Satyajit ; Sarker
email: s.sarker@wlv.ac.uk

Afaf Geroushi

Abdurazag A Auzi

Abdalla Salem Elhwuegi

Fawzi Elzawam

Akram Elsherif

Lutfun Nahar

Satyajit D Sarker

Anti-inflammatory sesquiterpenes from the root oil of Ferula hermonis

Keywords: Ferula hermonis, Apiaceae, daucane, antiinflammatory Ferula hermonis, Apiaceae, ferutinin, teferin, teferidin, antiinflammatory, carrageenan

Ferula hermonis Boiss. (Apiaceae), commonly known as 'Shilsh-el-zallouh', 'Hashishat-al-kattira' or 'The Lebanese viagra', is a small shrub that grows abundantly on the Hermon Mountain between Syria and Lebanon. The seeds and roots of this plant have long been used in the Middle East as an aphrodisiac, and for the treatment of frigidity and impotence for both men and women. The antiinflammatory properties of three major daucane esters, ferutinin (1) teferin (2) and teferidin (3), isolated from the root oil of Ferula hermonis, were assessed by the carrageenan-induced oedema model in rats. The anti-inflammatory effect of both 1 and 2 was observed with the dose of 100 mg/kg, while compound 3 did not show any anti-inflammatory activity; conversely it produced a significant proinflammatory effect 2 and 3 h after carrageenan injection.

INTRODUCTION

______________________________________________________________________

Ferula hermonis Boiss. (Apiaceae), commonly known as 'Shilsh-el-zallouh', 'Hashishat-al-kattira' or 'The Lebanese viagra', is a small perennial shrub that grows abundantly at more than 6000 feet on the high mountain areas of northern Lebanon, and on the biblical Mount Hermon in Southern Lebanon, at the joint borders of Syria and Israel [START_REF] El-Taher | Ferula hermonis "Zallouh" and enhancing erectile function in rats: efficacy and toxicity study[END_REF]GRIN Taxonomy Database, 2010). Middle East herbalists have used the seeds and roots of this plant for centuries as a folk remedy as an aphrodisiac to treat frigidity in women, and erectile and sexual dysfunction in men by increasing blood flow to sexual organs [START_REF] El-Taher | Ferula hermonis "Zallouh" and enhancing erectile function in rats: efficacy and toxicity study[END_REF][START_REF] Lev | Ethnopharmacological survey of traditional drugs sold in the Kingdom of Jordan[END_REF][START_REF] Said | Ethnopharmacological survey of medicinal herbs in Palestine, the Golan Heights and the West Bank region[END_REF]Hadidi et al., 2003). The antimicrobial activity of the crude extract and the isolated compounds has been reported (Hadidi et al., 2003;[START_REF] Hilan | Evaluation of the antibacterial activities of Ferula hermonis BOISS[END_REF]. Previous phytochemical studies on this plant have revealed the presence of various sesquiterpenes, mainly of daucane ester type [START_REF] Galal | Daucane sesquiterpenes from Ferula hermonis[END_REF][START_REF] Lhuillier | Daucane sesquiterpenes from Ferula hermonis[END_REF][START_REF] Auzi | Feruhermonins A-C: three daucane esters, from the seeds of Ferula hermonis (Apiaceae)[END_REF]. As part of our continuing phytochemical and pharmacological studies on F. hermonis [START_REF] Auzi | Feruhermonins A-C: three daucane esters, from the seeds of Ferula hermonis (Apiaceae)[END_REF][START_REF] Elouzi | Cytotoxicity study of Ferula Hermonis Boiss[END_REF], we now report on the antiinflammatory properties of the main daucane sesquiterpenes, ferutinin (1) teferin (2) and teferidin (3), isolated from the root oil of F. hermonis, assessed by the carrageenaninduced oedema model in mice.

MATERIALS AND METHODS

______________________________________________________________________

General experimental procedures. UV spectra were obtained using a Hewlett-Packard 8453 UV/vi spectrophotometer in MeOH. IR spectra (KBr) were taken on a JASCO FTIR-4000/6000 Spectrometer. NMR spectra were recorded in CDCl 3 on a Bruker AMX 400 MHz NMR Spectrometer (400 MHz for 1 H and 100 MHz for 13 C) using the residual solvent peaks as internal standard. MS analyses were performed on a Finnigan MAT95 spectrometer. Merck Silica gel 60 H was used for vacuum liquid chromatography (VLC). Silica gel 60G plates (0.5 mm thickness) were used for PTLC separation. HMBC spectra were optimized for a longrange J H-C of 9Hz and NOESY experiment was carried out with a mixing time of 0.8s.

