Luigi Menghini
email: lmenghini@unich.it

Francesco Epifano

Salvatore Genovese

Maria Carla Marcotullio

Silvio Sosa

Aurelia Tubaro

Guss ) Tammaro

Anti-inflammatory activity of coumarins

Keywords: Ligusticum lucidum, coumarins, Anti-inflammatory activity Ligusticum lucidum, coumarins, anti.inflammatory activity

published or not. The documents may come

Introduction

The Apiaceae family includes many plants distributed all over the world and a great number of these species are used as traditional ethnomedical remedies or food. Previous studies on coumarin occurrence in the family of Apiaceae, one of the richest source of these compounds, showed a profile comprising a wide range of phenylpropanoids, in particular substituted coumarins, fused furano-and pyranocoumarins (Wierzchowska-Renke 1974) and finally prenyloxycoumarins [START_REF] Curini | Chemistry and biological activity of natural and synthetic prenyloxycoumarins[END_REF]). Nevertheless, little information about the chemical constituents within the genus Ligusticum is available. In fact, a previous phytochemical investigation on the whole plant of Ligusticum lucidum Mill. reported only its fatty acid composition [START_REF] Kleiman | earch for new industrial oils: XVI. Umbelliflorae -seed oils rich in petroselinic acid[END_REF]. To the best of our knowledge, no other phytochemical studies have been carried out on this plant to date and in particular no The structure of compounds 1-4 was established by spectroscopic and spectrometry methods, particularly NMR and GC-MS techniques, and confirmed by comparison with the literature data [START_REF] Goswani | Selinidin: crystal structure generated by C-H•••O, C-H••• and -interactions[END_REF][START_REF] Liu | Preparative isolation and purification of coumarins from Peucedanum praeruptorum Dunn by high-speed counter-current chromatography[END_REF], Ikeshiro 1992[START_REF] Gray | New coumarins from Coleonema album[END_REF].

Materials and methods

Ligusticum lucidum

The topical anti-inflammatory activity of these natural compounds was evaluated using the Croton oil-induced ear oedema test in mice as experimental model of inflammation, in comparison to that of the non steroidal anti-inflammatory drug (NSAID) indomethacin (Tubaro 1685).

Male CD-1 mice (28-32 g; Harlan Italy, Udine, Italy) were anaesthetised with ketamine hydrochloride (145 mg/kg, intraperitoneally; Virbac, Milan, Italy). Inflammation was induced on the right ear (surface: about 1 cm 2 ) applying 80 µg of Croton oil (Sigma Chemical Co., St. Louis, USA) dissolved in acetone. Control animals received only the

Results and discussion

We report herein the isolation, structural characterization and anti-inflammatory activity of four coumarins from aerial parts of L. lucidum subsp. cuneifolium Compounds 1-4 (Figure 1) were isolated for the first time from the genus Ligusticum.

Among them selinidin 1 is known to occur in other species of Ligusticum [START_REF] Gupta | Coumarins of Ligusticum elatum[END_REF].

Selidinin 1, (+)-Praeruptorin A 2 and visnadin 3 are common in other genera belonging to the family of Apiaceae, such Ammi [START_REF] Duarte | Effects of visnadine on rat isolated vascular smooth muscles[END_REF], Angelica [START_REF] Akihisa | Chalcones, coumarins, and flavanones from the exudate of Angelica keiskei and their chemopreventive effects[END_REF], Peucedanum [START_REF] Chen | Coumarins and antiplatelet aggregation constituents from Formosan Peucedanum japonicum[END_REF] and Selinum [START_REF] Seshadri | Absolute configuration of selinidine, vaginidin, and related compounds[END_REF]). On the contrary, (R)-(+)-7-(2',3'-epoxy-3'methylbutoxy)-coumarin 4 has been previously isolated only form three species belonging to genus Coleonema (Rutaceae), namely C. album [START_REF] Gray | New coumarins from Coleonema album[END_REF], C. aspalathoides and C. calycinum [START_REF] Gray | Coumarins from two Coleonema species[END_REF], being identified in the genus Ligusticum for the first time. Results obtained for topical anti-inflammatory activity tests revealed that each coumarin reduced the oedematous response to a certain extent at the dose of 0.3 µmol/cm 2 (Table 1).

In particular, the most active compound, (R)-(+)-7-(2',3'-epoxy-3'-methylbutoxy)-coumarin 4, induced 68 % oedema reduction, exerting an effect higher than that of the same dose of the NSAID indomethacin (58 % oedema reduction). Oedema reductions provoked by the other coumarins ranged between 22% [(+)-praeruptorin A 2] and 43 % (visnadin 3) Thus, the anti-inflammatory activity of these compounds appears to be modulated by the substituents on their aromatic ring. In particular, the presence of the 7-(2',3'-epoxy-3'methylbutoxy) group (coumarin 4) is accompanied by a high anti-inflammatory activity (68 % oedema reduction) whereas that of a saturated pyrano-fused ring (coumarins 1-3) is associated to a lower effect (22-43 % oedema reduction). The substituents on the pyrane ring seem to slightly influence the antiphlogistic effect of these compounds as well. Indeed, a significant increase of activity can be observed in visnadin 3 (43 % oedema reduction),

Experimental procedures

Air-dried and finely powdered aerial parts (100 g) of L. lucidum subsp. cuneifolium were defatted with n-hexane (2 x 200 mL) and subsequently extracted with CH 2 Cl 2 (2 x 400 mL) by exhaustive maceration to give 2.1 g of dry extract. The extract was subjected to silica gel chromatography using CH 2 Cl 2 and increasing polarity mixtures of CH 2 Cl 2 /MeOH (99.5:0.5 to 97:3) as eluents. Two main fractions were obtained (180 mg and 130 mg, respectively) and subsequently purified by preparative TLC on 20 x 20 cm silica 60 F 254 gel-coated glass sheets with CH 2 Cl 2 /MeOH 99.5:0.5 and 99:1 mixtures as mobile phases respectively, yielding compounds 1 (16 mg), 2 (11 mg), 3 (8 mg) and 4 (11 mg).

Selinidin (1): white solid; mp: 97 -98°C; IR (KBr): 1655, 1630 cm -1 ; 1 H NMR; 6 13 C NMR; 6 Anal. Calcd. for C 19 H 20 O 5 : C,69.50;H,6.14;O,24.36. Found C,69.47,H,6.12;O,24.38.

(+)-Praeruptorin A (2): white solid; mp: 152 -154°C; IR (KBr): 1680, 1650, 1635 cm -1 ; 1 H NMR; 7 13 C NMR; 7 Anal. Calcd. for C 21 H 22 O 7 : C,65.28;H,5.74;O,28.98. Found C,65.27,H,5.72;O,28.95 6,112.2,112.6,113.3,128.5,143.1,155.5,160.2,160.9;Anal. Calcd. for C 14 H 14 O 4 : C,68.28;H,5.73;O,25.99. Found C,68.26,H,5.71;O,25.98

  on the subsp. cuneifolium.

  Mill. subsp. cuneifolium (Guss.) Tammaro (Apiaceae) is a perennial herb living on rocky calcareous gravels of the Majella Mountains (Abruzzo, Italy), at altitudes ranging from 1000 to 1300 m. It's an endemic entity and to date there is no record about its use in local ethnomedical traditions. Aerial parts of L. lucidum subsp. cuneifolium were collected at flowering stage in its native "locus classicus" (Tammaro 1989), at the end of July 2005. Identification of the plant was carried out by Prof. Fernando Tammaro from the Botanical Section of the Faculty of Biotechnology of University of L'Aquila, Italy. A voucher specimen (No. 2871/1) was deposited in the Herbarium (AQUI) of University of L'Aquila, Italy.

  the others received both the irritant and the compounds under study, dissolved in acetone. After 6 hours, mice were killed to excise a plug (6 mm ) from both the treated and untreated ears. Oedema was quantified as weight difference between the two plugs. The anti-inflammatory activity was expressed as percent of oedema reduction in mice treated with the compounds under test with regard to control mice. Oedema values, expressed as mean ± standard error of the mean, were analyzed by one-way analysis of variance followed by Dunnett's test for multiple comparison of unpaired data. Significance was considered for probability levels lower than 0.05. Experiments complied with the Italian D.L. n. 116 of January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986.

  characterised by the presence of 3'--O-dihydroangeloyl and 4'--O-acetyl residues, with respect to selinidin 1 or (+)-praeruptorin A 2 (27 % or 22 % oedema reduction, respectively), characterized by an -acyl configuration in the same positions [3'--Oangeloyl (1 and 2) and 4'-O-acetyl (2)].In conclusion, these findings suggest (R)-(+)-7-(2',3'-epoxy-3'-methylbutoxy)-coumarin 4 as a potential lead compound of a novel class of anti-inflammatory agents. Studies to elaborate a synthetic route to obtain 4 in sufficient amounts to get further insights into its mechanism of action and to synthesize structural analogues of 4 for in vitro and in vivo studies on their anti-inflammatory properties are in progress in our laboratories.

  Figure 1

  .

	Visnadin (3): white solid; white solid; mp: 86 -87°C; IR (KBr): 1685, 1680, 1630 cm -1 ; 1 H
	NMR; 8 13 C NMR; 8 Anal. Calcd. for C 21 H 24 O 7 : C, 64.94; H, 6.23; O, 28.83. Found C, 64.97,
	H, 6.22; O, 28.80.

Table 1 .

 1 . Anti-inflammatory activity of coumarins 1-4 p<0.05 at the analysis of variance, as compared with controls;..**p <0.05 at the analysis of variance, as compared with compounds 1 or 2.

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Acknowledgements

Authors wish to acknowledge the financial support from Regione Abruzzo (L.R. 35/97) Project "Tutela della Biodiversita", MIUR (Rome, Italy) and Prof. Fernando Tammaro for plant identification.


Katharina Heimes

Hauk

Florian

Dr Eugen J Verspohl

Mode of action of peppermint oil and (-)-menthol with respect to 5-HT 3 receptor subtypes: binding studies, cation uptake by receptor channels and contraction of isolated rat ileum

Keywords: Peppermint oil, (-)-Menthol, Peppermint oil, (-)-menthol, 5-HT 3 receptor

Mode of action of peppermint oil and (-)-menthol with respect to 5-HT3 receptor subtypes: binding studies, cation uptake by receptor channels and contraction of isolated rat ileum

Eugen J Verspohl

Peppermint (Mentha x piperita L. (Lamiaceae)) contains 0.5 -4 % volatile oil with the main component (-)-menthol. The volatile oil should consist of 30 -55 % (-)-menthol. Peppermint has numerous effects. A tea is used against acute and chronic gastritis, enteritis, disturbances of the GI-tract and gall bladder. An antispasmodic effect of an ethanolic peppermint extract was observed: contractions of guinea pig ileum induced by acetylcholine and histamine were inhibited [START_REF] Forster | Antispasmodic Effects of Some Medicinal Plants[END_REF]. The mechanism of action of menthol was investigated [START_REF] Della Loggia | Evaluation of some pharmacological activities of a peppermint extract[END_REF]. For the antispasmodic effect of menthol partly a Cachannel modifying effect was observed (Grigoleit and Grigoleit 2005a).

