Characterization and biological activity of Solidago canadensis complex

International Journal of Biological Macromolecules 52 (2013) 192–197

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Characterization and biological activity of Solidago canadensis complex a a ˇ ˇ M. Sutovská , P. Capek b,∗ , M. Kocmálová a , S. Franová , I. Pawlaczyk c , R. Gancarz c a b c

Department of Pharmacology, Jessenius Faculty of Medicine, Comenius University, SK-03753, Martin, Slovakia Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38, Bratislava, Slovakia ˙ Wyspia´ Division of Organic and Pharmaceutical Technology, Chemistry Department, Wrocław University of Technology, Wybrzeze nskiego 29, 50-370 Wrocław, Poland

a r t i c l e

i n f o

Article history: Received 2 July 2012 Received in revised form 11 September 2012 Accepted 25 September 2012 Available online 3 October 2012 Keywords: Medicinal plant Solidago canadensis L. Polysaccharide–polyphenolic–protein complex Chemical analysis Antitussive activity

a b s t r a c t Polyphenolic–polysaccharide–protein complex has been isolated from flowers of Solidago canadensis L. by hot alkaline extraction procedure. Compositional analyses of S canadensis complex revealed the presence of carbohydrates (43 wt%), protein (27 wt%), phenolics (12 wt%), uronic acids (10 wt%) and inorganic material (8 wt%). The carbohydrate part was rich in neutral sugars (81 wt%) while uronids were determined in lower amount (19 wt%). Monosaccharide analysis of carbohydrate part revealed the presence of five main sugar components, i.e. rhamnose (∼23 wt%), arabinose (∼20 wt%), uronic acids (∼19 wt%), galactose (∼17 wt%) and glucose (∼14 wt%), and indicated thus the presence of rhamnogalacturonan and arabinogalactan in S. canadensis complex. HPLC analysis of complex showed one single peak of molecule mass at 11.2 kDa. Antitussive activity tests, performed in three doses of Solidago complex, showed the reduction of the number of cough efforts in the dose-dependent manner. Higher doses (50 and 75 mg/kg b.w.) were shown to be by 15 and 20% more effective than that of lower one (25 mg/kg b.w.). However, the antitussive effect of the highest dose (75 mg/kg b.w.) was by 10% lower in comparison with that of codeine, the strongest antitussive agent. Besides, the highest dose of the complex (75 mg/kg b.w.) significantly decreased values of specific airways resistance and their effect remained longer as that of salbutamol, a representative of classic antiasthmatic drugs. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Solidago canadensis L. (Canada goldenrot) is a herbaceous perennial plant of the Asteracea family native to North America and nowadays it is widely spread in Asia (China, Russia–Caucasia and Siberia, Japan, Taiwan), Europe, Australia or New Zealand where it is considered as an invasive weed [1,2]. In some China provinces the invasion of this weed has reached pandemic levels and therefore has caused serious concerns. Plant is a highly variable species and its taxonomic status is not clear and difficult to assess. It grows mainly in meadows and pastures, along roads, ditches, upland forests, savannas, limestone glades, etc. Plant is recognized by its golden flowers with hundreds of small capitula and length of stems between 60 and 150 cm (sometimes up to 210 or 250 cm). S. canadensis is a highly aggressive plant, once established, it can reduce the species diversity or locally out-compete all native plants. Its invasion success is due to allelopathic compounds which plant can release [3]. The allelopathic compounds suppress also the local soil pathogens [4]. It has been found that acetone extracts of S. canadensis showed allelopathic effects on the growth of other

∗ Corresponding author. Tel.: +421 2 59410209; fax: +421 2 59410 222. E-mail addresses: [email protected], [email protected] (P. Capek). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.09.021

weeds [5]. Moreover, it has been used in traditional medicine as a urological and antiphlogistical medicament [6]. From economic point of view, large fields of S. canadensis flowers are important for honey production. Many herbal preparations exploited in the traditional medicine contain significant amount of polysaccharides or their glycoconjugates. It has been found that polyphenolic–polysaccharide–protein complexes isolated from medicinal plants of Asteraceae and Rosaceae families showed anticoagulant activity [7]. The aim of the present work was to investigate composition and structural features of the complex isolated from flowers of S. canadensis, and to verify the possible ability of this plant isolate to affect chemically induced cough reflex and airway smooth muscle activity in guinea pigs.