Plant material. The roots of Ferula hermonis Boiss. were collected from the Mount Hermon at a height of 2500 metres above the sea level close to the border between Lebanon and Syria in October 2002. The Plant material was identified at the Botany Department, Faculty of Sciences, University of Jordan, where a voucher specimen (FHS1999) has been maintained.

Extraction and isolation.

The dried and powdered roots (500 g) of F. hermonis were macerated in a 1:1 mixture of hexane and ethyl acetate (EtOAc) for 24 h. The extract was concentrated under reduced pressure and a temperature not exceeding 50 ˚C to obtain an oil (10.5 g). A portion (4.7 gm) of the oil was fractionated by liquid-liquid partition using a mixture of immiscible solvents of increasing polarity (hexane:water:methanol -4:3:1) to obtain two layers: a lipophilic portion (hexane layer) and hydrophilic portion (alcoholic layer). The solvents from these two layers were evaporated using rotary evaporator to obtain concentrated organic and aqueous extracts.

The hexane layer was further subjected to both preparative thin layer chromatography (PTLC) and column chromatography [START_REF] Auzi | Feruhermonins A-C: three daucane esters, from the seeds of Ferula hermonis (Apiaceae)[END_REF] yielding three major sesquiterpenes, ferutinin (1) [START_REF] Saidkhodzhaev | The structure of ferutinin[END_REF][START_REF] Miski | 6 Sesquiterpene alcohol esters from Ferula elaeochytris[END_REF], teferin (2) [START_REF] Khasanov | Structure of teferin -a new ester from Ferula tenuisecta roots[END_REF] and teferidin (3) [START_REF] Saidkhodzhaev | The structure of teferidin[END_REF][START_REF] Miski | 6 Sesquiterpene alcohol esters from Ferula elaeochytris[END_REF].

Animals.

Wister rats weighing between 100-230 g of both sexes were used throughout this study for both test and control groups. The animals were obtained from the local animal house of the Pharmacology Department, Faculty of Pharmacy, El-Fatah University of Medical Sciences, Tripoli, Libya. The animals were obtained one week before the experiment. They were housed in cages of 5 animals each and were maintained under controlled laboratory conditions (25ºC ± 5). Standard animal food and tap water were provided ad libitum. The animals were overnight fastened before each experiment. The studies involving rats were approved by the Ethical Review Committee, El-Fateh University, Libya, and the experiments were carried out strictly in accordance with the guidelines provided by the World Health Organization.

Carrageenan-induced rat paw oedema. Three groups of overnight fasting Wister rats weighing (100-170 g) (n=5) received separately oral doses of 100 mg/kg of compounds solublized in Tween 80 (5-10%) in water, and in 2% DMSO. A fourth group had received the vehicle only and served as a control and the fifth group was considered as the reference group where the animals were treated with aspirin (100 mg/kg) orally. Treatments were given one hour before carrageenan sub-plantar injection (1% in a volume of 0.1 mL) into the right hind paw of each rat [START_REF] Winter | Carrageenan induced edema in hind paw of the rat as an assay for aniinflammatory drug[END_REF][START_REF] Falodun | Phytochemical screening and antiinflammatory evaluation of methanolic and aqueous extracts of Euphorbia heterophylla (Euphorbiaceae)[END_REF]. The thickness of each hind paw of each animal was measured by means of a micrometer before any treatment (zero time, control values) and later one, two and three hours after carrageenan injection. The percentage increase in o was used for statistical comparison.

Statistical analysis. Data generated from the above studies were statistically analyzed by the SPSS, a computerized statistical program (version 10.0). Results were expressed as mean ± S.E. The results were also analyzed for normality of distribution (i.e. if the results obtained are parametric or non-parametric), using Kolmogorove-Smirnov test [START_REF] Fras | A new goodness of fit test for histograms regarding network traffic packet size process[END_REF]. Paired and unpaired t-Tests were used when comparing two means.

The one-way analysis of variance (ANOVA) was used for comparing more than two means of a parametric data followed by the LSD`s (Least Significant Difference) post hoc multiple comparisons to determine which population means were different. The differences between data are considered to be significant if the P<0.05 and a highly significant if P<0.01. 2) and teferidin (3), (Figure 1) on the basis of their spectroscopic data, particularly NMR spectroscopy (Table 1) and also by comparison with respective published data.