(-)-Menthol, menthon and similar compounds stereoselectively modulate the ionotropic channels of GABA A -und glycin receptors (Hall et al., 2004). Since it influences gastric motility, it is used against IBS (irritable bowel syndrome) (Grigoleit and Grigoleit 2005a).

Acid-resistant preparations (e.g. Colpermin ® ) are marketed and daily doses of 3 times 0.2 -0.4 mL peppermint oil are used. It is combined in many preparations for its effect as a carminative, cholagoge or sedative. It is marketed as a prokinetic, e.g. Carminativum-Hetterich ® , which includes additional constituents. Topical (-)-menthol induces a sensation of coldness by which itching (urticaria and pruritus) and pain are reduced. This effect is mediated by the activation of TRPM8 receptors [START_REF] Peier | A TRP channel that senses cold stimuli and menthol[END_REF]. Topically applicated peppermint oil is also used against migraine and tension headache.

In the context of this paper most importantly peppermint tea is used as an antiemetic; the mechanism of this action is not fully understood. It is a remedy in pregnant women besides ginger and cannabis [START_REF] Westfall | Use of anti-emetic herbs in pregnancy: women`s choices, and the question of safety and efficacy[END_REF]. Interestingly 5-HT 3 -receptor antagonists, 5-HT 4 receptor agonists and anticholinergics are not showing a better effect than peppermint oil.

Recently the ability was shown for ginger extracts and some of its compounds to inhibit 5-HT 3 mediated [ 14 C]guanidinium influx into N1E-115 cells [START_REF] Abdel-Aziz | 5-HT 3 receptor blocking activity of arylalkanes isolated from the rhizome of Zingiber officinale[END_REF]. Based on these results, the present work describes similar experiments to elucidate the possible mode of antiemetic action of peppermint oil and (-)-menthol with respect to 5-HT 3 receptors. Since 5-HT 3 receptors are also involved in the pathogenesis of IBS, a disease already mentioned above, the outcome could also help to understand the peppermint oil effectiveness in this disease. It was therefore, aimed to investigate a possible interaction of peppermint oil and of (-)-menthol with the 5-HT 3 -receptor channel system.

MATERIALS AND METHODS

Materials

The peppermint oil was from Caelo, Hilden (Germany) and (-)-mernthol from Fluka, Buchs (Switzerland). Serotonin creatinine sulphate monohydrate (5-HT) was from Fluka, Germany, veratridine and acetylcholine from Sigma-Aldrich, Steinheim (Germany). Tropisetron was a gift from Novartis, Basel (Switzerland). SR57227A ((4-amino)-(6-chloro-2-pyridyl)L-piperidine hydrochloride) was purchased from Tocris, UK, [ 14 C]guanidinium HCl from Biotrend, Cologne (Germany) (specific activity: 55 mCi/mmol) and

[ 3 H]GR65630 (3-(5-methyl-1H-imidazol-4-yl)- 1-(1-methyl-1H-indol-3-yl)-1-propanone) from Perkin-Elmer, Boston (USA) (specific activity: 85.5 mCi/mmol).

Preparation of Solutions

The peppermint oil and (-)-menthol were dissolved in DMSO (dimethylsulfoxide) and then diluted to the required concentration with the respective assay buffer. The final concentration of DMSO in any of the experiments was less than 0.3 %. In this concentration (and in a 10fold higher one) DMSO did not show any effect on either test system used. Veratridine, which was used as a control agent to activate fast voltage gated Na + channels, was dissolved in ethanol and then diluted to the required concentration with assay buffer (final concentration of ethanol was 1 %). Ethanol was shown not to affect the veratridine-induced [ 14 C]guanidinium influx into N1E-115 cells [START_REF] Barann | Increasing effect of ethanol on 5-HT 3 receptor-mediated 14C-guanidinium influx in N1E-115 neuroblastoma cells[END_REF], and, therefore, 1 % ethanol was used in the experiments with [ 14 C]guanidinium and veratridine. All other substances were dissolved directly in the respective assay buffer.

N1E-115 Cell Culture

Mouse neuroblastoma cells of the clone N1E-115 express 5-HT 3 receptors [START_REF] Bönisch | Characterization of 5-HT 3 receptors of N1E-115 neuroblastoma cells by use of the influx of the organic cation [14C]guanidinium[END_REF]. Passage numbers 40-70 were grown in Dulbecco's modified Eagle's medium (DMEM) containing sodium pyruvate, glucose (1000 mg/L) and pyridoxine; the growth medium was supplemented with penicillin (100 I.U./mL), streptomycin (100 µg/mL) and 10 % foetal calf serum (PAA, Colbe, Germany). Cells were cultured in a humified atmosphere containing 5 % CO 2 at 37 °C in cell culture flasks (Sarstedt, Nuembrecht, Germany) and were sub-cultured twice a week.

[ 14 C]Guanidinium Influx

Three days before starting equilibrium competition binding or [ 14 C]guanidinium influx experiments N1E-115 cells were sub-cultured in 24-well cell culture clusters (2.5 x 10 4 cells in 1000 µL/well) (Biochrom, Berlin, Germany). After removal of the growth medium, cells were washed and pre-incubated for 20 min with 37 °C incubation buffer (300 µL/well) containing (in mM): HEPES 25, Tris 25, KCl 5.4, MgSO 4 x 7 H 2 O 0.98, D-glucose 5.5, choline chloride 135 and bovine serum albumin 1 mg/mL and the drugs to be tested for inhibition of [ 14 C]guanidinium influx. Pre-incubation was carried out in a humified atmosphere containing 5 % CO 2 at 37 °C. After pre-incubation, the cells were incubated for a further 2.5 min at room temperature with the same incubation buffer, which, in addition, contained 5 µM [ 14 C]guanidinium chloride, the drug to be tested, and 100 µM serotonin creatinine sulphate monohydrate (5-HT) or 300 µM veratridine. Incubation was terminated by removing the incubation buffer and rapidly washing the cells twice with ice-cold washing buffer of the following composition (mM): HEPES 25, Tris 25, KCl 5.4, MgSO 4 x 7 H 2 O 0.98, D-glucose 5.5, NaCl 135. Thereafter, the cells were dissolved in 0.5 mL 0.1 % Triton X 100 and the [ 14 C]guanidinium content of the solution was determined by liquid scintillation counting using a Beckman ß-counter, LS 6000 LL (Beckman, Harbor, USA). All experiments were carried out in duplicate or triplicate. 100 µM Tropisetron was used as a positive control. From the counts obtained, the cpms of blanks were subtracted and the resulting data expressed as a percentage of the values obtained from serotonin -or veratridine-induced [ 14 C]guanidinium influx (100 µM or 300 µM, respectively).

Equilibrium Competition Binding Experiments using N1E-115 Cells

After removal of the growth medium, cells were washed and incubated with Krebs-Ringer-HEPES buffer containing 0.4 nM of the selective 5-HT 3 receptor antagonist [ 3 H]GR65630 and the drug to be tested in a total volume of 200 µL/well for 30 min at room temperature. Nonspecific binding was determined using an excess of serotonin creatinine sulphate (10 mM).

The incubation was terminated by sucking off the incubation buffer and rapidly washing the cells twice with ice-cold Krebs-Ringer-HEPES buffer. Thereafter, the cells were dissolved in 0.5 mL 0.1 % Triton X 100 and the [ 3 H]GR65630 content of the sample was determined by liquid scintillation counting using a ß-counter. All experiments were carried out in duplicate.

Studies using isolated rat ileum

Wistar rats of either sex, each weighing 240-350 g, were killed using ether anaesthesia. The ileum was excised as rapidly as possible and flushed with warm Krebs-Henseleit solution.

Whole ileal segments, 3-4 cm in length, were suspended in a 10 mL organ bath under 1.0 g tension in carbogen-aerated Krebs-Henseleit solution, maintained at pH 7.4 and 37° C. The ileal segments were allowed to equilibrate for 30 to 60 min till a straight base line was obtained. Before testing the antagonistic effect on serotonin receptors mediated by the oil and compound under investigation, a concentration-response-curve with serotonin was obtained for each segment and a submaximal concentration evoking 50-75 % of the maximal response was chosen.

The extract or substance to be tested was then added to the organ bath and left for a contact time of 60 s, before repeating to add the chosen sub-maximal concentration of the agonist.

The ileum was washed several times after each drug addition till it returned to its previous base line. The washing time was at least 20 min for serotonin. To abolish any effects resulting from muscle tiring, 1 µM acetylcholine was added twice at the beginning and the end of each experiment and the response was recorded for control. Experiments in which the average response at the end of the experiment was less than 90 % of the one at the beginning were discarded. Tropisetron was used as a positive control.

The effect of the different drugs was expressed as follows: __response to the agonist after addition of the drug under investigation x 100 response to the agonist without previous addition of the drug under investigation.

RESULTS

[ 14 C]Guanidinium influx experiments

Experiments using the N1E-115 cell model showed that peppermint oil is able to completely inhibit 5-HT 3 receptor mediated [ 14 C]guanidinium influx into N1E-115 cells in a concentration- dependent manner (Fig. 1). The same is obvious when (-)-menthol was used (Fig. 1). 100 µM Tropisetron served as a positive control.

In this figure the concentrations of (-)-menthol (lower x-axis) are shown as equivalents to the concentrations of volatile peppermint oil. The closer the concentration-response curve of (-)menthol is located to the curve of peppermint oil, the more important would be the participation of (-)-menthol on the main effect of the volatile oil.

Veratridine experiments (with respect to validity of [ 14 C] guanidinium experiments)

To ensure that this 5-HT 3 receptor channel effect (Fig. 1) is not based on a non-selective ion channel blockade, the ability of peppermint oil and (-)-menthol to block the veratridineinduced [ 14 C]guanidinium influx into N1E-115 cells was tested and compared to its effect on the serotonin-induced [ 14 C]guanidinium influx. The peppermint oil at a concentration of 0.32 µL/mL decreases the veratridine-induced influx to the same extent as serotonin (Table 1). Its main compound (-)-menthol, however, at a concentration of 1 mM shows no inhibition of veratridine effect. At a concentration of 0.32 µL/mL peppermint oil consists of approx. 750 µM (-)-menthol. (-)-Menthol is not involved in the nonspecific ion channel blockade of peppermint oil which also holds for tropisetron (Table 1).

5-HT 3 receptor binding

Using equilibrium competition binding experiments, peppermint oil and (-)-menthol rather failed to displace the radioligand [ 3 H]GR65630 (a selective 5-HT 3 receptor antagonist; 3-(5methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone) from the serotonin binding site on 5-HT 3 receptors of N1E-115 cells (Fig. 2). Only at very high concentrations of 1 µL/mL peppermint oil was able to decrease the specific binding to 85.4 ± 4.8 % (p = 0.0322). This effect could be mediated by an interaction of a compound other than (-)-menthol in the peppermint oil or a general lipophilic effect of the volatile oil may be the reason.