2. Materials and methods 2.1. Plant material and animals Air-dried flowers of S. canadensis L. were purchased from local market in Wroclaw, Poland. Healthy awaken male TRIK strain guinea-pig, weighing 200–350 g were used in experimental procedure. The animals were obtained from the animal breeding facility VELAZ, Prague, Czech

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republic and located in faculty animal house for one week of quarantine, food and water were available ad libitum with a standard air conditioning system. During subsequent several days, animals were daily placed into the bodyplethysmograph box to achieve 60 min time interval of undisturbed breathing. 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. Citric acid, codeine (Codeinium dihydrogenphosphoricum) and salbutamol were obtained from Sigma–Aldrich (Lambda life, Slovakia). Citric acid was dissolved in saline, antitussive agents codeine and salbutamol were dissolved in water for injection.

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of the column (Gearing Scientific, Polymer Lab., Hertfordshire, UK) and peak molecular mass (Mp ) was calculated from a calibration curve using formula log Mp = K + a·Ve (K and a are parameters calculated from calibration curve and Ve represents an elution volume of a sample). 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 Sc were recorded in D2 O at 60 ◦ C on Varian 400 MR spectrometer on direct 5 mm PFG AutoX probe. Sample of Sc was twice freeze-dried from D2 O before measurements. For 1 H and 13 C NMR spectra, chemical shifts were referenced to internal standard – acetone (ı 2.22 and 31.07, respectively). For the assignment of signals one-dimensional (1 H NMR) and two-dimensional Heteronuclear Single Quantum Correlation experiment (HSQC) were used.

2.2. Isolation of Solidago complex 2.4. Method of citric acid-induced cough reflex The isolation of S. canadensis complex (Sc) was performed according to already described procedures [8,9]. Shortly, dried flowers of plant were minced and suspended in 0.1 M NaOH at room temperature for 24 h, and refluxed for 6 h at 97 ◦ C. The nonextracted part was removed by centrifugation (1850 × g; 20 min) and the supernatant was neutralized by 1 M HCl, it was concentrated to a lower volume and gradually extracted twice with hexane (water:hexane 1:1, v/v) for 6 h at 69 ◦ C, diethyl ether (1:1) for 6 h at 34 ◦ C, chloroform (water:chloroform 1:1, v/v) for 6 h at 61 ◦ C, and with similar proportions of chloroform and ethanol mixture (chloroform:ethanol 3:2, v/v) for 6 h, at 70 ◦ C. All 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 residue was dissolved in distilled water, dialyzed (MWCO 12 kDa) and freeze-dried to give a S. canadensis complex (Sc). 2.3. General methods Concentrations were performed under reduced pressure at bath temperature not exceeding 40 ◦ C. Total carbohydrate content in the samples was estimated by the phenol–sulfuric acid assay [10]. The content of phenolics was measured by Folin–Ciocalteu assay, using gallic acid as a standard, and the result was expressed as percentage of gallic acid equivalent [11]. The protein content was determined by Lowry method [12]. The content of neutral sugars in samples was estimated by gas chromatography using heptitol as an internal standard. The uronic acid content was determined by m-hydroxybiphenyl reagent [13]. Glycoconjugate samples were hydrolyzed with 2 M trifluoroacetic acid for 1 h at 120 ◦ C and the quantitative determination of the neutral sugars was carried out in the form of their alditol acetates [14], by gas chromatography on a Trace GC Ultra coupled with ITQ 900 (Thermo Scientific, USA) equipped with a Restek RT2330-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. Elemental analysis was performed with EA 1108 apparatus (FISONS Instruments, East Grinstead, UK) and protein content was calculated as well from the nitrogen content (% N × 6.25). 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 calibration

