysis. Consequently, they afforded dominant signals in NMR spectra. Characteristic 1H/13C chemical shifts of signals found in HSQC spectrum (Fig. 2) were used for identification of sugars, their linkage types as well as types of carbohydrate polymers present in Echinacea complex. In the α-anomeric region the most important signals in form of singlets were due to Araf at δ 5.21/110.25 and 5.04/108.62. All other signals were of low intensity and very broad. The most intensive overlapped β-anomeric H1/C1 cross peaks at characteristic chemical shifts at δ 4.48/104.34 and 4.44/104.10 have been identified as 1,3,6-β-Gal and 1,6-linked βGal. The presence of 6-linked βGal units was evident from H6/C6 cross peaks with characteristic chemical shifts at δ 4.01, 3.88/70.61. These data are in a very good agreement with data published for arabinogalactan type II isolated from C. arabica (Capek et al. 2010) with a highly branched 1,3-β-galactan backbone with short 1,6-linked βGal side chains, which were also
13
branched at O3 by βGal and αAraf. The 1,3-linked βGal signals were not found. Characteristic chemical shifts of Araf units confirmed two types of linkages: one as βGal O3-linked (δ 5.21/110.25) and the other as Araf, branched at O5 (δ 5.04/108.62). A downfield shift of αAraf C5 signal due to 1,5-linkage was confirmed by H5/C5 cross peaks at δ 3.885, 3.795/67.84. These facts indicate that one component in the Echinacea complex is the highly branched arabinogalactan. A doublet H6 signal at δ 1.214 (6Hz, d) with C6 chemical shift δ 17.56 gave a clear evidence about Rhap presence. It can form a branching element in arabinogalactans (Nunes et al. 2008), but due to a high content of GalA in EP rhamnose should be also a component of rhamnogalacturonan which is usually a homopolymer formed by 1,4-linked αGalA. It was found that HSQC contains also signals of two O-methyl groups at H/C δ 3.45/58.60 and 3.45/60.78. For their location identification the comparison of cross peaks chemical shifts in the HSQC spectrum of Echinacea complex with published NMR data of not substituted, acetylated and O-methylated 1,4-linked galacturonans was used for analysis. Chemical shift of OMe group at C6 in GalA ester was identified at δ 3.80/54.1 and H5/C5 at δ 5.13/72.0 (Popov et al. 2011). Based on OMe chemical shifts and missing signal at H5/C5 at δ 5.13/72.0 a conclusion was made that GalA in Echinacea complex is not C6 esterified by OMe. Further analysis has shown that OMe H/C chemical shifts values are in agreement with data published for substituted 1,6-linked β-galactan at C3 by OMe isolated from Salvia officinalis L. (Capek, 2008). Thus the location of OMe on βGal units is highly probable. The presence of acetyl groups in NMR spectra was reveal by CH3 signals of OAc cross peaks at H/C 2.020/23.527, 2.060/21.28 and 2.158/21.28 and downfield shifted signals due to acetylation (broad signals at δ 4.675/72.87 and 4.60/72.32). Cross peak at δ 3.933/43.40 was characteristic chemical shifts of CH2-N group. Together with cross peaks due to CH2 at δ 2.009/28.49, 2.226/34.28 and CH3
14
groups at δ 0.857/15.84, 0.867/18.69 and 0.846/22.78 they are the most important signals due to proteic part of the complex. All other signals are too broad and of low intensity to be detected at given experimental conditions in HSQC spectrum. NMR data suggest the presence of complex mixture of arabinogalactan type II and rhamnogalacturonans polymers, O-methylated and acetylated, as the main carbohydrate components. Chemical shifts of Araf units do not indicate the presence of arabinans, but galactans can’t be excluded. Polyphenolics show large signals in 1H NMR spectrum which not allow their better identification.