Isotonic contractions of the rat ileum

Both peppermint oil and (-)-menthol were tested directly (curves starting at 0 %) and in the presence of 10 µM serotonin (curves starting at 100 %). In Fig. 3 both the concentrations of peppermint oil and of the equivalent concentration of (-)-menthol (both X-axis) are plotted versus the contraction (Y-axis). Both peppermint oil and (-)-menthol relax the smooth muscle in a concentration-dependent manner; the maximum effect is approx. -40 % (lower part of Fig. 3). Both antagonize the serotonin-induced stimulation by approx. 80 % (upper part of Fig. 3).

DISCUSSION

Peppermint oil and (-)-menthol are used for various indications in which 5-HT 3 receptors may be involved. This possible mechanism via 5-HT 3 receptors was not clearly investigated yet.

[ 14 C]Guanidinium-Influx-Assay

Experiments using the [ 14 C]guanidinium influx assay show that both peppermint oil and (-)menthol inhibit the serotonin-induced cation influx into N1E-115 cells in a concentrationdependent manner. The effect of peppermint oil at concentrations higher than 0.1 µL/mL is partly mediated (40-57 %) by (-)-menthol. Especially at low concentrations of 0.032 µL/mL volatile oil, (-)-menthol inhibits the cationic flux already by 19.8 % ± 6.5 %, whereas the oil itself does not posses an inhibitory effect at this concentration. The rightward shift of the (-)menthol concentration-response curve can be interpreted in the way that in addition to menthol other constituents are involved in the inhibitory effect of the peppermint oil.

Peppermint oil, therefore, shows a 5-HT 3 receptor channel antagonistic effect which may positively influence the disturbed motility and visceral sensitivity during symptoms of IBS and emesis and may contribute besides other mechanisms to the overall effect of peppermint oil.

2 Specificity of [ 14 C]guanidinium assay and validity of [ 14 C]guanidinium approach

It should be stressed that various compounds/extracts were already described to show a non-specific inhibition of 5-HT 3 receptors (e.g. steroids, local anaesthetics) [START_REF] Barann | Inhibition by anaesthetics of 14Cguanidinium flux through the voltage-gated sodium channel and the cation channel of the 5-HT 3 receptor of N1E-115 neuroblastoma cells[END_REF]1999). In addition the N1E-115 cell line has been used extensively to also study other compounds including NMDA receptor antagonists, barbiturates, cannabinoids, chlorpromazine etc., which were able to inhibit 5-HT 3 receptor function, albeit by different mechanisms (competitive and non-competitive antagonism, desensitisation, allosteric modulation and through interaction with sigma-2-binding sites) [START_REF] Molderings | Inhibition of 5-HT 3 receptor function by imidazolines in mouse neuroblastoma cells: potential involvement of sigma 2 binding sites[END_REF] [START_REF] Barann | Inhibition by anaesthetics of 14Cguanidinium flux through the voltage-gated sodium channel and the cation channel of the 5-HT 3 receptor of N1E-115 neuroblastoma cells[END_REF]1999). Two possible mechanisms have been proposed by Barann et al. (1993) to explain this non-specific inhibition: 1. non-specific hydrophobic interaction with membrane lipids or hydrophobic regions of the receptor proteins, in which case the potency of the compounds is correlated with their lipophilicity and 2. nonspecific inhibition of ion channels in general. Since the N1E-115 cells, being neuronal cells, are also endowed with fast voltage-gated Na + channels, which can be activated by veratridine and which are permeable to [ 14 C]guanidinium ions as well, the same cell line can be used to investigate this type of (nonspecific) interaction by comparing the potency of compounds in inhibiting cation influx through both channels (5-HT 3 receptor channels and fast voltage-gated Na + channels).

To investigate whether or not the presented data are a result of a non-specific inhibitory effect on ion channels in general, the ability of peppermint oil and (-)-menthol to inhibit veratridine-induced [ 14 C]guanidinium influx into N1E-115 cells (mediated through fast voltage-dependent Na + channels) was compared to its inhibitory effect on serotonin-induced influx (mediated through 5-HT 3 receptor channels). The peppermint oil at a concentration of 0.32 µL/mL decreases the veratridine-induced influx to the same degree as the serotonininduced cationic influx indicating a broad, nonselective effect on ion channels. Its main compound (-)-menthol, however, at a concentration of 1 mM showed no inhibition of the veratridine effect. At a concentration of 0.32 µL/mL peppermint oil contains approx. 750 µM (-)-menthol. (-)-Menthol, therefore, is not involved in the nonspecific ion channel blockade induced by peppermint oil. This could be interpreted in the way that additional compounds of peppermint oil contribute to the nonspecific blockade of ion channels and the 5-HT 3 -receptor channel.

5-HT 3 receptor binding site

The specific binding of the receptor antagonist [ 3 H]GR65630 as a radioligand is not influenced by (-)-menthol up to concentrations of 1 mM, albeit peppermint oil at an extremely high concentration of 1 µL/mL shows a small, significant inhibition of binding. Eventually other substances in peppermint oil interact with the binding site of the radioligand. It can be assumed that high oil concentrations may influence the binding of the radioligand by their high lipophilic effects.

Comparable to the volatile oil of ginger [START_REF] Abdel-Aziz | Mode of action of gingerols and shogaols on 5-HT 3 receptors: Binding studies, cation uptake by the receptor channel and contraction of isolated guinea-pig ileum[END_REF] it may be assumed that there exists a 5-HT 3 receptor channel antagonistic effect of peppermint oil by an allosteric modulating binding site. The presence of such modulatory binding sites seems to be typical for receptors belonging to the superfamily of ligand-gated ion channels like the 5-HT 3 receptor. Members of this family, which include the nicotinic acetylcholine receptor, the GABA A and the glycine receptor [START_REF] Miller | The nature of sites of general anaesthetic action[END_REF], share many structural and functional similarities. They all exist as homo-or hetero-pentamers with each subunit consisting of an extracellular N-terminal domain which encodes the ligand-binding site and four transmembrane regions. They also possess several modulatory binding sites for GABA A receptors, benzodiazepines, barbiturates, ethanol, neurosteroids, some general anaesthetics [START_REF] Mehta | An update on GABA A receptors[END_REF], and in extension for nicotinic acetylcholine receptors, substance P, serotonin, methysergide, spiperone and quinacrine [START_REF] Arias | Noncompetitive inhibition of nicotinic acetylcholine receptors by endogenous molecules[END_REF], and for anandamide and other cannabinoid receptor agonists [START_REF] Barann | Direct inhibition by cannabinoids of human 5-HT 3 A receptors: probable involvement of an allosteric modulatory site[END_REF] as well as nimodipine [START_REF] Hargreaves | Direct inhibition of 5hydroxytryptamine3 receptors by antagonists of L-type Ca2+ channels[END_REF]. This includes that those compounds inhibit 5-HT 3 receptor function without affecting the specific binding of [ 3 H]GR65630 to its binding site. The results obtained in the present work support the possible existence of modulatory binding sites on 5-HT 3 receptors.

For other ion channel receptors it was already demonstrated that (-)-menthol interacts in this way: Hall et al. ( 2004) have shown that (-)-menthol is a potent positively acting allosteric modulator on GABA A -and glycin receptors. Additionally (-)-menthol has a local anesthetic (Galeotii et al., 2001) and an antinociceptive effect [START_REF] Galeotti | Menthol: a natural analgesic compound[END_REF] which is presumably mediated according to [START_REF] Haeseler | Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol[END_REF] via blocking voltage-dependent neuronal Na + channels. (-)-Menthol activates TRPM8 channels from the family of vanilloid receptors and induces via the subsequent cationic influx a sensation of coldness [START_REF] Peier | A TRP channel that senses cold stimuli and menthol[END_REF].

Antiserotoninergic effects of peppermint oil and (-)-menthol on rat ileum

The spasmolytic and antispasmodic effect of peppermint oil was established by various investigations: for review see Grigoleit and Grigoleit (2005b). To confirm the results obtained with the N1E-115 cell system, the isolated rat ileum was used as a second assay. This preparation contains neuronally located 5-HT 3 receptors. In our experiments both the spontaneous contraction and the serotonin-induced contraction were diminished. Most of the effect was mediated by (-)-menthol. The shift of the curve to the right can only be interpreted in the way that additional components of the oil contribute to the overall effect. [START_REF] Hills | The mechanism of action of peppermint oil on gastrointestinal smooth muscle. An analysis using patch clamp electrophysiology and isolated tissue pharmacology in rabbit and guinea pig[END_REF] observed in contraction studies in the isolated taenia coli of the guinea-pig that serotonin-, histamine-and substance P-induced contractions are inhibited by peppermint oil in a non-competitive manner. Peppermint oil inhibits the serotonin-induced motoric answer much stronger than that of the other stimuli. Our experiments enlarged the outcome with respect to (-)-menthol and the 5-HT 3 -receptor.

With respect to the results from [ 14 C]guanidinium-influx experiments also a 5-HT 3 -receptor antagonistic effect could be involved in the effect of the volatile oil.

Biological relevance of the peppermint oil concentrations

In these experiments concentrations of peppermint oil of 0.01 and 1 µL/mL were used. In the IBS therapy the recommended dose is 3 times daily 0.2-0.4 mL peppermint oil. The highest dose would be 1.2 mL peppermint oil daily. Under the assumption of a distribution volume of 70 L/70 kg and a complete absorption of the compounds of the volatile oil, there will be theoretically a concentration of volatile oil of 0.017 µL/mL in the body volume which would be in the lower range of concentrations used in our experiments. Because of the acid-resistant formulation of peppermint preparations much higher concentrations may exist in the gastrointestinal tract than in plasma (body volume).

Summary and conclusion:

• Peppermint oil and its main constituent (-)-menthol inhibit the cationic influx through 5-HT 3 receptor channels in a concentration-dependent manner. (-)-Menthol is responsible for rather half of the effect of the volatile oil.

• Both do not compete with the radioligand [ 3 H]GR65630 for the 5-HT 3 binding site and, therefore, does not act directly.

• Peppermint oil shows nonspecific ion channel effect; (-)-menthol, however, does not.

• Both the volatile oil as well (-)-menthol reduce the serotonin-induced contraction of the rat ileum and posses a direct relaxant effect. The effect of the volatile oil is partly mediated by (-)-menthol; additionally there are other components involved in the overall effect of peppermint oil.

• Their action, therefore, may be mediated by binding to a modulatory site distinct from that of serotonin, an allosteric binding site not yet identified.

• This binding site, once exactly identified, might represent a valuable future target for the development of a new class of anti-emetics lacking the typical side effects caused by setrones due to their direct action on 5-HT 5 receptors.

  was used as positive control. All results were expressed as a percentage of maximum specific binding.