Three sesquiterpenes (1-3), isolated from the root oil of F. hermonis, were assessed for the anti-inflammatory properties using the carrageenan-induced oedema model. The anti-inflammatory effect of both ferutinin (1) and teferin (2) was observed with the dose of 100 mg/kg, while teferidin (3) did not show any anti-inflammatory activity; conversely it produced a significant pro-inflammatory effect 2 and 3 hours after carrageenan injection (Figure 2). Ferutinin (1) significantly reduced the inflammation only at the initial phase (up to 2 hours after carrageenan injection), a phase that is said to be mediated by histamine and serotonin [START_REF] Marsha-Lyn | Preliminary investigation of the anti-inflammatory properties of an aqueous extract from Morinda citrifolia (Noni)[END_REF]. Thus, it can be assumed that the anti-inflammatory effect of ferutinin (1) might have resulted from an antagonism of histamine and/or serotonin actions. It is noteworthy that the antiinflammatory effect of 1 was significantly more potent than that produced by the selected dose of the positive control, aspirin, at this phase. On the other hand, when teferin (2), dissolved in 2% DMSO, was administered, it showed significant decrease in inflammation induced by carrageenan at both the initial, the intermediate and even the start of the third phase (3-6 hours after carrageenan injection) (Figure 3). This indicated that the anti-inflammatory effect of teferin (2) might have involved all inflammatory mediators including PGs, histamine, 5hydroxytryptamine, bradykinin or nitric oxide, all of which have been reported in carrageenan-induced oedema [START_REF] Marsha-Lyn | Preliminary investigation of the anti-inflammatory properties of an aqueous extract from Morinda citrifolia (Noni)[END_REF]. Teferidin (3) showed insignificant decrease in oedema only at the start of the initial phase, while it revealed significant pro-inflammatory effect at the later phases of inflammation.

These results might be because of the structural differences in these compounds (1-3) in relation to the substitution of the benzene ring. Teferidin (3) has no substitution in its aromatic ring and lacks any anti-inflammatory effect. On the other hand, the monosubstituted benzene ring is present in ferutinin (1) and it displayed a considerable antiinflammatory activity. Teferin (2), having a disubstituted benzene ring, proved to be the most potent anti-inflammatory agent among the three compounds. It is obvious that the degree of oxygenation on the benzene ring could be directly linked to the activity of these compounds. Substitution on the benzene ring is expected to affect the degree of ionization of the compound at biological fluids. It is well known that the ionization constants play a significant role in interpretation of the mechanisms of drug action. In particular ionization may influence the selectivity of drug action and the adsorption of drugs on the surface of receptors. The findings of the present study support the traditional use of the genus Ferula in the management of pains, e.g. headache and arthritis. Anti-inflammatory effect of ferutinin (1), teferin (2) and teferidin (3) using Tween 80 (5%) at the dose (100 mg/kg) on the rat paw oedema compared with aspirin 1,2 and 3 hours after carrageenan injection.

  column chromatography and preparative thin layer chromatography of the root oil of Ferula hermonis afforded three daucane esters sesquiterpenes, which were identified as ferutinin (1) teferin (

  Figure 3.Anti-inflammatory effect of the compound teferin (2) (100 mg/kg) using

Table 1 . 1 H NMR (500 MHz, coupling constant J in Hz in parentheses) and 13 C NMR (125 MHz) data of daucane esters 1-3 a

 1

	Position	1 H NMR chemical shifts (δ Η ) in ppm	13	C NMR chemical
					shifts (δ C ) in ppm
		1	2	3	1	2	3
	1	-	-	-	46.0 46.1	46.0
	2	1.41 m; 1.62 m 1.40 m; 1.60 m	1.42 m; 1.61 m	41.5 41.6	41.6
	3	1.95 m; 2.05 m 1.95 m; 2.05 m	1.95 m; 2.05 m	31.2 31.1	31.3
	4	-	-	-	85.8 85.5	85.8
	5	2.01 d (10.5)	2.00 d (10.5)	2.01 d (10.5)	52.2 52.3	52.3
	6	5.89 m	5.90 m	5.92 m	71.1 71.2	71.1
	7	2.54 m; 2.14 m 2.54 m; 2.11 m	2.52 m; 2.14 m	49.1 48.9	49.0
	8	-	-	-	139.4 139.4	139.4
	9	5.56 bt	5.57 bt	5.55 bt	126.3 126.3	126.3
	10	2.22 bd	2.24 bd	2.28 bd	41.1 41.1	41.1
	11	1.80 m	1.80 m	1.80 m	37.1 37.1	37.0
	12	0.85 d (6.4)	0.85 d (6.4)	0.85 d (6.4)	17.4 17.5	17.5
	13	0.96 d (6.4)	0.96 d (6.4)	0.96 d (6.4)	18.5 18.5	18.3
	14	1.83 s	1.84 s	1.83 s	22.0 22.1	22.0
	15	1.11 s	1.11 s	1.12 s	20.5 20.5	20.4
	1'	-	-	-	122.8 122.6	130.5
	2'	7.91 bd (8.6)	7.55 d (1.8)	8.06 m	132.9 112.1	130.0
	3'	7.04 bd (8.6)	-	7.47 m	116.2 146.3	128.7
	4'	-	-	7.60 m	160.8 150.6	133.4
	5'	7.04 bd (8.6)	6.96 d (8.3)	7.47 m	116.2 114.5	128.7
	6'	7.91 bd (8.6)	7.58 dd (8.3,	8.06 m	132.9 124.5	130.0
			1.8)
	7'	-	-	-	168.0 166.9	166.9
	OMe	-	3.96 s	-	-	56.3	-
	a Spectra obtained in CDCl 3

http://mc.manuscriptcentral.com/ptr

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


Marko ; Toivanen
email: marko.toivanen@uef.fi

Sanna Huttunen

Seppo Lapinjoki

Carina Tikkanen-Kaukanen

Inhibition of adhesion of Neisseria meningitidis to human epithelial cells by berry juice polyphenolic fractions