3.2. The influence of Echinacea and control drugs on defence reflexes of the airways
In this study the attention was focused on the pharmacodynamic profile of E. purpurea complex using animal model of allergic airway inflammation induced by exposure of guinea pigs to ovalbumin (OVA+). Repetitive exposure of animals to allergen leads to complex of changes that almost mimic the asthma phenotype in human, e.g. airway hyperreactivity (AHR) and allergic inflammation of the airways characterized by typical histological features and changes in cytokines and mediators production (Franova et al. 2013, Sutovska et al. 2015). Results of in vivo measurements showed the changes in basal specific airway resistance values and sRaw induced by citric acid (AC), histamine and methacholine (Fig. 3) used as mediators, each producing bronchoconstriction through different pathway involved in AHR. The significant increase in basal hyperreactivity and in the response on contracting mediators is the common feature in asthma and it was confirmed in sensitized, saline-treated animals (OVA+). Echinacea complex significantly decreased bronchoconstriction induced by AC, histamine or methacholine (AC and histamine,
15
p<0.001; methacholine, p<0.01). Furthermore, Echinacea complex significantly reduced basal hyperreactivity (p<0.001) and its effect was similar to that of salbutamol, a standard bronchodilatory antiasthmatic drug. The measurement of sRaw is widely used by paediatric pulmonologists and important therapeutic decisions are based on this approach. According to Mahut et al. (2009) sRaw is very sensitive parameter appropriate to detect mild levels airway obstruction. The long-term treatment of sensitized animals by Echinacea complex significantly decreased sRaw induced by bronchoconstrictors used as well as basal sRaw. These values characterized basal hyperreactivity. These findings strongly support possible effectiveness of Echinacea complex in allergic asthma. In vitro studies were based on a contraction change of tracheal and pulmonary smooth muscles after treatment with acetylcholine and histamine, used as exogenous contractile agents in cumulative concentrations. In the Figs. 4 and 5 the highest amplitudes are observed in sensitized (OVA+) tissues, while the lowest ones are in tissues of animals’ long term treated by Echinacea complex. Moreover, amplitudes are even significantly lower than those due to salbutamol treated animals, especially at low concentration of histamine (10-6 M) added directly into the pulmonary muscle strips or at high concentration of histamine (10-4 and 10-3 M) in the case of tracheal smooth muscle. The inhibition of airway smooth muscles (ASM) reactivity by Echinacea complex evaluated in vitro was consistent with in vivo findings. Furthermore, in comparison to salbutamol effect, the long-term administration of Echinacea complex results in a greater reduction in histamine-induced contraction of pulmonary smooth muscle tissue. It is generally accepted, that asthma is a disease of small diameter intraparenchymal bronchi and thus a good response of the lung tissue to Echinacea complex be regarded as a very positive outcome.
16
The evaluation of a ciliary beat frequency (CBF) of healthy and treated animals is shown in Fig. 6. Results showed that allergic inflammation induced by repetitive exposure to ovalbumin is associated with significant enhancement of CBF (p<0.05). Furthermore, long-term treatment of animals with the Echinacea complex did not change the frequency of cilia beating, that is, Echinacea complex has no effect on the natural cilia clearance. The control drug budesonide, tested under the same condition, changed CBF only insignificantly. The cilia of respiratory epithelium are a relatively vulnerable structures targeted by various stimuli, e.g. serotonin or TNF-α caused the increase in the frequency of cilia beating (Weiterer et al. 2014) and IL-13 inhibits not only CBF but also alter the morphology of the ciliated cells (Laoukili et al. 2001) . CBF is a key parameter of mucociliary clearance and ciliary transport efficiency is linearly dependent on the CBF (Braiman and Priel, 2001). Our study showed that both, budesonide and Echinacea do not affect the effectiveness of mucociliary clearance.
3.3. The influence of Echinacea and control drugs on allergic inflammation of airways
Two ways, i.e. indirect and direct were used to evaluate the influence of Echinacea on allergic inflammation of airways. The indirect method of exhaled nitrogen oxide (eNO) measurement is based on the fact, that the level of eNO is higher in animals with a developed inflammation of airways. As can be seen from the Fig. 7, the levels of eNO measured in animals’ long term treated by Echinacea complex was significantly lower than that of sensitized (diseased) animals. It was found that levels of eNO in animals treated by Echinacea complex were comparable with those of animals treated by classic anti-inflammatory drug budesonide.