Fig. 1 :

 1 Fig. 1: Concentration response curves for the effect of peppermint oil and (-)-menthol and tropisetron on serotonin-induced [ 14 C]guanidinium influx into N1E-115 cells. Cells were incubated for 2.5 min at room temperature with 100 µM serotonin in the presence of various concentrations of indicated oil or compound or 100 µM tropisetron (as control). Prior to stimulation with serotonin, cells were preincubated with the same concentration of oil or (-)-menthol for 20 min. Each point of the curves represents the mean value + S.E.M. from 4-7 independent experiments expressed as percentage of maximum [ 14 C]guanidinium influx. The scaling of (-)-menthol is an equivalent to the peppermint oil concentrations.

Fig. 2 :

 2 Fig. 2: Displacement of [ 3 H]GR65630 from 5-HT 3 receptors on N1E-115 cells by different concentrations of peppermint oil and (-)-menthol. Cells were incubated for 30 min at room temperature with different concentrations of indicated oil or (-)-menthol or 100 µM tropisetron in the presence of 0.4 nM [ 3 H]GR65630. Each point of the curves represents the mean value + S.E.M. of specific binding from 4-5 independent experiments expressed as percentage of maximum [ 3 H]GR65630 binding. The scaling of (-)-menthol is an equivalent to the peppermint oil concentrations.

Fig. 3 :

 3 Fig. 3: Effect of different concentrations of peppermint oil and (-)-menthol on the isotonic contractions of the isolated rat ileum to serotonin. The compounds were left to act for 60 s before measuring the response to 10 µM serotonin. Each point of the curves represents the mean value + S.E.M. from 3-7 experiments expressed as a percentage of the response to 10 µM SR57227A.

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http://mc.manuscriptcentral.com/ptr Phytotherapy Research

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Acknowledgement: We thank Novartis Pharma, Basel, Switzerland for supplying tropisetron.

Serotonin-induced contraction after pretreatment with peppermint oil Direct efffect after pretreatment with peppermint oil Serotonin-induced contraction after pretreatment with (-)-menthol Direct effect after pretreatment with (-)-menthol Fig. 3:


Eva Calviño

Lucía Pajuelo

Jon A Ochoa

Eribe Casas

María José Luis Manjón

Cristina Tejedor

Angel Herráez

Manuel Díez

José C Diez

Eva Calviñoº

Lucía Pajueloº

Cristina Tejedorº

Angel Herráezº

CYTOTOXIC ACTION OF GANODERMA LUCIDUM ON INTERLEUKIN-3 DEPENDENT LYMPHOMA DA-1 CELLS: INVOLVEMENT OF APOPTOSIS PROTEINS

Keywords: Ganoderma, Lymphoma, Apoptosis Bax, Bcl-2, Ganoderma lucidum (Curtis)

INTRODUCTION

In recent times, a search for new therapeutic agents more effective and with more selective action on tumor cells is being conducted. The fungus Ganoderma lucidum (common names: Reishi, Lingzhi) is a well known mushroom in traditional Chinese medicine. Extracts from different parts (mycelia, spores and fruiting bodies) from Ganoderma lucidum have been used as a source of active compounds against tumor processes [START_REF] Hu | Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell[END_REF][START_REF] Liu | Antitumor activity of the sporoderm-broken germinating spores of Ganoderma lucidum[END_REF]Hong, 2004, Jiang, 2004b;Jiang, 2004a;Lu, 2004;;[START_REF] Sliva | Cellular and physiological effects of Ganoderma lucidum (Reishi)[END_REF]. Among the different compounds present in Ganoderma lucidum, nucleosides, proteins, polysaccharides, fatty acids, sterols and triterpenes have been described as molecules which could be responsible for the antitumor activity [START_REF] Yeung | Chemical and Biochemical basis of the potential anti-tumor properties of Ganoderma lucidum[END_REF]. Ganoderma lucidum extracts inhibit growth of human prostate and bladder cancer cell lines [Jiang, 2004a;Lu, 2004] and cell proliferation inducing apoptosis in human colon carcinoma and breast cancer cell lines [START_REF] Hu | Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell[END_REF]Hong, 2004;Jiang, 2004b].

Polysaccharides present in Ganoderma lucidum may be responsible of growth delay of sarcoma cells [START_REF] Cao | Antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides peptide[END_REF]. In part, their antitumor activity may be a consequence of their immunomodulatory properties [START_REF] Wilasrusmee | In vitro immunomodulatory effects of ten commonly used herbs on murine lymphocytes[END_REF][START_REF] Williamson | Synergy and other interactions in phytomedicines[END_REF]Ling, 2005]. Also, triterpenes present in Ganoderma lucidum inhibit growth of hepatoma cells by altering intracellular phosphorylation pathways [START_REF] Lin | Triterpene-enriched extracts from. Ganoderma lucidum inhibit growth of hepatoma cells via suppressing protein kinase C, activating mitogen-activated protein kinases and G2-phase cell cycle arrest[END_REF].

We have previously studied the apoptotic action of two antitumor compounds, etoposide and hydroxyurea, on DA-1 lymphoma cells [Olmos, 2005a;Olmos, 2005b] as a model for cytotoxicity by inducing apoptosis. These cells, derived from a murine lymphoma, could be sensitive to the action of antitumor and toxic compounds of therapeutical interest.

Additionally, this model shows the advantage of being modulated by interleukin 3 [START_REF] Gottlieb | Down-regulation of wildtype p53 activity interferes with apoptosis of IL-3-dependent hematopoietic cells following IL-3 withdrawal[END_REF][START_REF] Gottlieb | Relationship of sequence-specific transactivation and p53-regulated apoptosis in interleukin 3-dependent hematopoietic cells[END_REF][START_REF] Gottlieb | p53 facilitates pRb cleavage in IL-3-deprived cells: novel proapoptotic activity of p53[END_REF]]. Thus, antiproliferative and apoptotic effects of antitumor compounds can be studied in this cell model. We have demonstrated the possible F o r P e e r R e v i e w differential involvement of Bax and Bcl-2 factors in the apoptotic death of these cells after treatment with etoposide or hydroxyurea [Olmos, 2005a;Olmos, 2005b]. Thus, in the present work we have studied the cytotoxicity of different Ganoderma lucidum extracts, prepared as described by other authors [START_REF] Sliva | Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells[END_REF][START_REF] Stanley | Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-beta1 from prostate cancer cells[END_REF] on these interleukin 3 dependent lymphoma cells. Aqueous extracts and a methanol-extracted semipurified fraction from Ganoderma lucidum demonstrated ability to reduce cell viability in DA-1 cells and also induced DNA fragmentation. Changes in protein expression of apoptosis factors were also observed after treatments with these extracts showing activation of apoptosis cascades.

Some authors have previously shown the activation of intracellular kinase cascades by

Ganoderma extracts in induced neuronal differentiation in rat PC12 cells [START_REF] Cheung | Ganoderma extract activates MAP kinases and induces the neuronal differentiation of rat pheochromocytoma PC12 cells[END_REF].

Jiang, 2004b also showed inhibition of Akt/NF-kB signalling by Ganoderma lucidum in breast cancer cells. Thus, we have also focused our interest on activation and phosphorylation of intracellular kinases which are altered in other cellular model systems [Jiang, 2004b[START_REF] Cheung | Ganoderma extract activates MAP kinases and induces the neuronal differentiation of rat pheochromocytoma PC12 cells[END_REF]. Changes in expression and phosphorylation of Akt have been studied after treatment of DA-1 cells with aqueous extracts and the methanol-extracted semipurified fraction from Ganoderma lucidum.

MATERIALS AND METHODS

Cell lines

Two cell lines were used:

Mouse interleukin 3 dependent lymphoma DA-1 cells were kept at 37º C and 5% CO 2 , in Iscove´s MDM medium containing 10 % foetal calf serum, 2 mM L-glutamine and 2.5 x 10 -5 M β-mercaptoethanol.

WEHI-3B cell line was employed and applied as a source of conditioned medium containing interleukin 3, which is required for the survival of DA-1 cells. The supernatant containing the medium with secreted IL-3 was obtained by centrifugation at 1200 x g for 5 min and subsequent filtration. DA-1 cells were grown in the presence of 5% of this conditioned medium.

Source of Ganoderma lucidum

Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] strain was isolated from the fruit body tissue (context) of a wood-inhabiting filamentous parasitic fungi growing in a natural habitat on Quercus ilex roots in Cáceres (Spain). A small part of the tissue context developed Ganoderma lucidum mycelium that was cultured and subcultured in a Malt extract solid medium (MA). In order to produce fruiting bodies it was necessary to cultivate the fungus on lignocellulosic solid medium substrate [START_REF] Chang | Mushrooms: cultivation, nutritional value, medicinal effect, and environmental impact[END_REF], adequately hydrated and autoclaved. Once the lignocellulosic medium was inoculated with G. lucidum mycelia, the substrate was incubated until a proper mycelial colonization.

Subsequently, the bulk mycelium developped fruit bodies that reached maturity in 1-2 months.

Extracts of Ganoderma lucidum

Fruiting bodies of G. lucidum were resuspended in sterile water to a concentration of 50 mg/ml [START_REF] Sliva | Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells[END_REF][START_REF] Stanley | Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-beta1 from prostate cancer cells[END_REF], homogenized and then centrifuged at 14,000 rpm (in a Beckman J2.21 centrifuge using JA-20.1 rotor) for 5 min, at room temperature, to obtain a supernatant called extract 1 (E1). The pellet was resuspended in the same volume of sterile water, boiled for 5 min, sonicated for 20 seconds at 50% intensity, centrifuged at 14,000 rpm for 5 min and the supernatant obtained was called extract 2 (E2). Afterwards, these extracts were lyophylized and disolved in 1/10 the initial volume, giving ten-fold concentrated extracts.

Methanolic extracts of G. lucidum were also obtained. Fruiting bodies (2 g) were disrupted with liquid nitrogen and the resulting material was resuspended at 50 mg/ml concentration in 10% methanol and extracted three times with 10% methanol for 24h with agitation. The combined extracts were evaporated to drynewss in a rotary evaporator, and dissolved in absolute methanol. To purify this extract, it was adsorbed on 0,2g of silicagel to be applied onto a silica gel column (1x15.5 cm) and subsequently eluted with 3:1:1 butanol: acetic acid: water. Column fractions (0.2 ml) with an Rf near 0.74 were combined, the solvent was evaporated and the final sample (1.6 mg) was resuspended in 0,01 ml DMSO to obtain the extract 3 (E3).

