Authors Accepted Manuscript

Echinacea complex chemical view and anti-

asthmatic profile

Martina utovsk, Peter Capek, Ivana

Kazimierov, Lenka Pappov, Marta Jokov,
Mria Matulov, Soa Fraov, Izabela Pawlaczyk,
Roman Gancarz
www.elsevier.com

PII: S0378-8741(15)30132-X
DOI: http://dx.doi.org/10.1016/j.jep.2015.09.007
Reference: 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 utovsk, Peter Capek, Ivana Kazimierov, Lenka
Pappov, Marta Jokov, Mria Matulov, Soa Fraov, 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
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Echinacea complex chemical view and anti-asthmatic profile

Martina utovska, Peter Capekb*, Ivana Kazimierova, Lenka Pappova, Marta Jokova,

Mria Matulovb , Soa Fraova, Izabela Pawlaczykc, Roman Gancarzc

a
Department of Pharmacology, Jessenius Faculty of Medicine Comenius University,

Martin's Biomedical Center (BioMed) Mal Hora 11161 4C, Martin, Slovakia
b
Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dbravsk

cesta 9, Bratislava, Slovakia

c
Division of Organic and Pharmaceutical Technology, University of Technology, Wrocaw,

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 asthma-

related 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 anti-

inflammatory 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, Anti-

asthmatic effect, Bronchodilatory effect, Anti-inflammatory effect

____________________

*Corresponding author. Tel.:+421 2 59410209; fax: +421 2 59410222

E-mail address: chemcape@savba.sk (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 immune-

3
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-, IL-6 , IL-10, and IL-1-,

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, anti-

inflammatory 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

4
 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 Wrocaw University, Poland and a

voucher specimen (No. 011493) has been deposited in the Botanical Garden of Wrocaw

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 manufacturers 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 phenolsulfuric acid, FolinCiocalteu 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 C 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 C (12 min)160 C (8 C /min)250 C (4 C /min, 25

min at 250 C)265 C (20 C /min, 10 min at 265 C) 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 mm250 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 cm1 resolution. NMR spectra of conjugate were recorded in D2O at

60 C 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 two-

dimensional 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 animals 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; Prisenkov et al., 2005;

utovsk et al., 2014) and following the manufacturers 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-Henseleits buffer saturated by pneumoxide (95 % O2 + 5 %

CO2), held at the temperature 36 0.5 C, 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 10-810-3 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 04-

1210-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

2124 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 3738 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 streptavidin-

phycoerythrin (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

12
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 96 reveal the presence of aromatic rings

in phenolic compounds; carbohydrate signals were present mainly in the region at 5.53,

while those due CH2, CH3 signals of deoxy sugars, proteins, phenolics and acetyl groups

were located in the region at 30.6. 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 -anomeric region the most important

signals in form of singlets were due to Araf at 5.21/110.25 and 5.04/108.62. All other

signals were of low intensity and very broad. The most intensive overlapped -anomeric

H1/C1 cross peaks at characteristic chemical shifts at 4.48/104.34 and 4.44/104.10 have

been identified as 1,3,6--Gal and 1,6-linked Gal. The presence of 6-linked Gal units

was evident from H6/C6 cross peaks with characteristic chemical shifts at 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--galactan backbone with short 1,6-linked Gal side chains, which were also

13
branched at O3 by Gal and Araf. The 1,3-linked Gal signals were not found.

Characteristic chemical shifts of Araf units confirmed two types of linkages: one as Gal

O3-linked ( 5.21/110.25) and the other as Araf, branched at O5 ( 5.04/108.62). A

downfield shift of Araf C5 signal due to 1,5-linkage was confirmed by H5/C5 cross

peaks at 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 1.214 (6Hz, d)

with C6 chemical shift 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 GalA.

It was found that HSQC contains also signals of two O-methyl groups at H/C

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 3.80/54.1

and H5/C5 at 5.13/72.0 (Popov et al. 2011). Based on OMe chemical shifts and missing

signal at H5/C5 at 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 -galactan at C3 by

OMe isolated from Salvia officinalis L. (Capek, 2008). Thus the location of OMe on Gal

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 4.675/72.87 and

4.60/72.32). Cross peak at 3.933/43.40 was characteristic chemical shifts of CH2-N

group. Together with cross peaks due to CH2 at 2.009/28.49, 2.226/34.28 and CH3

14
groups at 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 cant 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- 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 . 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- 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 rhinovirus-

transfected cell lines. Reduced BALF levels of IL-4, IL-5, IL-13 and TNF- 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.

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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 vehicle-

treated 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 (10-810-3 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 (10-810-3 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.

26
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-

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,

ultravioletvisible; 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; Gal, -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;

28
 Influence on airway
defense mechanisms
Ovalbumin
sensitized
Echinacea flowers
PPP complex

0.1 M NaOH

in vivo/in vitro
tests
PPP: polysaccharide-polyphenolic-protein

Influence on allergic
inflammation

Graphical abstract

29
Figure

Fig. 1. Sutovska et al., J Ethnopharm

Figure

Fig. 2. Sutovska et al., J Ethnopharm

Figure

Fig. 3.Sutovska et al., J Ethnopharm

Figure

Fig. 4. Sutovska et al., J Ethnopharm

Figure

Fig. 5.Sutovska et al., J Ethnopharm

Figure

Fig. 6.Sutovska et al., J Ethnopharm

Figure

Fig. 7.Sutovska et al., J Ethnopharm

Figure

Fig. 8.Sutovska et al., J Ethnopharm