17
Indicative results of Echinacea complex on allergic inflammation of airways were confirmed by measurement of cytokine levels in BALF and plasma. TH1/TH2 panel Human Cytokine allows us to evaluate changes in cytokines that are responsible for key features of allergic asthma: IL-4, IL-5, IL-13 and TNF α. Results showed that the treatment of ovalbumine sensitized animals (OVA+) by Echinacea complex decreased cytokine levels in both, BALF and plasma (Fig. 8). Furthermore, the induced decrease by Echinacea complex was more significant than the budesonide, with the exception of plasma levels of IL-5 and IL-13. The reduction in the BALF cytokines appears to be the most important results with regard to allergic inflammation, as this shows that Echinacea complex is able to very effectively reduce the local, epithelium-mediated pulmonary inflammation. Exhaled NO levels were measured to estimate the degree of inflammation in response to repeated exposure to ovalbumin, as well as anti-inflammatory action of Echinacea complex. As reported earlier, significantly increased eNO was regularly found in the developed allergic inflammation of the airways and eNO is normally reduced by corticosteroids administration (Kocmalova et al., 2015; Sutovska et al., 2013). The presented study confirmed increase in eNO of ovalbumin – sensitized untreated animals and showed a significant reduction in the levels of eNO in animals sensitized with ovalbumin and treated long-term with Echinacea complex. Several studies have shown an enhanced in exhaled NO levels due to upregulated inducible NO-synthase (iNOS) in human subjects suffering from untreated asthma and have found correlations between eNO and airway eosinophilia (Redington, 2006). Eosinophils, T cells and mast cells have been shown not only as a source of iNOS, but also the mediators and cytokines, e.g., IL-4, IL-5, IL-13 and TNF-α by stimulated increased of iNOS expression in airway epithelial
18
cells (Benson et al., 2011; Paoliello-Paschoalato et al., 2005) and played a key role in asthma (Feske et al., 2012). The significant anti-inflammatory activity of Echinacea complex, determined with eNO was supported also by the results of the Bio-Plex® assay, which showed a significant decrease in levels of key cytokines mediating asthma in plasma as well as in BALF. The similar changes in cytokine production were reported by Altamirano-Dimas et al. (2009) and Sharma et al. (2009). Authors studied profile of produced cytokines and chemokines and changes induced by Echinacea preparations on human bronchial rhinovirustransfected cell lines. Reduced BALF levels of IL-4, IL-5, IL-13 and TNF-α evidenced the Echinacea complex suppressive effect on secretory functions of respiratory epithelium, which represents the most important tissue-organizing inflammatory response in asthma (Holgate, 2011).
4. Conclusion
It can be concluded that long-term administration of Echinacea complex, composed of carbohydrate, phenolic and protein components, showed significant bronchodilator and anti-inflammatory effects and maintains unchanged frequency of cilia beating in guinea pigs with experimentally induced allergic airway inflammation. Using animal model of allergic asthma, Echinacea showed potency to suppress both, airway hyperreactivity and airway inflammation. Bronchodilation was similar to the effect of salbutamol, a classic antiasthmatic bronchodilator and suppression of allergic inflammation was comparable with that of budesonide, an anti-inflammatory corticosteroid agent. Preliminary studies confirmed that Echinacea complex is beneficial to patients suffering from allergic
19
disorders of the airways, such as asthma and thus provide a scientific basis for the application of this herb in traditional medicine.
Acknowledgements
Studies were supported by the project BioMed co-financed from EC sources and by the grants VEGA No. 1/0165/14 and 2/0018/15, MZ No. 2012/35-UKMA-12 and APVV project No. 0305-12.