The method of chemically-induced cough was used for assessing the cough reflex [15–17]. Awaken guinea pigs were individually placed in a bodyplethysmograph box and were exposed to citric acid aerosol in concentration 0.3 M for 2 min. The citric acid aerosol was generated by a jet nebulizer (PARI jet nebulizer, Paul Ritzau, Pari-Werk GmbH, Germany, output 5 L/s, particles mass median diameter 1.2 ␮m) and delivered to the head chamber of the bodyplethysmograph. The following methods for detection of cough were used to distinguish the cough efforts from sneezing and movements: (i) the changes of the expiratory airflow interrupting the basic respiratory pattern during cough effort were measured by pneumotachograph connected to the head chamber of bodyplethysmograph, (ii) the typical cough reflex movements and sounds were recognized by two independent trained observers. Discrepancies in their evaluation were reanalyzed by experienced and educated person absent from an experiment. The number of coughs was evaluated on the basis of sudden enhancement of expiratory flow accompanied by a typical cough movement and sound during 3 min inhalations of the tussigen. The cough response was measured before administration of any agents (baseline measurement; N value in graphs) and then after their application in confirmed time intervals (60, 120 and 300 min). Minimal time difference between two measurements was 2 h to prevent cough receptors adaptation on that kind of irritation. The tested complex was administered in the doses of 25, 50 and 75 mg/kg b.w. and the peroral dose of control antitussive agent codeine (10 mg/kg b.w.) was selected according to previous experiments [15–17]. 2.5. The airway smooth muscle reactivity, in vivo conditions The airway smooth muscle reactivity was expressed as values of specific airway resistance (sRaw) calculated by Pennock [18]. This non-invasive plethysmograph technique is commonly used for evaluation of bronchoactive substances effect [19]. Conscious adult male TRIK strain guinea pigs were individually placed in a double chambers bodyplethysmograph box for laboratory animals (HSE type 855, Hugo Sachs Elektronik, Germany) consisting of head and body chambers. The nasal airflow is registered in head chamber and the thoracic airflow in body chamber. The value of specific airway resistance is proportional to phase difference between nasal and thoracic respiratory airflow, which means the bigger phase difference the higher value specific airway resistance and also more significant degree of bronchoconstriction. The values of sRaw were measured consecutively after the citric acid exposure and cough response registration during 1 min interval. Their intensity prior to administration of polysaccharides and control bronchodilating drug salbutamol was considered as

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Fig. 1. The 1 H NMR spectrum of S. canadensis (Sc) complex.

baseline (N value in graphs). The next values were measured 60, 120 and 300 min time intervals. Between the cough response recording and measurements of airways specific resistance was an interval of minimum 5 min. During intervals, fresh air was insufflating into the nasal chamber. The dose of salbutamol (10 mg/kg b.w., intraperitoneally) was selected according to our previous results [20]. 2.6. Statistics Student-t test was used for the statistical analysis of the obtained results. Data are presented as mean ± standard error of the mean (SEM). p < 0.05 was considered statistically significant. Significance of p < 0.05 and p < 0.01 is shown by one and two asterisks, respectively. 3. Results and discussion 3.1. Isolation and chemical composition of S. canadensis complex The hot alkaline extraction of air-dried S. canadensis flowers followed by neutralization and multi-step extractions with organic solvents afforded a crude plant material which was further dialyzed against distilled water and freeze-dried to give a dark brown S. canadensis isolate (Sc). Chemical analyses of Sc isolate revealed the presence of carbohydrates (43 wt%), protein (27 wt%), phenolics (12 wt%, 1 g of Sc contained about 0.7 mM of gallic acid equivalent), uronic acids (10 wt%) and inorganic material (∼8 wt%). Its carbohydrate part was composed of neutral sugars (81 wt%) and uronic acids (19 wt%). Monosaccharide analysis of carbohydrate part of Sc complex revealed the presence of rhamnose (∼23 wt%), arabinose (∼20 wt%), uronic acids (∼19 wt%), galactose (∼17 wt%) and glucose (∼14 wt%) while other sugars, i.e. xylose, mannose and fucose were found in lower amounts only (∼7 wt%). In Sc complex were observed relatively high protein and glucose contents in comparison with that of isolated from L. salicaria flowering parts [21]. Uronic acids and rhamnose contents (∼42 wt%) and the presence of galactose and arabinose residues (∼37 wt%) indicate the prevalence of pectic polysaccharide, i.e. rhamnogalacturonan and arabinogalactan in this complex, similarly as in L. salicaria conjugate, however,