Keywords: berry polyphenols, meningococci, antiadhesion, epithelial cells, antibacterial berry polyphenols, meningococci, epithelial cells, antiadhesion, 33 antibacterial

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

point-prevalence nasopharyngeal carriage rates have been estimated to range from 10 to 40 35% in young adults (Caugant and Maiden, 2009). There is no vaccine against the 41 serogroup B which together with serogroup C accounts for a large majority of cases in 42 Europe and in the Americas [START_REF] Schwartz | Global epidemiology of meningococcal 286 disease[END_REF]. Clinically the most important 43 meningococcal phenotype is encapsulated (Carbonnelle et al., 2009). In encapsulated 44 strains meningococcal adhesion to epithelial and endothelial cells occurs through type 45 IV pili (Nassif et al., 1999;[START_REF] Pujol | Interaction of Neisseria 278 meningitidis with a polarized monolayer of epithelial cells[END_REF]. The major neisserial pilus subunit, the 46 pilin [START_REF] Nassif | Interaction of pathogenic Neisseriae with nonphagocytic cells[END_REF] and the tip-located PilC1 protein (Nassif et al., 1994) are the 47 potential adhesins.

48

One strategy to avoid bacterial infections could be antiadhesive agents which may 49 prevent the attachment of infection causing bacteria to human cells. Unattached bacteria 50 can be swept away by natural cleansing mechanisms of the host. Berries could be used 51 as antiadhesives against binding of pathogenic bacteria to human epithelia. Cranberry 52 polyphenols have been shown to be capable to inhibit the attachment of Escherichia coli 53 (Gupta et al., 2007), Helicobacter pylori [START_REF] Shmuely | Susceptibility of Helicobacter pylori isolates to the 289 antiadhesion activity of a high-molecular-weight constituent of cranberry[END_REF] and Streptococcus 54 mutans [START_REF] Weiss | A high molecular mass cranberry constituent reduces mutans 300 streptococci level in saliva and inhibits in vitro adhesion to hydroxyapatite[END_REF]. Isolation of berry juice polyphenols. Berry juice polyphenols were isolated as 82 described before [START_REF] Toivanen | Screening of binding 293 activity of Streptococcus pneumoniae, Streptococcus agalactiae and Streptococcus suis 294 to berries and juices[END_REF]. Briefly, berry juice concentrates were 83 fractionated into three fractions according to their molecular size, i.e. <10 kDa fraction 84 (referred as FI), 10-100 kDa fraction (FII) and >100 kDa fraction (FIII) using 85 centrifugal filtering devices (Millipore Corp., Bedford, MA). The fractions FIII of the 86 bilberry, cranberry and crowberry juices and the fraction FII of the lingonberry juice 87 were further subfractionated using solid-phase extraction with C-18 SPE cartridge 88 (Waters Corp., Milford, MA). Subfractions were eluted with ethyl acetate, water and 89 methanol. The eluted subfractions were analysed using RP-HPLC-DAD. Subfractions 90 (here polyphenolic fractions), which were eluted with water and contained anthocyanins 91 and proanthocyanidins [START_REF] Toivanen | Screening of binding 293 activity of Streptococcus pneumoniae, Streptococcus agalactiae and Streptococcus suis 294 to berries and juices[END_REF] were used in the inhibition experiments 92 of the cell culture studies. epithelial cells as a model (Nassif et al., 1994). HEC-1B human epithelial cell line was 104 obtained from X. Nassif (INSERM U570, Paris) and used between passages 11 and 28. fraction was significant between the concentrations of 5-50 µg/mL and peaked at the 154 concentration of 10 µg/mL with the inhibition of 63% (Fig. 1a). The cranberry juice 155 polyphenolic fraction inhibited significantly meningococcal adherence to epithelial 156 cells, the inhibitory percent was 60-75% over the studied range of 1-50 µg/mL (Fig. 157 1b). Crowberry juice polyphenolic fraction showed significant inhibition from 47% to 158 63% between the concentrations of 5 µg/mL and 50 µg/mL, respectively (Fig. 1c).