We have used aqueous extracts E1 and E2 prepared as described by other authors. It is highly probable that sugars are present in these extracts. Several polysaccharides present in G. lucidum have been shown to have immunomodulatory and antitumor activities [Cao & Lin, 2006;[START_REF] Sliva | Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells[END_REF][START_REF] Stanley | Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-beta1 from prostate cancer cells[END_REF]. With respect to the E3 fraction, isolated in column chromatography from a methanolic extract of G. lucidum, based on the solvents used it may be possible that compounds similar to terpenoids could be present in this fraction [START_REF] Li | Ganoderic acid X, a lanostanoid triterpene, inhibits topoisomerases and induces apoptosis of cancer cells[END_REF][START_REF] Lin | Triterpene-enriched extracts from. Ganoderma lucidum inhibit growth of hepatoma cells via suppressing protein kinase C, activating mitogen-activated protein kinases and G2-phase cell cycle arrest[END_REF][START_REF] Min | Triterpenes from the spores of Ganoderma lucidum and their cytotoxicity against meth-A and LLC tumor cells[END_REF].

Cell treatments

Cultured DA-1 cells were incubated at concentrations of 0,5 x 10 6 cells/ ml at 37°C in an atmosphere with 5% CO 2 in the presence of IL-3 conditioned medium. DA-1 cells were E1 and E2 extracts and E3 fraction were added separately to 1 ml aliquots of cell culture ( 0.5

x 10 6 cell/ml) and allowed to act for 13, 19 and 24 hours.

Lyophylized E1 extract (14.9 mg dried weight) was dissolved in 0.9 ml of sterile distilled water and 60 µl were added per ml of cell culture. Lyophylized E2 extract (3 mg dried weight) was dissolved in 0.9 ml of sterile distilled water and 60 µl were added per ml of cell culture.

Fraction 3 (E3) (2.08 mg) dissolved in 13 µl of DMSO was added per ml of cell culture (0.5 x 10 6 cell/ml). DMSO (13 µl) was also added to cells as a negative control and no toxic effect was observed on cell culture (i.e. no reduction of cell viability, no DNA fragmentation, etc.).

Cell viability, permeability of treated cells to propidium iodide and flow cytometry analyses

Cell viability of DA-1 cells incubated in the presence of IL-3 and treated with Ganoderma lucidum extracts was determined by flow cytometry by measuring the impermeability to propidium iodide. 2.5 x 10 5 treated cells were collected, washed in PBS, pelleted at 1,200 rpm for 5 min, resuspended in 500 µl of PBS and stained with propidium iodide at 5 µg/ml final concentration and, analyzed by flow cytometry either in FACScan or FACScalibur (Becton Dickinson, San José, CA, USA). For each sample the acquisition was finished at 10.000 counts. Data analysis was done using the program Win-MDI (Windows Multiple Document Interface for Flow Cytometry, version 2.8, available from Scripps Research Institute, FACS Core Facility, http://facs.scripps.edu/software.html) [START_REF] Trotter | WinMDI (Windows Multiple Document Interface for Flow Cytometry)[END_REF]. Cell fragments were discriminated from the non viable cells on FSC/FL-2-H dot plots, where FL-2-H corresponds to propidium iodide fluorescence. Cell Disruptor B15 Sonifier and then centrifuged (14,000 rpm, 5 min, 4ºC) and the supernatants were analyzed by electrophoresis and blotting. Proteins (20 µg/ well) were loaded on a 10% polyacrylamide gel with SDS, electrophoretically separated and transferred to nitrocellulose membranes as described [START_REF] Towbin | Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications[END_REF]. Membranes were blocked with 5% powder milk in TTBS (50 mM Tris pH 7.2, 140 mM NaCl, 0.06 % Tween 20). Afterwards, Anti-p53 mouse monoclonal IgG antibody (Ab-1, OP03; 100 µg/ml) was purchased from Calbiochem (Oncogene Research Products, MA, USA) and diluted 1:50.

Monoclonal anti-β-actin antibodies were purchased from Sigma A5441 and used at a dilution of 1:5000.

Polyclonal caspase 3 antibodies were purchased from Cell Signalling Technology (Reference number 9662) and used at a dilution of 1:1000.

After incubation of the nitrocellulose membranes with specific antibodies overnight at 4º C the bands were revealed using goat anti-mouse horseradish peroxidase-conjugated polyclonal antibodies (1:2000) from Promega (Madison, WI) in an enhanced chemiluminescence (ECL) detection kit (Amersham). The intensities of the bands were corrected with respect to the intensity of actin band in the blot, as a control for constitutive expression, and quantified relative to the intensity of the band in control cells without any treatment, which were considered as 100%.

All the experiments were repeated three or four times. The mean and SEM were graphically represented. In order to determine the significance of the effect, Student's t test was used. The asterisk * indicates p < 0,05.

RESULTS

Cell viability analyses of DA-1 cells

Cell viability of DA-1 lymphoma cells was determined after treatment with Ganoderma lucidum extracts obtained as described in Methods. As can be observed, etoposide (Figure 1) at concentrations of 100 µM induced a decrease in cell viability, with values of 70, 58 and 51% viability in cells treated with etoposide for 13, 19 and 24 hours. Also, different extracts obtained from Ganoderma lucidum were used: Both E1 and E3 show an effective reduction in cell viability with reduction of up to 70 and 36% at 24 hours of treatment, respectively (Figure 1). On the contrary, E2 extract shows not so clear action on viability. In the case of E1 the effects are clearly visible after 19 hours. E3 showed an even more effective action than E1 extract (Figure 1). Thus, the analyses of cell viability clearly demonstrate the action of these extracts (E1 and E3) on cell membrane permeability which allows the entrance of propidium iodide.

Figure 2 shows some representative diagrams where it is possible to observe the increment in the peak corresponding to cells permeant to propidium iodide due to the alteration of the membrane (see the increased peak of higher fluorescence in FL2-H in samples treated with Ganoderma lucidum extracts). The toxic action of E3 is more clearly visible than that of E1 both at 13 and 24 hours.

Cell death induction in DA-1 cells

Previously, we have shown that either of two antitumor compounds (hydroxyurea or etoposide) induces apoptosis in DA-1 cells [Olmos, 2005a;Olmos, 2005b], as tested by permeability alteration analyses, DNA fragmentation and annexin-FITC studies. The apoptotic action of these compounds can be related to changes in cell cycle. Thus, we studied the effects on cell cycle as well as on the presence of subdiploid DNA. In figure 3 Ganoderma lucidum. As it can be observed, E1 extract showed a level of cell death induction (as tested by production of subdiploid DNA) similar to that of 100 µM etoposide. At 24 hours, the induction of subdiploid DNA was even higher in the case of E1 than etoposide. E3 fraction showed too an important induction of subdiploid DNA in DA-1 cells, an effect that is more clearly visible at 24 hours. On the contrary, E2 extract showed a very limited induction of cell death in DA-1 cells even at 24 hours. Figure 4 shows representative diagrams of DNA content of control and E1 and E3 treated DA-1 cells. The effects of E1 extract are clearly visible at 24 hours (with an important peak corresponding to subdiploid DNA) although they are already apparent at 13 hours. Methanol-extracted semipurified E3 fraction also induced DNA fragmentation (see the panels on the right side of Figure 4). In this case, it is possible to observe a prominent peak with high fluorescence in FL2-H at 13 and 24 hours, that might indicate an arrest in S phase of the cell cycle as a consequent of the treatment.

Analyses of expression of apoptosis related factors

Since some factors such as p53, Bax, Bcl-2 and Mdm-2 are involved in apoptosis processes, we have analysed the levels of these proteins (Figures 5,6, 7 and 8) by Western-blot using specific antibodies.

Figure 5 shows representative plots corresponding to the Western-blot analyses of DA-1 cells treated with E1 extract. p53 expression is reduced with respect to control in cells after 13 hours of treatment with E1, an effect which later reverts reaching 160% at 19 hours of treatment (Figures 5 and8). Bax protein increases earlier, doubling the levels of control at 13 and for 19 hours of treatment with E1. Analysis of Bcl-2 expression reveals a decrease at 13 hours. Longer treatments (19 and 24 hours) recover the level to values similar to untreated cells. Mdm2 showed also a clear reduction at 13 hours and an increase at 19 hours with restoration of the level at 24 hours to those found in control cells (Figure 5). The effect of E2 extract is shown in Figure 6. At 13 hours, treated DA-1 cells showed increments in Bax and Mdm2, while Bcl-2 showed a reduction. In the case of Mdm2, a high level of this protein was observed also at treatments for 19 hours.

Figure 7 shows the response to E3 fraction. Increments in p53, Mdm and Bcl-2 were observed after treatment for 19 hours (see also Figure 8).

We also checked variations in the expression of some other factors such as the kinases Akt and NFkB (Figures 5,6,7 and 8).

Akt was increased after 19 or 24 hours treatment with E1 extract (Figures 5 and8) while

NFkB was reduced at 13 hours of E1 treatment (Figure 5).

In the case of E2 treatments, minor variations in Akt and slight increments of pAkt were observed as time passed by (Figures 6 and8) and NFkB showed values of 80% of control at all time points (Figure 6).

After treatment of DA-1 cells with E3 fraction, the levels of Akt remain close to controls after 13 hours, with a slight increase at 19 hours and then a decrease. Its phosphorylated form p-Akt remains nearly constant through the treatment although lower than in untreated cells (61%, 60% and 65% at 13, 19 and 24 hours, respectively) (Figures 7 and8). NFkB was reduced by E3 treatment (Figure 7).

To further confirm an apoptosis effect induced by either of these extracts we quantitated uncleaved caspase 3 in DA-1 treated cells in comparison to untreated cells (Figure 9). As it can be observed, both E1 extract and semipurified E3 fraction produced a clear reduction of inactive caspase 3 in Western-blots analyses (Figure 9), and effect clearly visible at 24 hours.

To the contrary, no effect was manifested by treatment with E2. to show antitumor properties. Some authors have shown inhibition of the growth of solid tumors of sarcoma by using aqueous extracts from G. lucidum [START_REF] Sone | Structures and antitumor activities of the polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum[END_REF]. For instance, Xie, 2006 observed significant changes in the induction of cell death in breast cancer and lymphoma cells alter treatment with a polysaccharide extract from G. lucidum. In contrast, [START_REF] Sliva | Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells[END_REF]or Jiang, 2004b in breast and prostate cancer cells, [START_REF] Cao | Antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides peptide[END_REF] in endotelial cells [START_REF] Müller | Ganoderma lucidum causes apoptosis in leukemia, lymphoma and multiple myeloma cells[END_REF] in different lymphoma cell lines did not observe significant changes in cell viability alter treatment with different extracts from G. lucidum.