the content of rhamnogalacturonan was significantly lower [21]. HPLC analysis of Sc complex showed one single peak of Mp (peak molecule mass) at 11.2 kDa. Its molecule mass was significantly lower compared with that of L. salicaria conjugate (Mp ∼ 3.6 and 212 kDa), although the same procedure for isolation of both isolates was used [21]. It seems that these differences in molecule masses could be due to different plant sources or organs used (flowers and flowering parts) for their isolations. 3.2. NMR spectroscopy of S. canadensis complex The complexity of the 1 H NMR spectral pattern (Fig. 1) of S. canadensis isolate resembled to that of L. salicaria one [21]. In the spectrum of Sc except signals of carbohydrates, observed at ı 3.0–5.5 also those due to protein at ı 3.0–0.7 and phenolics at ı 6.5–7.5 (as a broad hump of signals) were found. In the anomeric region two main signals only were found at ı 5.26 and 5.09 while other resonances in this region (at ı 5.3–4.5) were of low intensities. Besides, in the spectrum signals due to CH3 protons in the region at ı 1.26–1.37 could derive from rhamnose residues. Sugar analysis revealed five main monosaccharide components in Sc, i.e. rhamnose, arabinose, uronic acid, galactose and glucose residues. However, in the anomeric region of the 1 H spectrum two main signals were found only and the HSQC spectrum of Sc (not shown) revealed in the anomeric region four cross peaks, two derived from H1/C1 resonances of arabinose residues at ı 5.26/110.22 (indicate the terminal position of Araf residues) and 5.09/108.53 (indicate 1,5-linked Araf residues), and two at ı 4.53/104.10 and 4.48/104.37 derived from H1/C1 resonances of ␤-galactose and ␤-glucose residues, respectively [22]. No anomeric cross peaks derived from galacturonic acid or rhamnose were found in the spectrum of Sc. However, relatively intensive H6/C6 cross peaks in the range at ı 1.37–1.26/19.28–17.77 confirmed the presence of rhamnose residue in Sc complex [22]. The absence of galacturonic acid and rhamnose anomeric signals could be due to lower solubility of rhamnogalacturonan part of Sc complex and broad variabilities of their linkages. However, relatively intensive cross peaks due to CH2 groups (not involved in 1,6-linkages) were detected in the spectrum at ı 3.91/3.78/62.17 and 3.83/3.73/62.37 derived from glucose

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983

1261

1369

1800

1600

1400

1200

1000

810

881

914

1515

1557

1443

1718

Absorbance

1615

1152

1647

1047

1070

Sc

195

800

Wavenumbers (cm-1) Fig. 2. The FT-IR spectrum of S. canadensis (Sc) complex.