References Altamirano-Dimas, M., Sharma, M., Hudson, JB., 2009. Echinacea and anti-inflammatory cytokine responses: Results of a gene and protein array analysis. Pharm. Biol. 47, 500– 508. Barrett, B., 2003. Medicinal properties of Echinacea: A critical review. Phytomed. 10 (1), 66– 86. Benson, R.C., Hardy, K.A., Morris, C.R., 2011. Arginase and arginine dysregulation in asthma. J. Allergy (Cairo), 736319. doi: 10.1155/2011/736319. Blumenkrantz, N., Asboe-Hansen, O., 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54, 484–489. Braiman, A., Priel, Z., 2001. Intracellular stores maintain stable cytosolic Ca2+ gradients in epithelial cells by active Ca2+ redistribution. Cell Calcium, 30, 361–371. Capek, P., Matulova, M., Navarini, L., Suggi Liverani, F., 2010. Structural features of an arabinogalactan-protein isolated from instant coffee powder of Coffea arabica beans. 20
Carbohydr. Polym. 80, 180–185. Capek, P., 2008. An arabinogalactan containing 3-O-methyl-D-galactose residues isolated from the aerial parts of Salvia officinalis L. Carbohydr. Res. 343, 1390–1393. Currier, N.L., Lejtenyi, D., Miller, S.C., 2002. The effect with time of administration in vivo, of the polysaccharide, arabinogalactan, on the immune and hemopoietic cell lineages in murine spleen and bone marrow. Phytomed. 10, 145–153. Currier, N.L., Miller, S.C., 2000. Natural killer cells from aging mice treated with extracts from Echinacea purpurea are quantitatively and functionally rejuvenated. Exp. Gerontol. 35, 627–639. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Biochem. 28 (3), 350– 355. Englyst, H.N., Cummings, J.H., 1984. Simplified method for the measurement of total nonstarch polysaccharides by gas-liquid chromatography of constituent sugars as alditol acetates. Analyst, 109, 937-942. Feske, S., Skolnik, E.Y., Prakriya, M., 2012. Ion channels and transporters in lymphocyte function and imunity. Nat. Rev. Immunol. 12, 532-547. Fonseca, F.N., Papanicolaou, G., Lin, H., Lau, C.B.S., Kennelly, E.J., Cassileth, B.R., Cunningham-Rundles, S., 2014. Echinacea purpurea (L.) Moench modulates human Tcell cytokine response. Int. Immunopharm. 19, 94–102. Franova, S., Joskova, M., Sadlonova, V., Pavelcikova, D., Mesarosova, L., Novakova, E.,
21
Sutovska, M., 2013. Experimental model of allergic asthma. Ad. Exp. Med. Biol. 756, 4955. Goel, V., Lovlin, R., Barton, R., Lyon, M.R., Bauer, R., Lee, T.D.,Basu, T.K., 2004. Efficacy of a standardized echinacea preparation (Echinilin) for the treatment of the common cold: a randomized, double-blind, placebo-controlled trial. J. Clin. Pharm. Ther. 29, 75–83. Hargaš, L., Koniar, D., Štofan, S., 2011. Sophisticated Biomedical Tissue Measurement Using Image Analysis and Virtual Instrumentation, in LabVIEW – Practical Applications and Solutions, in: Folea, S. (Editor), InTECH, pp. 155-180. Holgate, S.T., 2011. The sentinel role of the airway epithelium in asthma pathogenesis. Immunol. Rev. 242, 205-219. Kocmalova, M., Oravec, M., Adamkov, M., Sadlonova, V., Kazimierova, I., Medvedova, I., Joskova, M., Franova, S., Sutovska, M., 2015. Potassium ion channels and allergic asthma. Adv. Exp. Med. Biol. 838, 35-45. Laoukili, J., Perret, E., Willems, T., Minty, A., Parthoens, E., Houcine, O., Coste, A., Jorissen, M., Marano, F., Caput, D., Tournier, F., 2001. IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells. J. Clin. Invest. 108, 1817–1824. Lowry, O., Rosebrough, N., Farr, L., Randall, R., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Mahut, B., Trinquart, L., Bokov, P., Le Bourgeois, M., Waernessyckle, S., Peiffer, C., Delclaux, C., 2009. Relationships between Specific Airway Resistance and Forced 22
Expiratory Flows in Asthmatic Children. Plos One, 4 (4), e5270. Doi: 10.1371/journal.pone.0005270. Mali, R.G., Dhake, A.S., 2011. A review on herbal antiasthmatics. Orient. Pharm. Exp. Med. 11, 77-90. Nakagome, K., Nagata, M., 2011. Pathogenesis of airway inflammation in bronchial asthma. Auris Naxus Larynx, 38, 555-563. Nunes, F.M., Reis, F.M., Silva, V.A., Domingues, A.M.S., Coimbra, M.A., 2008. Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee arabinogalactans. Phytochem. 69, 1573–1585. Paoliello-Paschoalato, A.B., Oliveira, S.H., Cunha, F.Q., 2005. Interleukin 4 induces the expression of inducible nitric oxide synthase in eosinophils. Cytokine, 30, 116-124. Pawlaczyk, I., Czerchawski, L., Pilecki, W., Lamer-Zarawska, E., Gancarz, R., 2009. Polyphenolic-polysaccharide compounds from selected medicinal plants of Asteraceae and Rosaceae families: chemical characterization and blood anticoagulant activity. Carbohydr. Polym. 77, 568–575. Pennock, B.E., Cox, C.P., Rogers, R.M., Cain, W.A.,Wells, J.H., 1979. A non-invasive technique for measurement of changes in specific airway resistance. J. Appl. Physiol. 46, 399-406. Percival, S.S., 2000. Use of echinacea in medicine. Biochem. Pharm. 60, 155-158. Popov, S.V., Ovodova, R.G., Golovchenko, V.V., Popova, G.Y., Viatyasev, F.V., Shashkov, A.S., Ovodov,Y.S., 2011. Chemical composition and anti-inflammatory activity of a pectic polysaccharide isolated from sweet pepper using a simulated gastric medium. Food Chem. 124, 309–315. 23