159

Significant inhibition against meningococcal attachment with 56-57% inhibition over 160 the studied range was seen with the lingonberry juice polyphenolic fraction, except 48% 161 inhibition at 25 µg/mL (Fig. 1d). Inhibition of the bacterial attachment to human cells may offer a way to avoid infections 173 [START_REF] Ofek | Anti-adhesion therapy of bacterial diseases: 276 prospects and problems[END_REF]. In the present study we showed bilberry, cranberry, crowberry and 174 lingonberry juice polyphenolic fractions containing anthocyanins and proanthocyanidins 175 [START_REF] Toivanen | Screening of binding 293 activity of Streptococcus pneumoniae, Streptococcus agalactiae and Streptococcus suis 294 to berries and juices[END_REF] acting as antiadhesives. These fractions inhibited the attachment pili bound significantly to 0.39-12.5 µg/mL of these fractions [START_REF] Toivanen | Screening of binding 293 activity of Streptococcus pneumoniae, Streptococcus agalactiae and Streptococcus suis 294 to berries and juices[END_REF].

184

None of the studied berry juice polyphenolic fractions gave total inhibition for the 185 attachment of meningococci and the highest inhibition was achieved up to 75% with the 186 cranberry juice polyphenolic fraction with the concentration of 5 µg/mL. In the binding 187 assays with pili constant binding activity level was achieved at 50 µg/mL of 188 polyphenolic fractions except 1.56 µg/mL of lingonberry juice polyphenolic fraction 189 [START_REF] Toivanen | Screening of binding 293 activity of Streptococcus pneumoniae, Streptococcus agalactiae and Streptococcus suis 294 to berries and juices[END_REF].

190

The important role of type IV pili is in colonisation and infection in encapsulated N.

191 meningitidis strains (Nassif 2000). N. meningitidis has complex adhesion mechanisms 192 and carries 2 potential adhesins in its type IV pili, the tip-located PilC1 (Rudel et al., 193 1995) and pilin (PilE) subunit proteins (Nassif et al., 1994). The mechanisms of PilE 194 and PilC1 in epithelial cell binding are complicated, when encapsulated N. meningitidis 195 interact with host cells in a multistep process (Nassif et al., 1999;[START_REF] Pujol | Interaction of Neisseria 278 meningitidis with a polarized monolayer of epithelial cells[END_REF].

196

The concept of two different binding specificities located in two different components

204

Berries and their phenolics have been shown to inhibit the growth of human 205 pathogenic bacteria [START_REF] Puupponen-Pimiä | Berry phenolics selectively inhibit 282 the growth of intestinal pathogens[END_REF]. In the present study the bacterial 206 survival rates were above 90% for all samples with the exception that cranberry juice 207 polyphenolic fraction at 1 µg/mL induced 85% survival and at 50 µg/mL induced 75% 208 survival (Table 1). At these concentrations adhesion inhibition was 74% and 60%, 209 respectively. Thus, the inhibitory effect of cranberry juice polyphenolic fraction can 210 partly result from the antibacterial effect of the fraction. For the other samples the 211 inhibitory effect achieved did not result from antibacterial effect but from the inhibition 212 of bacterial attachment.

Antiadhesive agents do not act by killing the pathogens so the spread of bacterial 214 strains resistant to antiadhesive agents will likely occur at a significantly lower rate 215 compared to antibiotic-resistant strains [START_REF] Ofek | Anti-adhesion therapy of bacterial diseases: 276 prospects and problems[END_REF]

  Neisseria meningitidis is a serious public health problem 36 worldwide. The bacteria are transmitted from person to person through droplets of 37 respiratory or throat secretions. N. meningitidis primarily infects human mucosal cell 38 surfaces and colonises the nasopharyngeal epithelium. In Europe and the United States

  39

55

  In our previous studies we have shown that N. meningitidis pili, Streptococcus 56 pneumoniae bacterial cells and Streptococcus agalactiae bacteria bind to berry 57 molecular size fractions especially from Vaccinium species and they inhibit 58

  culture conditions. Neisseria meningitidis serogroup C class I 95 strain 8013 (Nassif et al., 1993) (X. Nassif, INSERM U570, Paris) was cultured at 37 96 °C in CO 2 atmosphere for 18 h on GCB agar (Oxoid Ltd., Basingstoke, England) 97 containing Kellogg's supplement I and II (Kellogg et al., 1963.). After three washings 98 with PBS (2000 × g at 4 °C for 10 min) the absorbance of the bacterial suspension was 99 adjusted to A 600 = 0,770 and the bacteria were suspended to DMEM and diluted serially 100 to 1:1000.