and galactose residues. The lower intensity cross peaks due to CH2 groups involved in linkages were observed at ı 3.89/3.81/67.92 and indicated that arabinose residues are linked by 1,5-linkages, and cross peaks at ı 4.06/3.94/73.32 indicating 1,6-linkage of ␤galactose or ␤-glucose residues in Sc. 3.3. FT-IR spectroscopy of S. canadensis complex In order to confirm the main structural components in Sc conjugate, it was analyzed by FT-IR spectroscopy. The FT-IR spectrum of Sc conjugate (carboxyl groups of uronic acids were in acidic COOH form) was recorded in the region of 1850–700 cm−1 (Fig. 2). This region reflects the presence of functional bands characteristic for carbohydrate, protein or polyphenolics moieties. The bands typical for polysaccharide moiety were found at 1152, 1070, 1047 and 983 cm−1 [23]. From Fig. 2 it is evident that vibration bands characteristic for carbohydrate moiety in Sc were overlapped while in the conjugate isolated from L. salicaria flowering parts were all above mentioned vibration bands clearly distinguishable [21]. Moreover, the distinctive band at 1718 cm−1 is due to C O resonances of COOH groups derived from galacturonic acid. Its lower intensity in the spectrum is in good agreement with the chemical analysis of uronides (∼10 wt%). As it can be seen from the spectrum, the distinctive bands in the region at 1516 −1261 cm−1 could be clearly recognized and indicate the presence of phenolics in Sc. The bands found at 1647 and 1557 cm−1 arise from protein part, i.e. from stretch vibrations of peptide bonds ␯(C O) (Amide I) and from bending ␦(N H) vibrations (Amide II), respectively). However, the band around 1650 cm−1 can be overlapped with bending ␦(O H) vibrations of water [24]. The presence of protein in Sc is supported as well with the results of elemental analysis in which about 4.4 wt% of N was determined. The FT-IR spectrum indicated the presence of three structural components in Sc complex, i.e. carbohydrates, protein and phenolics. 3.4. Antitussive activity of S. canadensis complex Many experimental trials were performed to declare the empiric biological activities of different plant isolates on the scientific base using various animal models in order to mimic conditions in human. Cough reflex was studied in various animal species, e.g. mice, cats or guinea pigs [25–27]. However, there are reservations for the use of mice model of cough given that mice do not have rapidly adapting receptors (RARs), which play an important role in the cough reflex [28]. Moreover, mice lack intraepithelial nerve endings and thus are thought to be devoid of any cough reflex [29]. In animals, cough reflex has been provoked by mechanical,

Fig. 3. The changes of number of cough efforts (NE) on administration of S. canadensis complex (Sc 25, 50 and 75 mg/kg b.w.) compared to codeine (10 mg/kg b.w.). N – represents baseline value before agent administration, t (min) – time interval (min), *p < 0.05; **p < 0.01 and ***p < 0.001 (t-test).

chemical or by electrical stimulation of the airways mucosa. Chemical or mechanical stimulus is more similar to the physiological condition of cough onset and also the experimental models generally used in man [30]. Our work examined the influence of Sc and control drug codeine on cough reflex using citric acid aerosol to induce cough in healthy guinea pigs. This method is the most generally accepted animal model, because distribution of airways receptors and arcs of cough reflex in guinea pigs and humans are very similar [31]. Peroral application of Sc resulted in reduction of number of cough efforts (NE) in a dose-dependent manner. According to results presented in Fig. 3, it is evident that the first statistically significant decrease of NE within 60 min indicated on prompt onset of the complex effect, regardless of dose used. The significance of decline lasted till measurement 2 h after application of Sc in dose 25 mg/kg b.w., while duration of cough suppression of higher tested doses (50 and 75 mg/kg b.w.) remained till 5 h after administration of Sc. From the Fig. 3 it is apparent that the suppression of experimentally induced cough by different doses of Sc was lower in comparison with the efficacy of opioid agonist codeine, tested under same conditions. Besides cough suppression, the effect of Sc complex (tested in three different doses) on airways smooth muscle reactivity was expressed as values of specific airways resistance (sRaw), which is a valuable predictor of airways smooth muscle reactivity in vivo conditions [19]. Statistically significant decrease of sRaw was recorded 5 h after perorally administered dose −75 mg/kg b.w., while the dose 50 mg/kg b.w. increased sRaw values and the lowest dose of Sc (25 mg/kg b.w.) did not show any influence on sRaw (Fig. 4). It is evident that dose required to achieve the bronchodilator effect is three times higher than dose of complex, which lead to antitussive activity. This fact is also important in terms of clinical practice. It is use as a bronchodilator drug should be considered in those diseases in which the inhibition of cough could lead to clinical deterioration, e.g. bacterial superinfection in asthmatic patients. Salbutamol, short acting beta-agonist, as a representative of classic antiathmatic drugs, administered via intraperitoneal route, resulted in statistically significant decline of sRaw values measured in two time intervals (sRaw 60, p ≤ 0.05, sRaw 120, p ≤ 0.05). In general, practices of traditional medicine vary greatly from country to country, and from region to region, as they are influenced by different factors. In many cases, their theory and application are quite different from those of conventional medicine. Water extracts or various decoctions from medicinal plants have been used in traditional medicine to treat a large scale of