On the basis of previous studies [START_REF] Gottlieb | Down-regulation of wildtype p53 activity interferes with apoptosis of IL-3-dependent hematopoietic cells following IL-3 withdrawal[END_REF][START_REF] Gottlieb | Relationship of sequence-specific transactivation and p53-regulated apoptosis in interleukin 3-dependent hematopoietic cells[END_REF][START_REF] Gottlieb | p53 facilitates pRb cleavage in IL-3-deprived cells: novel proapoptotic activity of p53[END_REF]Olmos, 2005a;Olmos, 2005b], we have studied the activity of aqueous and alcoholic Ganoderma lucidum extracts on DA-1 lymphoma cells. A reduction in viability was observed after treatment with an unboiled aqueous extract (E1) that was obtained similarly to that described by others [START_REF] Sliva | Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells[END_REF][START_REF] Stanley | Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-beta1 from prostate cancer cells[END_REF] (Figs. 1 and2). Thus, aqueous extraction from Ganoderma lucidum renders some compounds with toxicity on DA-1 lymphoma cells (Figs. 1 and2). E3 fraction also showed toxic effects on lymphoma cells reducing cell viability up to 36% (Figs. 1 and2). These results are in accordance with those results described by other authors [START_REF] Lin | Cellular and molecular mechanisms of immuno-modulation by Ganoderma lucidum[END_REF] that showed inhibition of cell growth in liver cancer cells (Huh-7) after treatment with alcohol extracts from G. lucidum.

Our results shown in this work (Figures 3 and4 In the present work, we have checked whether the observed cell death may be mediated by changes in apoptosis factors. p53 is known to play an essential role in apoptosis induced by some antitumor compounds [Lowe, 1994;[START_REF] Newcomb | P53 gene mutations in lympoid diseases and their possible relevance to drug resistance[END_REF]. In some cell types, p53 is actually required for an apoptotic response [START_REF] Messmer | Attenuation of p53 expression and Bax down-regulation during phorbol ester mediated inhibition of apoptosis[END_REF][START_REF] Palacios | The role of p53 in death of IL-3-dependent cells in response to cytotoxic drugs[END_REF]. p53 has been identified in many cases as an initiator of apoptotic signals [START_REF] Merino | Molecular bases of the programmed cell death process: implications of tumor suppressor protein p53 and other proteins in the control of cell cycle. Mechanisms of apoptotic action[END_REF] being a regulator of cell cycle progression and a mediator of apoptosis in response to DNA damage. Tang, 2006 observed an increase in p53 levels in lung cancer 95-D cells alter treatment with T ganoderic acid assuming that this activation of p53 could be stimulated by DNA damage induced by this compound [START_REF] Li | Ganoderic acid X, a lanostanoid triterpene, inhibits topoisomerases and induces apoptosis of cancer cells[END_REF]. Cerebrosides isolated from G. lucidum inhibited DNA polymerases and induced DNA damage [START_REF] Mizushina | A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases[END_REF]. In the present study, treatments of DA-1 cells with E1 or E3 fraction produced increments in p53 levels after 19 and 24 hours (Figs. 567), particularly for E3. Thus, this protein might be involved on cell death induced in DA-1 lymphoma cells by G. lucidum (Figs. 5,6 and 7). In contrast, the modulation of p53 by E2 extract is quite modest. p53 regulates the expresión of Mdm2 and Bcl-2 [START_REF] Barak | Mdm2 expression is induced by wild type p53 activity[END_REF] both of which inhibit the apoptosis mediated by p53 [Chiu, 1994;[START_REF] Chen | Mdm-2 inhibits the G1 arrest and apoptosis functions of the p53 tumor suppressor protein[END_REF]]. In the case of Mdm2, the pattern of expression showed a reduction for E1 and E3 treatments at 13 hours. One common factor between E1 and E3 appears to be the up/down-regulation of p53/Mdm2 whereas this was not the case for E2 extract. p53 could also regulate the expression of bax [START_REF] Miyashita | Tumor supressor p53 is a direct transcriptional activator of the human bax gene. Cell 80: 293-299. interleukin-3-dependent lymphoma cells[END_REF], which promotes cell death [START_REF] Allen | Mechanisms controlling cellular suicide: role of Bcl-2 and caspases[END_REF]. We observe a reduction of Bcl-2 levels in DA-1 cells after treatment for 13 hours with E1 or E2 extracts or E3 fraction from Ganoderma lucidum (Figs. 5 and7). Cao, 2004 also observed a reduction of the levels of Bcl-2 and an increment of the levels of Bax, in HUVEC cells treated with polysaccharides from G. lucidum. Tang, 2006 found that Bax expression increased in lung cancer 95-D cells after treatment with T ganoderic acid maintaining the levels of Bcl-2. These authors suggested apoptosis induction by T ganoderic acid via alteration of the Bax/Bcl-2 ratio. Jiang, 2004 found that an aqueous extract containing polysaccharides and triterpenes from G. lucidum reduced the expression of Bcl-2 in prostate cancer PC-3 cells and increased the levels of Bax, correlating changes in Bax/Bcl-2 ratio with apoptosis induction [Raisova, 1998]. The increase of Bax in cells treated for 13 hours with Ganoderma lucidum E1 or E2 extract could suggest a role of Bax in the apoptosis induced by this extracts (Figures 5 and6). In the case of E3 fraction (Figure 7), Bax disminished slightly with respect to untreated cells while Bcl-2 increased, showing low values for Bax/Bcl-2 ratio which could be characteristic of resistant cells. NF-kB is a factor related to cell growth, survival, angiogenesis, adhesion and migration [START_REF] Mayo | The transcription factor NF-kappaB: control of oncogenesis and cancer therapy resistance[END_REF][START_REF] Shibata | Inhibition of NF-kappaB activity decreases the VEGF mRNA expression in MDA-MB-231 breast cancer cells[END_REF]. Jiang, 2004b studied the effect of an aqueous extract containing polysaccharides and triterpenes from G. lucidum in MDA-MB-231 breast cancer cells showing inhibition of NF-kB. A similar result has been described in PC-3 prostate cancer cells [Jiang, 2004a]. In cultures of rat cortical neurons exposed to hypoxia, Zhao, 2004 also Stanley, 2005 also showed a reduction of the level of p-Akt-Ser 473 in treatments for 24 hours with different doses of an aqueous extract from Ganoderma lucidum containing polysaccharides and triterpenes. E2 extract induced a reduction of Akt and pAkt Ser 473 at 13 hours (Figures 6 and8) with an increase of Akt at longer times. The correlation of the effects of E1, E2 extracts or E3 fraction on cell viability and DNA fragmentation in DA-1 cells (Figures 1 and3) with the involvement of Akt expression and its phosphorylation on the cytotoxic action of Ganoderma lucidum in DA-1 cells can not be yet clearly established.

  presence of IL-3 conditioned medium alone or supplemented with either Ganoderma lucidum extracts or 100 µM etoposide as a positive control for toxicity and apoptosis induction.

  Cytometric analysis of cell populations with subdiploid DNA and cell cycleApoptotic cells were counted on the basis of DNA content per cell after permeabilization of cells with NP40. After treatments with 100 µM etoposide or G. lucidum extracts, 2.5 x 10 5 cells were collected and washed with PBS. The pellet was resuspended in 475 µl of a solution containing 0.5 mg/ml RNase, 0.1% of NP-40 in PBS and incubated for 30 min in order to extract low molecular weight DNA from cell nuclei. The remnant DNA in cells was stained with 0.05 mg/ml propidium iodide, immediately before measuring the fluorescence in the cytometer. Cells with hypodiploid DNA (apoptotic cells) were distinguished from those containing diploid DNA (non apoptotic cells) on the basis of a different fluorescence intensity of propidium iodide in flow cytometer. Cell cycle progress was studied in the same samples. Histograms of the untreated cells were used to define the positions of the different peaks for the G1 and G2/M phases in the cell cycle. Western blot 5 x 10 6 cells treated with the extracts during 13, 19 or 24 hours were collected by centrifugation at 1,200 rpm for 5 min, resuspended in 200 µl of lysis buffer (50 mM Tris/HCl pH 8.0, 150 mM NaCl; 5 mM EDTA; 0.5 % NP-40; 1 mM PMSF) and incubated for 20 min at 4º C. Cells were sonicated for 20 s (duty cycle 100%, output control 50%) in a Branson

  with TTBS and incubated with antibodies specific against several apoptosis factors diluted in TTBS containing milk.All the following antibodies were obtained from Santa Cruz Biotechnology, CA, USA, and used at the dilution indicated: Anti-Bax mouse monoclonal IgG 2b (B-9; sc-7480; 200 µg/ml) 1:100; anti-Bcl-2 mouse monoclonal IgG 1 antibody (C-2; sc7382; 200 µg/ml) 1:200; anti-MDM2 (SMP14) 1:100; anti-Akt1/2/3 (H-136) sc-8312 antibodies, 1:400; anti-pAkt1/2/3 (Ser 437) sc-7985-R antibodies 1:400; anti Erk1/2 sc-154 antibodies 1:3000; anti p-Erk1/2 (Tyr 204 ) sc-7383 antibodies 1:200; anti-NFkB p50 and p105 (EA0) sc-8414 antibodies 1:200; anti-NFkB p65 (F-6) sc-8008 antibodies 1:200.

  , we show the results obtained for treatments of DA-1 cells in the presence of the extracts obtained from

  exert toxic effects on different kinds of cells and induce apoptosis in tumor cells. Ganoderma lucidum is a mushroom with components that have been claimed

  ) indicate that E1 extract and E3 fraction induced fragmentation of DNA, measured as subdiploid DNA. These data are similar to those described by Jiang, 2004a who showed programmed cell death induced by G. lucidum in prostate cancer PC-3 cells and by Cao, 2004 who described the apoptosis induction in endotelial cells from umbilical cord by a polysaccharidic extract from G. lucidum. Müller, apoptosis in lymphoma cells induced by methanolic extract from G. lucidum containing C2 ganoderic acid. Our data are also in accordance to that described by Hu, 2002 who showed inhibition of the proliferation of breast cancer MCF-7 in a timedependent way and induction of apoptosis after treatment with an alcoholic extract from G. lucidum. Our results may also be compared to those described by Tang, 2006 who studied the action of T ganoderic Acid in lung cancer 95-D cells finding induction of apoptosis in a timedependent way. Li, 2005 also observed induction of apoptosis in human cancer HuH-7 cells by triterpenes from Ganoderma amboinense, in particular by X ganoderic acid with inhibition of topoisomerases, what can be correlated to the presence of cytotoxic compounds in Ganoderma sp.

  activation of NF-kB as a consequence of a polysaccharidic extract from G. lucidum. From our results it can be inferred an inhibition of NF-κB, since E1, E2 or E3 reduced the levels of NF-kB in DA-1 cells (Figures5-7). Jiang, 2004b studied the implication of Akt on the inhibitory effect of an aqueous extract containing polysaccharides and triterpenes from G. lucidum on NF-kB in MDA-MB-231 cells finding an inhibition of Akt expression in a dose-and time-dependent way. Although they did not find a reduction in phosphorylation of Akt (pAkt) in Thr 308 , they observed a reduction in the levels of pAkt Ser 473 . Stanley, 2005 carried out a similar study in PC-3 cells, finding no changes either in Akt expression or in its phosphorylation in Thr 308 . Anyhow the levels of pAkt in Ser 473 disminished in a dose-dependent response. Our results show that independent treatments with any of the extracts or E3 fraction from G. lucidum slightly increased the levels of Akt at 19 hours. In the case of E1 extract and E3 fraction (Figures 5, 7 and 8) the levels of Akt decreased at 24 hours in accordance to Jiang, 2004a and b who carried out longer treatments (up to 96 hours). E1 extract treatment also produced a very slight reduction of pAkt in treatment for 24 hours what could correlate to that showed by Jiang, 2004 a and b.