  Adhesion of N. meningitidis has been tested using HEC-1B human 103

  105

  Cells were cultured in cell culture dishes (Costar®, Corning Inc., Corning, NY) by 106 using 10 mL of high-glucose DMEM supplemented with 10% heat-inactivated FBS 107 and 4 mM L-glutamine without addition of antibiotics at 37 °C in a humidified 5% CO 2 / 108 95% air incubator. Medium was changed 2-3 times a week and cells were passaged 109 once or twice a week at 1:2-1:4 ratio using 1 mL of trypsin-EDTA solution. For 110 adhesion-inhibition assays HEC-1B cell monolayers were prepared in six-well tissue 111 culture plates (Costar®, Corning Inc., Corning, NY). 10 5 cells were inoculated per well 112 to obtain confluence and were cultured overnight prior to the adhesion-inhibition assay. 113 114 Adhesion-inhibition assay. Berry juice polyphenolic fractions were diluted with cell 115 culture medium to different concentrations between 0.5-50.0 µg/mL. The diluted 116 polyphenolic fractions (1.5 mL) were added on the overnight cultured HEC-1B cells 117 and incubated over the cells for one hour at 37 °C in a humidified 5% CO 2 / 95% air 118 incubator. The control wells were prepared by adding culture medium without 119 polyphenolic fractions. Bacterial suspension (1.5 mL) was added to the wells (approx. 120 10 CFU per one HEC-1B cell) and the wells were incubated for one hour at 37 °C. Non-121 adherent bacteria were removed by washing the wells twice with 3 mL of PBS. The 122 cells were detached with trypsin-EDTA solution (0.5 mL) from the bottom of the wells. 123 The detached cells were diluted 1:10 with PBS and 100 µL of the diluted cell samples 124 was plated on the GCB agar plates. The plated bacteria were cultured overnight at 37 °C 125 in CO 2 atmosphere. The amount of the attached bacteria to the epithelial cells was 126 determined by counting the bacterial colonies. The inhibitory activity of the berry juice 127 polyphenolic fractions was calculated as follows: . Microtiter broth microdilution assay (Amsterdam, 1996) 131 was chosen for testing the antibacterial activity of cranberry, bilberry, lingonberry and 132 crowberry juice polyphenolic fractions against N. meningitidis because it has been 133 described most suitable for plant extracts and phenolic compounds (King et al., 2008). 134 Bacterial suspension and berry juice polyphenolic fractions in GC broth (1.5% of 135 proteose peptone #3, 23 mM K 2 HPO 4 , 7 mM KH 2 PO 4 , 85 mM NaCl, Kellogg's 136 supplements I and II) were mixed together using the same amount of bacteria per berry 137 polyphenolic fraction as in the adhesion-inhibition assay. The mixtures were incubated 138 in wells of the microtiter plate strips (Nunc TM , Brand Product, Roskilde, Denmark) at 37 139 °C in CO 2 atmosphere for 2 hours. In control wells bacterial suspension was incubated 140 with GC broth without adding berry juice polyphenolic fractions or by adding 141 ampicillin (100 µg/mL). The antibacterial activity of the samples was measured by 142 plating the incubation mixtures on GCB agar plates. The surviving colony forming units 143 were counted next day. All the tests were made in duplicate. Statistical analysis was performed using GraphPad Prism 4.03 for Windows. 146 One-way ANOVA followed by Dunnett's Multiple Comparison Test was performed to 147 compare the amount of bacterial colonies between the control and the samples in the 148 adhesion-inhibition assay. Inhibition of adhesion was considered significant at P values assay. Inhibitory activity of the bilberry juice polyphenolic 153

  meningitidis to epithelial cells in vitro. The results were generated using an 177 adhesion assay based on human epithelial cell line HEC-1B and living encapsulated 178 meningococcal cells. The significant antiadhesion activity was achieved with relatively 179 low concentrations, 1 µg/mL of cranberry and lingonberry, and 5 µg/mL of bilberry and 180 crowberry juice polyphenolic fractions. Although the methodologies used in the 181 experiments differ, the effective concentrations of the inhibition were on the same level 182 as the concentrations in our previous microtiter well assay studies, where the isolated 183

197

  of the pilus is complicated by the fact that PilE undergoes antigenic variation(Meyer 198 and van Putten 1989), which influences epithelial cell-specific adherence(Virji et al., 199 1993). Some pilin variants are more efficient than others in enhancing bacterial 200 large bundles with enhancement of adhesiveness (Marceau et 201 al., 1995). The inhibition of the adhesion achieved in the present study by the berry 202 juice polyphenolic fractions may result either from pilin and/or PilC1 mediated 203 adhesion.

FIGURE 1 .

 1 FIGURE LEGEND

Table 1 .