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cough suppression effect of Sc. Nevertheless, the role of other mechanism inclusive interaction with specific subcellular structures, e.g. plasma membrane receptors or ion channels cannot be excluded. 4. Conclusion

Fig. 4. The values of specific airways resistance (sRaw, mL/s) registered on administration of S. canadensis complex (Sc 25, 50 and 75 mg/kg b.w.) compared to salbutamol (10 mg/kg b.w.). Statistical significance vs. salbutamol is marked by asterisks. *p < 0.05; **p < 0.01 and ***p < 0.001 (t-test), t (min) – time interval (min), N – represents baseline value before agent administration.

respiratory tract illnesses, e.g. Adhatoda vasica, Trichilia emetica, Opilia celtidifolia, etc. These traditional medicines are used in household cough and cold remedies worldwide [25,32]. It has been found that polysaccharide constituents represent the dominant part of these water extracts and their ability to suppress cough was experimentally proven [25,32]. However, by alkaline extraction of S. canadensis flowers relatively complex polyphenolic–polysaccharide–protein isolate has been recovered. Biological activity studies on this isolate confirmed as well its ability to suppress experimentally elicited cough reflex in dose-dependent manner and moderate relaxation of airways smooth muscle without provoking any notable undesirable reactions. Similarly, a significant cough suppressive effect and insignificant decrease of sRaw of polysaccharide-proteins isolated from medicinal plants Cucurbita pepo and Glycyrrhiza glabra were noticed [33,34]. Unlike Sc complex the polyphenolic–polysaccharide conjugate isolated from Lythrum salicaria showed mild antitussive effect only while its bronchodilator effect was significant [21]. It seems that polyphenolic compound could be responsible for observed bronchodilation. Similar results ware reported on polyphenolic–polysaccharide complexes isolated from wheat bran [35]. Till now, the cough suppressive effect of polysaccharides is not sufficiently explained even though research activities increased in this field. Aqueous extracts of polysaccharides are widely used in therapy for irritated gastrointestinal mucus membranes [36]. There is an increased evidence for bioadhesive effects of polysaccharides to epithelial tissue [37]. Many antitussive acting herbs are claimed to work by an antispasmodic or bronchodilator actions resulting in either bronchial muscle relaxation in vitro, or decreases in airways’ resistance in vivo [38]. The relationship between bronchomotor tone and cough and cough sensitivity has been much studied. In general, bronchoconstriction causes or enhances the sensitivity of cough, while bronchodilation does the opposite [39]. Thus, bronchodilator herbs should be also antitussive. The previous pharmacological studies on L. salicaria conjugate (polyphenolic–polysaccharide–protein) proved its antitussive and bronchodilatory effects and suggested the participation of phenolic compound on airways smooth muscle relaxation [21]. S. canadensis complex did not significantly change the airways smooth muscle reactivity in vivo. However, the highest tested dose of Sc evoked significant decline of sRaw values and its effect was longer than the effect of control bronchodilator salbutamol. Experimental results indicated that bronchodilation should partly contribute to the