FinallyFigure 1 .

 1 Figure 1. Cell viability of DA-1 cells grown in different culture conditions.

Figure 2 .

 2 Figure 2. Propidium iodide permeability of DA-1 cells grown in different culture

Figure 3 .

 3 Figure 3. Apoptosis of DA-1 cells induced by extracts from Ganoderma lucidum. The

Figure 4 .

 4 Figure 4. DNA fragmentation on DA-1 cells induced by treatments with different

Figure 5 .

 5 Figure 5. Expression of apoptotic factors in DA-1 cells treated with E1 aqueous extract.

Figure 6 .

 6 Figure 6. Expression analyses of DA-1 cells levels of apoptotic factors in DA-1 cells

Figure 7 .

 7 Figure 7. Expression analyses of DA-1 cells levels of apoptotic factors in DA-1 cells

Figure 8 .

 8 Figure 8. Western blot analyses of p53, Akt and p-Akt in DA-1 cells treated

Figure 9 .

 9 Figure 9. Western blot analyses of procaspase 3 levels in DA-1 cells treated

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .

 123456789 Figure 1. Cell viability of DA-1 cells grown in different culture conditions. DA-1 cells were grown in the conditions indicated in Experimental section in the presence of conditioned medium containing interleukin 3, E1, E2 or E3 represent treatments with Ganoderma lucidum extracts. Etoposide 100 µM treatment was used as a control. Different times of treatment are shown by open, filled and striped bars. All the experiments were repeated three or four times and the mean and SEM are plotted. Significance was evaluated using student´s t test. The asterisk * indicate p < 0.05. 165x90mm (600 x 600 DPI)

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Acknowledgments

This work was supported in part by Grants from UAH PI2004/025, CAM-UAH 2005-040 and CCG06-UAH/SAL-0672. E. Calviño was supported by a "Miguel de Cervantes" fellowship from Universidad de Alcalá. We also want to thank the technical assistance of Isabel Trabado (C.A.I. Medicina-Biología. Unidad de Cultivos. Universidad de Alcalá) in cytometric analyses.

Ethics

This work accomplishes all the ethical institutional or national requirements.

Conflict of interest

The authors declare no conflict of interest with this work.


Sanna Huttunen
email: sanna.huttunen@uef.fi

Marko Toivanen

Satu Arkko

Marika Ruponen

& Carina

Tikkanen-Kaukanen

Inhibition activity of wild berry juice fractions against Streptococcus 1 pneumoniae binding to human bronchial cells 2 3

Keywords: Streptococcus pneumoniae, berries, anti-adhesion, antimicrobial, Calu Streptococcus pneumoniae, berries, anti-adhesion, antimicrobial, Calu-3

Bacterial adhesion to the cell surface is a crucial step before infection can take place.

22

Inhibition of bacterial binding offers a novel preventive approach against infections. 23 Cranberry (Vaccinium macrocarpon Ait.) juice has been found to have anti-adhesive 24 activity against different bacteria. Streptococcus pneumoniae is an important pathogen 25 and the most common cause for pneumonia, meningitis, and otitis media. In this study 26 the inhibitory activity of cranberry (Vaccinium oxycoccos L.), bilberry (Vaccinium 27 myrtillus L.), and crowberry (Empetrum nigrum and Empetrum hermaphroditum L.) 28 juice fractions against pneumococcal binding was tested using human bronchial cells 29 (Calu-3) as an adhesion model. In addition, antimicrobial activity of the berry juice 30 fractions was tested. It was found that the studied berry juice fractions had anti-adhesion 31 activity and cranberry juice was the most active. The adhesion inhibition activity of 32 cranberry juice was nearly 90 % at the concentration of 8.7 mg/g of soluble solids. 33 Antimicrobial activity of the studied berry juice fractions was found to be remarkable; 34 pneumococcal growth was inhibited totally at the concentration of ~86 mg/g. Both anti-35 adhesion and antimicrobial activities were reduced after solid-phase extraction of the 36 berry juices, which may suggest molecular synergistic effects of the berry juice 37 molecules against S. pneumoniae. The findings indicate that cranberry, bilberry, and 38 crowberry juices have potential against pneumococcal infections.

INTRODUCTION 43

Streptococcus pneumoniae is an important human pathogen and the most common 44 cause for pneumonia, meningitis, and otitis media (Feldman and Anderson, 2009). It 45 is estimated that up to 60 % of the population in the world carry the pneumococcus 46 in the nasopharynx [START_REF] Mitchell | The pathogenesis of streptococcal infections: from tooth decay to 384 meningitis[END_REF]. Nasopharyngeal colonisation is mostly 47 asymptomatic and it has a key role in pneumococcal disease and spread (Bogaert et 48 al., 2004). Pneumococcal infection is the cause of death for more than 1 million 49 children in the world per year, most of them in developing countries (Kadioglu et al., 50 2008). Risk factors for pneumococcal diseases include extremes of age, 51 immunodeficiency [START_REF] Bogaert | Streptococcus pneumoniae colonisation: 340 the key to pneumococcal disease[END_REF], as well as health habits and life style 52 factors such as smoking and contact with pets (Almirall et al., 2008).

53

There is a vaccine available against S. pneumoniae. The pneumococcal vaccine is 54 targeted against the capsular polysaccharide of S. pneumoniae. This 23-valent 55 vaccine has a theoretical coverage of 90 % of disease causing serotypes. For young 56 children a 7-valent capsular polysaccharide-protein conjugate vaccine is used due to 57 weak response to polysaccharide vaccination [START_REF] Bogaert | Streptococcus pneumoniae colonisation: 340 the key to pneumococcal disease[END_REF]. High costs of the 58 conjugate vaccine reduce its use in developing countries, where the need for 59 prevention of pneumococcal diseases is most urgent [START_REF] Kadioglu | The role of Streptococcus 372 pneumoniae virulence factors in host respiratory colonization and disease[END_REF].

60

Antibiotics of different classes, e.g. β-lactams, macrolides, aminoglycosides, and 61 fluoroquinolines, are used against pneumococcal infections [START_REF] Woodhead | Guidelines for the management of adult lower 419 respiratory tract infections[END_REF]. (Toivanen et al., 2010). These berry juice FI fractions composed mostly of 115 sugars and small amounts of small size phenolics [START_REF] Toivanen | Binding of Neisseria meningitidis pili to berry 406 polyphenolic fractions[END_REF]Toivanen et 116 al., 2010). testing, the subfractionation method previously described was used (Toivanen et al., 121 2009). Briefly, the FI fraction of berry juice (3 mL) was mixed with phosphate buffer 122 (pH 7.0) (J. T. Baker, Deventer, The Netherlands) at 1:1 ratio (v/v), and passed through 123 a C-18 SPE cartridge (Waters Corp., Milford, MA; cartridges of 3 cm 3 capacity filled 124 with 500 mg of adsorbent). The cartridge was conditioned with 15 mL of methanol 125 (HPLC grade) (VWR International Ltd., Leuven, Belgium) and equilibrated with 5 mL 126 of pH 7.0 phosphate buffer. After washing with 5 mL of diluted phosphate buffer (1:8, 127 v/v, with water) and with 5 mL of water (purified on a Millipore Milli-Q apparatus, 128 Molsheim, France) the cartridge was dried under vacuum for 30 sec. Then the elution 129 with 5 mL of ethyl acetate (Laboratory-Scan, Dublin, Ireland) was performed. The 130 cartridge was dried again under vacuum for 30 sec before the next elution with 3 mL of 131 water. After that, the elution with 3 mL of 10 % aqueous methanol was discarded and 132 the ones with 3 mL of 20 % and 3 mL of 60 % aqueous methanol were collected the methodology employed [START_REF] Toivanen | Binding of Neisseria meningitidis pili to berry 406 polyphenolic fractions[END_REF][START_REF] Sun | Fractionation of red wine 400 polyphenols by solid-phase extraction and liquid chromatography[END_REF] were chosen for the Finland). An atmosphere of increased CO 2 was provided for optimal growth by 156 using a candle-extinction jar [START_REF] Weiser | Phase variation in 412 pneumococcal opacity: relationship between colonial morphology and nasopharyngeal 413 colonization[END_REF]. antimicrobial activity of plant material [START_REF] King | Comparative evaluation of methods commonly 375 used to determine antimicrobial susceptibility to plant extracts and phenolic compounds[END_REF]. In this study, a previously [START_REF] Kontiokari | 378 Randomized trial of cranberry-lingonberry juice and Lactobacillus GG drink for the 379 prevention of the urinary tract infections in women[END_REF]. The anti-adhesive constituents in cranberry include 237 fructose [START_REF] Zafriri | Inhibitory activity of cranberry 421 juice on adherence of type 1 and type P fimbriated Escherichia coli to eukaryotic cells[END_REF], high molecular weight material (> 15 kDa) (Burger et al., 238 2000), and proanthocyanidins (Foo et al., 2000a;Foo et al., 2000b). In this study we [START_REF] Barthelson | Adherence of Streptococcus 337 pneumoniae to respiratory epithelial cells is inhibited by sialylated oligosaccharides[END_REF][START_REF] Andersson | Inhibition 334 of attachment of Streptococcus pneumoniae and Haemophilus influenzae by human 335 milk and receptor oligosaccharides[END_REF] and by fractions of 246 milk [START_REF] Andersson | Inhibition 334 of attachment of Streptococcus pneumoniae and Haemophilus influenzae by human 335 milk and receptor oligosaccharides[END_REF]. In our previous microtiter well assay, pneumococcal 247 adherence to cranberry and bilberry juice FI fractions was not due to sugar molecules 248 (Toivanen et al., 2010). It is known that proanthocyanidins from cranberry juice can 249 prevent the adhesion of oligosaccharide α-Gal (1→4)β-Gal recognizing adhesins in E.

250 coli (Foo et al.,2000a) and adhesion of Neisseria meningitidis pili can be blocked by 251 milk oligosaccharides (Hakkarainen et al., 2005) or by berry fractions containing 252 phenolic components [START_REF] Toivanen | Binding of Neisseria meningitidis pili to berry 406 polyphenolic fractions[END_REF]. Here as well the phenolic compounds, µg/mL of crowberry). The best activity was found with cranberry and bilberry.