 1 Nassif X,Pujol C, Morand P, Eugène E. 1999. Interactions of pathogenic Neisseria with 272 host cells. Is it possible to assemble the puzzle? Mol Microbiol 32: 1124-1132.Toivanen M, Ryynänen A, Huttunen S, Duricová J, Riihinen K, Törrönen R, Lapinjoki Survival of Neisseria meningitidis after incubation with berry juice polyphenolic fractions.

	220
	221	Therefore a single agent that has a broad spectrum of antiadhesion activity or a mix of
	222	different antiadhesive agents that target several adhesins may be necessary.
		Our results indicate that it could be possible to use berry juice polyphenols as

. Small proportion of the 216 bacteria may be mutants resistant to antiadhesive agent. In only a few people the mutant 217 propagate, while in majority of cases bacteria bind to antiadhesive agents and are swept 218 away, and hence slowing down the spread of resistant strains.

219

Bacteria may also start to express new adhesins which bind to different receptors. 223 antiadhesives against N. meningitidis. Molecules may provide multiple binding sites for

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

ACKNOWLEDGMENTS

This work was supported by the European Regional Development Fund and the Finnish 236 Funding Agency for Technology and Innovation (TEKES), grant number 70031/06 and 237 the Academy of Finland.


S Maltophilia Juraj Majtan

Lubica Majtanova

Jana Bohova

Viktor Majtan

Dr Juraj Majtan
email: juraj.majtan@savba.sk

Honeydew honey as a potent antibacterial agent in eradication of multi-drug resistant Stenotrophomonas maltophilia isolates from cancer patients

Keywords: Stenotrophomonas maltophilia, honey, multi-drug resistance, antibacterial, manuka, honeydew honey, Stenotrophomonas maltophilia, multi-drug resistance, antibacterial, manuka, honeydew

Honeydew honey as a potent antibacterial agent in eradication of multi-drug resistant Stenotrophomonas maltophilia isolates from cancer patients

Juraj Majtan, Lubica Majtanova, Jana Bohova, Viktor Majtan

Introduction

Nowadays, Stenotrophomonas maltophilia has been considered, as an important opportunistic nosocomial pathogen in immune-competent individuals associated with different diseases that include both acute and chronic infections. Especially susceptible to S. maltophilia are immunecompromised patients, for example, patients with cancer [START_REF] Khardori | Nosocomial infections due to Xanthomonas maltophilia (Pseudomonas maltophilia) in patients with cancer[END_REF][START_REF] Paez | Risk factors associated with mortality of infections caused by Stenotrophomonas maltophilia: a systematic review[END_REF]. S. maltophilia infection in hospitalized patients with cancer has significantly increased over the past two decades [START_REF] Safdar | Stenotrophomonas maltophilia: changing spectrum of a serious bacterial pathogen in patients with cancer[END_REF].

Very recent studies have revealed that S. maltophilia originating from external environment, the enteric tract or respiratory secretion may also participate in pathogenesis of persistent infections, particularly in chronic mucosal of skin surface infections [START_REF] Dowd | Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing[END_REF][START_REF] Guggenheim | Changes in bacterial isolates from burn wounds and their antibiograms: A 20-year study (1986-2005)[END_REF]Tan et al., 2009). It has been found that incidence of S. maltophilia in burn wounds markedly increased in last decade [START_REF] Guggenheim | Changes in bacterial isolates from burn wounds and their antibiograms: A 20-year study (1986-2005)[END_REF]. In addition, [START_REF] Dowd | Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing[END_REF] showed that S. maltophilia was a predominant bacterial type in biofilm associated with venous leg ulcers.

A characteristic feature of S. maltophilia is its low susceptibility to most agents currently used for treatment of human infections [START_REF] Looney | Stenotrophomonas maltophilia: an emerging opportunistic human pathogen[END_REF]. Moreover, S. maltophilia exhibits highlevel resistance to a variety of structurally unrelated antibiotics, including β-lactams, quinolones, aminoglycosides, tetracycline, disinfectants and heavy metals (Zhang et al., 2000). The treatment of choice for S. maltophilia infections is still co-trimoxazole alone, or in combination with other agents. However, isolates resistant to this agent have already been reported [START_REF] Nicodemo | Antimicrobial therapy for Stenotrophomonas maltophilia infections[END_REF]Vartivarian et al., 1994). The increasing lack of antimicrobials with activity against S. maltophilia is of concern and there is an urgent need for new drugs. Beside two well-characterized major antibacterial factors in honey, hydrogen peroxide and high osmolarity; methylglyoxal [START_REF] Adams | Isolation by HPLC and characterisation of the bioactive fraction of New Zealand manuka (Leptospermum scoparium) honey[END_REF][START_REF] Mavric | Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand[END_REF] and bee defensin-1 [START_REF] Kwakman | How honey kills bacteria[END_REF]) also act as antibacterial substances. In addition, phenolic compounds found in dark honeys are partially responsible for antibacterial activity [START_REF] Aljadi | Isolation and identification of phenolic acids in Malaysian honey with antibacterial properties[END_REF][START_REF] Estevinho | Antioxidant and antimicrobial effects of phenolic compounds extracts of Northeast Portugal honey[END_REF]Wahdan, 1998).