Structural characterization of S. canadensis complex revealed the presence of polysaccharides, phenolics and protein moieties. The carbohydrate part was rich in rhamnose, uronic acids, arabinose and galactose residues and indicated the dominance of rhamnogalacturonan and arabinogalactan moieties in this complex. Biological activity studies, aimed at the ability of S. canadensis complex to suppress cough reflex and airways smooth muscle reactivity, indicated the dose dependent cough suppressive effect without any side toxicity. Its antitussive effect was higher than that of L. salicaria polysaccharide–phenolic–protein conjugate, however, lower than that of centrally acting codeine. Besides, the highest dose only (75 mg/kg b.w.) was able to suppress the reactivity of airways smooth muscle. Considering the influence of S. canadensis complex on sRaw values and pharmacokinetic parameters e.g. a low gastrointestinal absorption and distribution a bronchodilator effect, and other peripheral mechanisms of action are expected. Acknowledgements This research was supported by the Centre of Experimental and Clinical Respirology II. – Project co-financed from EU sources – ERDF: European Regional Development Fund, the VEGA Grant Nos. 1/0062/11, 1/0020/11 and 2/0017/11, the APVV Project No. 0125/11, Wrocław University of Technology, Wrocław, Poland, and this contribution is the result of the project implementation: Centre of excellence for Glycomics, ITMS 26240120031, supported by the Research & Development Operational Programme funded by the ERDF. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ijbiomac.2012.09.021. References [1] J.L. Walck, J.M. Baskin, C.C. Baskin, American Journal of Botany 86 (1999) 820–828. [2] E. Weber, Flora (Switzerland) 195 (2000) 123–134. [3] D. Abilasha, N. Quintana, J. Vivanco, I.J. Josh, Journal of Ecology 96 (2008) 993–1001. [4] Z. Shanshan, J.Y. Jin, Ch Xin, Applied Soil Ecology 41 (2) (2009) 215–222. [5] P. Solymosi, Acta Phytopathologica et Entomologica Hungarica 29 (1994) 361–370. [6] P. Apáti, T.Z. Kristó, E. Szöke, A. Kéry, K. Szentmihályi, P. Vinkler, Acta Horticulturae 597 (2003) 69–73. [7] I. Pawlaczyk, L. Czerchawski, W. Pilecki, E. Lamer-Zarawska, R. Gancarz, Carbohydrate Polymers 77 (2009) 568–575. [8] R. Gancarz, I. Pawlaczyk, L. Czerchawski, Patent Application PL380474 (2006). [9] I. Pawlaczyk, L. Czerchawski, J. Kanska, J. Bijak, P. Capek, A. Pliszczak-Krol, R. Gancarz, International Journal of Biological Macromolecules 131 (2010) 63–69. [10] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Analytical Biochemistry 28 (1956) 350–355. [11] V.L. Singleton, R. Orthofer, R.M. Lamuela-Raventos, Methods in Enzymology 299 (1999) 152–178. [12] O. Lowry, N. Rosebrough, L. Farr, R. Randall, Journal of Biological Chemistry 193 (1951) 265–275. [13] N. Blumenkrantz, O. Asboe-Hansen, Analytical Biochemistry 54 (1973) 484–489. [14] H.N. Englyst, J.H. Cummings, Analyst 109 (1984) 937–942. [15] J. Mokry, G. Nosál’ová, Journal of Physiology and Pharmacology 58 (5) (2007) 419–426. ˇ ˇ [16] S. Franová, G. Nosál’ová, O. Pechanová, M. Sutovská, Journal of Pharmacy and Pharmacology 59 (2007) 727–732.

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