Prisenžňáková, L., Nosáľová, G., Hromádková, Z., Ebringerová, A., 2011. The pharmacological activity of wheat bran polysaccharides. Fitoterapia. 81, 1037-1044. Redington, AE., 2006. Modulation of nitric oxide pathways: therapeutic potential in asthma and chronic obstructive pulmonary disease. Eur. J. Pharm. 533, 263-276. Roesler, J., Steinmuller, C., Kiderlen, A., Emmendorffer, A., Wagner, H., LohmannMatthes, M.L., 1991. Application of purified polysaccharides from cell cultures of the plant Echinacea purpurea to mice mediates protection against systemic infections with Listeria monocytogenes and Candida albicans. Int. J. Immunopharmacol. 13, 27–37. Sharma, M., Schoop, R., Hudson, J.B., 2009. Echinacea as an antiinflammatory agent: the influence of physiologically relevant parameters. Phytother. Res. 23, 863- 867. Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M., 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods Enzymol. 299, 152–178. Spelman, K., Wetschler, M.H., Cech, N.B., 2009. Comparison of alkylamide yield in ethanolic extracts prepared from fresh versus dry Echinacea purpurea utilizing HPLCESI-MS. J. Pharm. Biomed. Anal. 49, 1141–1149. Steinmuller, C., Roesler, J., Grottrup, E., Franke, G., Wagner, H., Lohmann-Matthes, M.L., 1993. Polysaccharides isolated from plant cell cultures of Echinacea purpurea enhance the resistance of immunosuppressed mice against systemic infections with Candida albicans and Listeria monocytogenes. Int. J. Immunopharmacol. 15, 605–14. Sutovska, M., Kocmalova, M., Joskova, M., Adamkov, M., Franova, S., 2015. The Effect of Long-term Administered CRAC Channels Blocker on the Functions of Respiratory 24
Epithelium in Guinea Pig Allergic Asthma Model. Gen. Physiol. Biophys. 34(2), 167-76. Sutovska, M., Kocmalova, M., Adamkov, M., Vybohova, D., Mikolka, P., Mokra, D., Hatok, J., Antosova, M., Franova, S. (2013). The long-term administration of Orai1 antagonist possesses antitussive, bronchodilatory and anti-inflammatory effects in experimental asthma model. Gen. Physiol. Biophys. 32, 251-259. Šutovská, M., Capek, P., Fraňová, S., Pawlaczyk, I., Gancarz, R., 2012. Antitussive and bronchodilatory effects of Lythrum salicaria polysaccharide-polyphenolic conjugate. Int. J. Biol. Macromol. 51, 794-799. Šutovská, M., Capek, P., Kočmalová, M., Pawlaczyk, I., Zaczyńska, E., Czarny, A., Uhliariková, I., Gancarz, R., Fraňová, S., 2014. Characterization and pharmacodynamic properties of Arnica montana complex. Int. J. Biol. Macromol. 69, 214-221. Wagner, H., Kraus, S., Jurcic, K., 1999. Search for immunostimulating agents fromplants and other natural sources, in: Wagner, H. (Ed.), Immunomodulatory Agents from Plants. Birkhäuser, Verlag, Basel-Boston-Berlin, pp. 1–40. Weiterer, S., Schulte, D., Müller, S., Kohlen, T., Uhle, F., Weigand, M.A., Henrich, M. 2014. Tumor Necrosis Factor Alpha Induces a Serotonin Dependent Early Increase in Ciliary Beat Frequency and Epithelial Transport Velocity in Murine Tracheae. Plos One, 9, e91705. doi: 10.1371/journal.pone.009170. Whelan, C.J., Johnson, M., Vardey, C.J., 1993. Comparison of the anti-inflammatory properties of formoterol, salbutamol and salmeterol in guinea-pig skin and lung. Br. J. Pharmacol. 110, 613-618. 25
Figure legends
Fig. 1. 1H NMR spectrum of Echinacea complex.
Fig. 2. HSQC spectrum of Echinacea complex.
Fig. 3. The changes in specific airways resistance (sRaw) under the basal conditions and changes in this parameter induced by short-time inhalation of citric acid (AC), histamine (His) and methacholine (Met) measured in unsensitized (healthy) and sensitized vehicletreated animals (OVA+), and in groups of sensitized guinea pigs received long-term positive control drug salbutamol (Sal LT) or E. purpurea complex (Ep LT). ●p < 0.05 and
●●●
p<
0.001 vs healthy; ** p < 0.01 and *** p < 0.001 vs OVA+.