259

Cranberry juice subfraction inhibited significantly the pneumococcal adhesion at the 260 highest concentration tested (50 µg/mL); the adhesion inhibition was found to be 38 %

261

(Fig. 2A). As a comparison, potent anti-adhesion activity against E. coli was found from 262 ethyl acetate extracts of Sephadex LH20-purified cranberry proanthocyanidins at a 263 concentration of 75 µg/mL (Foo et al., 2000a). The adhesion inhibition activity of 264 bilberry with 50 µg/mL concentration was found to be 37 % and it was not significant 265 (Fig. 2B). Crowberry had the lowest activity; anti-adhesion activity was 19 % (50 266 µg/mL) (Fig. 2C).

267

Subfractions were prepared from juice FI fractions by SPE subfractionation in order to 268 extract the active phenolic compounds from FI fractions [START_REF] Toivanen | Binding of Neisseria meningitidis pili to berry 406 polyphenolic fractions[END_REF]Sun et 269 al., 2006). Adhesion inhibition activity of the cranberry subfraction was significant, but et al., 2003;[START_REF] Matsushima | Growth inhibitory action of cranberry on Helicobacter pylori[END_REF]

292

Both anti-adhesion and antimicrobial effects were minor after subfractionation when 293 compared to anti-adhesion and antimicrobial effects of the F1 berry juice fractions.

294

Our findings showed that small molecular size FI fractions from cranberry, bilberry, and 295 crowberry juices had significant effect on growth of S. pneumoniae. In addition,

296

cranberry juice FI fraction inhibited significantly binding of S. pneumoniae to human 297 bronchial cells, while the FI fractions from bilberry and crowberry juices had a lower 298 anti-adhesion effect. Compared to significant anti-adhesion activity of cranberry juice

299

FI fraction, the level of the anti-adhesion activity of the FI phenolic subfraction was 300 substantially lower.

301

Here we examined adhesion inhibition in cell culture conditions by using excess amount 302 of pneumococcal bacteria. The anti-adhesion activity was achieved with the cranberry 303 juice FI fraction at the concentration of 8.7 mg/g of soluble solids, which contain small 304 amount of polyphenols [START_REF] Toivanen | Binding of Neisseria meningitidis pili to berry 406 polyphenolic fractions[END_REF]Toivanen et al., 2010). The cranberry 305 polyphenolic subfraction had inhibitory activity at the concentration of 5 mg/100ml.

62

  However, antibiotic resistance to various classes of antibiotics has made the 63 treatment of pneumococcal infections even more difficult (Feldman and Anderson, (Vaccinium myrtillus L.), cranberry (Vaccinium oxycoccos 90 L.), and crowberry (Empetrum nigrum and hermaphroditum L.) juices against S. 91 pneumoniae. Adhesion and anti-adhesion experiments were conducted using Calu-3 92 cells (human bronchial epithelial cell line) as a model for adhesion. In addition, 93 antimicrobial activity of the studied berry juice fractions was tested. Bilberry (Vaccinium myrtillus L.), cranberry (Vaccinium oxycoccos L.), 96 and crowberry (Empetrum nigrum and Empetrum hermaphroditum L.) (65 °Brix, 97 content of soluble solids g/ 100 g of solution) juice concentrates were purchased from 98 Kiantama Ltd (Suomussalmi, Finland). Juice concentrate of apple (mix of cultivars) (70 99 °Brix) was purchased from VIP-Juicemaker Ltd (Kuopio, Finland). Additive-free juice 100 concentrates were stored at -20 °C. 101 Preparation of Berry Juice Molecular Size Fractions. Berry juice concentrates were 102 diluted with water (1:4) and fractionated into three different molecular size fractions as 103 described before (Toivanen et al., 2009). Briefly, 15 mL of berry juice was loaded onto 104 a 100 kDa cut-off Biomax Ultrafree-15 filter device (Millipore Corporation, MA, USA) 105 and centrifuged (2000g at 4 o C) until the volume was 1.5 mL. Top fraction (> 100 kDa) 106 was collected and stored at -20 o C. The filtrate was loaded onto a 10 kDa cut-off Biomax 107 Ultrafree-15 centrifugal filter device (Millipore Corporation, MA, USA) and 108 centrifuged as described above. Both the top fraction (10-100 kDa) and filtrate (< 10 109 kDa, referred here as FI) were stored at -20 o C. The content of soluble solids in the FI 110 fractions (mg/g) was analyzed by determining the °Brix values by using a refractometer 111 (ATAGO NAR-IT, Tokyo, Japan). FI fractions of cranberry, bilberry, and crowberry 112 were chosen for the present study according to our previous results, where 113 was detected to FI fractions of cranberry, bilberry, and 114 crowberry

  Extraction (SPE) Subfractionation of Berry Juice FI Fractions. In 119 order to isolate phenolic compounds from the berry juice FI fractions for activity 120

  133separately. Solvents in the subfractions were evaporated by a rotary evaporator at +30 134 ºC. Finally, the solids were reconstituted with water to the concentration of 0.5 or 1.0 135 mg/mL. Subfractions eluted with water containing phenolic components according to

  The human bronchial epithelial cell line (Calu-3) was a kind gift from 139 Professor Jouni Hirvonen (University of Helsinki, Finland), and it was used between 140 passages 28 and 45. Cells were cultured in Eagle's Minimum Essential Medium 141 (BioWhittaker, Cambrex, Verviers, Belgium) (10 % v/v) supplemented with heat 142 inactivated fetal bovine serum (Gibco, Paisley, U.K) (10-20 % v/v), L-glutamine 100x 143 (Euroclone, Pero, Italy) (1 % v/v), non-essential amino acid solution 100x (Euroclone, 144 Pero, Italy) (1 % v/v), sodium pyruvate (BioWhittaker, Cambrex, Verviers, Belgium) (1 145 % v/v), penicillin-streptomycin 100x (Euroclone, Pero, Italy) (1 % v/v) and NaHCO 3 146 (Merck, Darmstadt, Germany) (0.15 % w/v). Cells were cultivated in 75 cm 2 flasks 147 containing 10-20 mL medium and maintained in a humidified 5 % CO 2 -95 % 148 atmospheric air incubator at 37 °C. The culture medium was changed every 2-3 days 149 and cells were passaged once or twice per week at a 1:2-1:5 split ratio using trypsin-150 EDTA solution (Gibco, Paisley, UK). 151 Bacterial Strain and Culture Conditions. Streptococcus pneumoniae clinical strain 152 SB 53845 (isolated from lung) was received from Sauli Haataja (University of Turku, 153 Finland). Bacteria were cultivated at 37 °C for 18 h on sheep blood agar plates 154 (Microbiological Laboratory, Kuopio University Hospital, Finland or Labema Inc., 155 Kerava,

1578

  Isolation of S. pneumoniae. S. pneumoniae were harvested from ten plates and 158 suspended in 40 mL of sterile PBS (Gibco, Paisley, U.K.) at 0 °C. The suspension was 159 centrifuged at 2000g at 4 °C for 10 min and washed three times with cold sterile PBS. The density of the bacterial suspension was standardized to absorbance value of 0.420 at 161 A 600 . After the last washing, bacteria were suspended to prewarmed (37 °C) Eagle's 162 Minimum Essential Medium and diluted to the density of 10 8 /mL. 163 Binding of S. pneumoniae to Calu-3 Cells. A previously described method (Rytkönen 164 et al., 2004; Todoriki et al., 2001) was used with modifications. Calu-3 cells were 165 seeded on 6-well plates with the cell density of 10 5 per well 20-24 hours before the 166 experiment, and were let grown into confluence. A culture medium without antibiotics 167 was used. Diluted bacterial suspension (1.5 mL) prepared as described above was 168 incubated over the cells for one hour at 37 °C. Nonadherent bacteria were removed by 169 three washings with sterile PBS and the infected cells were detached with 0.5 mL of 170 trypsin-EDTA solution. Infected cells were diluted and appropriate dilutions were 171 plated (100 µl) on the sheep blood agar plates and bacteria were cultivated overnight at 172 37 °C in a CO 2 atmosphere. Attachment of bacteria to Calu-3 cells was determined from 173 duplicate cultures on sheep blood agar plates. Colony forming units (CFU) were 174 counted next day. 175 Inhibition of the Binding of S. pneumoniae to Calu-3 Cells. The assay was done as Microtiter Plates. Antimicrobial activities of the berry juice 188 F1 fractions and subfractions were tested against S. pneumoniae. According to 189 literature, a microtiter broth microdilution method is most preferable for testing 190

239

  found adhesion inhibition against S. pneumoniae from low molecular size fractions of 240 cranberry, bilberry and crowberry juices (< 10 kDa). We have shown that FI fractions 241 contain only low-molecular-weight molecules, mainly sugar molecules and small 242 amounts of lower molecular weight phenolic compounds (Toivanen et al 2009; 243 Toivanen et al 2010). Carbohydrate receptors mediate the adhesion of S. pneumoniae to 244 respiratory epithelial cells and pneumococcal adherence can be blocked by 245 oligosaccharides (Bartehlson

253

  such as proanthocyanidins and anthocyanins of the berry juice fractions(Toivanen et 254 al., 2009), could be the active molecules against the pneumococcal adherence.255Anti-adhesion activity of the water subfractions prepared from the berry juice FI 256 fractions was also tested (Fig.2). Adhesion inhibition achieved plateau in very low 257 concentrations of the subfractions (1 µg/mL of cranberry, 5 µg/mL of bilberry, 15

270

  it was lower than adhesion inhibition caused by the cranberry FI fraction. The detected 271 reduction in activity of the F1 subfractions against the adhesion of S. pneumoniae may 272 result from loss of a combination of different phenolic molecules and/ or combination 273 with sugars or loss of a certain type of proanthocyanidins (Foo et al., 2000b; Määttä-274 Riihinen et al., 2005) present in FI berry juice fractions and needed for the adhesion 275 inhibition. Detailed structural elucidation of the active phenolic molecules and the 276 possible combination of different molecules participating in the inhibition remains to be 277 carried out in future. 278 Antimicrobial Activity of Berry Juice FI Fractions and Subfractions against S. 279 pneumoniae. Berries and especially their polyphenols are known to inhibit the growth 280 of many human pathogens (Cavanagh

Figure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Inhibition of adhesion (mean ± SEM, n = 6) of S. pneumoniae to Calu-3

Figure 3 .

 3 Figure 3. Antimicrobial activity of FI berry juice fractions (< 10 kDa) of cranberry,

Table 1 .

 1 In vitro and in vivo anti-adhesion activity of cranberry (Vaccinium

	306

* Vaccinium oxycoccos L.; UTI, urinary tract infection

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[START_REF] Andersson | Identification of an active disaccharide unit of a glycoconjugate receptor for 332 pneumococci attaching to human pharyngeal epithelial cells[END_REF]. In order to test the anti-adhesion activity of selected wild berry

widely in vitro against several bacteria (table 1). In vivo clinical trial has also been

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used for culinary purposes. J Med Food 6: 57-61.

Contract/ grant sponsor: the European Regional Development Fund; Contract/ grant 16 sponsor: the Finnish Funding Agency for Technology and Innovation (TEKES), 17 contract/ grant number: 70031/06; Contract/ grant sponsor: the Academy of Finland