Fig. 4. The contractile response of tracheal smooth muscle (mN) on cumulative concentrations of acetylcholine and histamine (10-8–10-3 M) added to isolated strips to bath chamber. The tracheal reactivity of animals treated long-term by E. purpurea complex (Ep LT) was compared to contractile response of control sensitized groups treated by vehicle (OVA+) and salbutamol (Sal LT). *p < 0.05 and **p < 0.01 vs OVA+; +p < 0.05 vs salbutamol.
Fig. 5. The changes in pulmonary smooth muscle (mN) contractile response on cumulative concentrations of acetylcholine and histamine (10-8–10-3 M) registered in animals treated long-term by Echinacea complex (Ep), vehicle (OVA+) and salbutamol (Sal). *p < 0.05; **p < 0.01 and ***p < 0.001 vs OVA+; +p < 0.05 vs salbutamol.
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Fig. 6. The changes in ciliary beat frequency (CBF) evaluated in unsensitized (healthy) animals and following sensitized groups: Echinacea complex (Ep LT), negative control (OVA+) and positive budenoside control (Bud LT) as the response on long-term administration of Echinacea complex, vehicle and budesonide. ●p < 0.05 vs healthy animals.
Fig. 7. The variations in values of exhaled NO (eNO) measured in unsensitized (healthy) guinea pigs, sensitized negative control group treated by vehicle (OVA+), experimental group of animals treated long-term by Echinacea complex (Ep LT) and positive control group received long-term budesonide (Bud LT). ●●p < 0.01 vs healthy; *p < 0.01 vs OVA+.
Fig. 8. The comparison of changes in levels of cytokines IL-4, IL-5, IL-13 and TNF-α measured in BALF (part A) and plasma (part B) obtained from unsensitised (healthy) guinea pigs (healthy), ovalbumin-sensitized negative control (OVA+), animals treated long-term by Echinacea complex (Ep LT) and budesonide (Bud LT).●p<0.05, ●●p<0.01 and ●●●p<0.001 vs healthy animals; *p<0.05, **p<0.01 and ***p<0.001 vs OVA+; ++p<0.01 and +++p<0.001 vs budesonide.
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Abbreviations
Ep, Echinacea complex; sRaw, specific airway resistance; CBF, ciliary beat frequency; NO, nitrogen oxide; BALF, bronchoalveolar lavage fluid; Th2 cytokine, T helper cell cytokine; IL-1-, IL-4, IL-5, IL-6, IL-10, IL-13, interleukins 1, 4, 5, 6, 10, 13; TNF-, tumor necrosis factor alpha; NMR, nuclear magnetic resonaces; AC, Citric acid; HCl, hydrochloric acid; TFA, trifluoroacetic acid; HPLC, high-performance liquid chromatography; UV-vis, ultraviolet–visible; NaCl, sodium chloride; DTGS detector, deuterated-triglycine sulfate detector; D2O, deuterium oxide; HSQC, heteronuclear single quantum correlation experiment; OVA+, ovalbumin-sensitized animals; i.p., intraperitoneally; Sal LT, salbutamol long term; Bud LT, budenoside long term; Ep LT, Echinacea complex long term; Al(OH)3, aluminium hydroxide; eNO, exhaled nitric oxide; ASM, airway smooth muscle; ppb, particle per billion; ROI, region of interest; FFT, Fourier transform algorithm; GalA, galacturonic acid; OMe, O-methyl; βGal, β-linked galactose; Araf, arabinofuranose; AHR, airway hyperreactivity; iNOS, inducible NO-synthase; Gal, galactose; Ara, arabinose; Rha, rhamnose; Xyl, xylose; Glc, glucose; Man, mannose; Fuc, fucose; GalA, galacturonic acid;
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Influence on airway defense mechanisms Ovalbumin sensitized
Echinacea flowers PPP complex 0.1 M NaOH
PPP: polysaccharide-polyphenolic-protein
in vivo/in vitro tests
Influence on allergic inflammation
Graphical abstract
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Figure
Fig. 1. Sutovska et al., J Ethnopharm
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Fig. 2. Sutovska et al., J Ethnopharm
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Fig. 3.Sutovska et al., J Ethnopharm
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Fig. 8.Sutovska et al., J Ethnopharm