In our study, we investigate the antibacterial effect of Slovak honeydew honey against 20 nosocomial multi-drug S. maltophilia isolates from cancer patients and compare it with the activity of manuka honey.

Materials and Methods

Bacterial isolates

Honey samples

The antibacterial activity of Slovak honeydew honey against 20 nosocomial clinical isolates of S. maltophilia was determined by comparison to the commercially available active manuka honey imported from New Zealand (Natur's Nectar, UMF 15+). Honeydew honey used in this study is monofloral honey derived from the floral source of Abies spp. localized in Bardejov (Slovakia) and was obtained from an apiary of Mr. Jozef Volansky (Medar s.r.o., Bardejov, Slovakia).

To distinguish the effect of the antibacterial components of honey from any osmotic effect, artificial honey, a control solution with sugar content similar to that of natural honey, was also used for comparison. The artificial honey was prepared by dissolving 39 g D-fructose, 31 g Dglucose, 8 g maltose, 3 g sucrose and 19 g distilled water. All honey samples were stored in darkness at 2-5°C when not in use.

Fifty percent (w/v) stock solution of each type of honey was prepared by weighing 20 g of honey and bringing the volume up to 40 ml of Trypton soya broth (TSB). Further dilutions of stock solution of natural honeys were done to obtain honey concentrations of 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 11.25%, 10%, 8.75%, 7.5%, 6.25%, 5% and 3.75%. Dilution range of 50%, 25% and 12.5% of artificial honey was used.

Antibiotic susceptibility test

Results

Antibiotic susceptibility testing

All 20 clinical isolates were multi-drug resistant against 11 to 19 antibiotics. Among all isolates, four strains (20%) were resistant against co-trimoxazole (trimethoprim-sulfamethoxazole). The resistance against co-trimoxazole was associated with isolates resistant at least to 15 antibiotics. Ciprofloxacin resistance was found in two isolates (10%). Antimicrobial resistance profiles of all isolates are summarized in Table 1.

Determination of MIC of honey

The MIC values of the two natural honeys and artificial honey are shown in Table 2. The MICs for honeydew honey ranged from 6.25% to 17.5%, while those for active manuka honey ranged from 7.5% to 22.5%. Honeydew honey had the lower MICs than manuka honey against 16 of tested S. maltophilia isolates, where the MIC values of honeydew honey were below 10% in 14 isolates. No difference was observed in the antibacterial activity of both natural honeys against two isolates. Artificial honey inhibited the growth of all isolates at concentration higher than 25%. S. maltophilia has emerged as an important pathogen, whose eradication is often problematic due to resistance to broad spectrum of antibiotics. When the administration of co-trimoxazole for the treatment of patients with S. maltophilia infections is not possible, there may be other effective alternative treatments using ciprofloxacin as a monotherapy or in combination with other antibiotics, or ticarcillin-clavulanate alone or in combination with other agents. An increasing trend of resistance to ciprofloxacin and emerging co-trimoxazole resistance in S. maltophilia was reported in last decade [START_REF] Gales | Emerging importance of multidrug-resistant Acinetobacter species and Stenotrophomonas maltophilia as pathogens in seriously ill patients: geographic patterns, epidemiological features, and trends in the SENTRY Antimicrobial Surveillance Program (1997-1999)[END_REF][START_REF] Gabriel | Antimicrobial susceptibility and synergy studies of Stenotrophomonas maltophilia isolates from patients with cystic fibrosis[END_REF]. We found that 20% of isolates used in this study were resistant to co-trimoxazole, an agent still recommended for treatment of S. maltophilia infections. In addition, 10% of isolates exhibited the resistance to ciprofloxacin, as a second therapeutic option. Novel or re-emerged antibacterial agents are urgently needed for treatment of infections caused by multi-drug resistant S. maltophilia. Honey could be a potential natural candidate to help eradicate of S. maltophilia infections.

It has been demonstrated that medical-grade manuka honey is effective against broad range of microorganisms including multi-resistant clinical strains [START_REF] George | Antibacterial honey (Medihoney TM ): in vitro activity against clinical isolates of MRSA, VRE and other multiresistant gram-negative organisms including Pseudomonas aeruginosa[END_REF]. Until now, it has not been shown that honey is able to inhibit the growth of multi-drug clinical S. [START_REF] Adams | Isolation by HPLC and characterisation of the bioactive fraction of New Zealand manuka (Leptospermum scoparium) honey[END_REF][START_REF] Mavric | Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand[END_REF]. On the other hand, antibacterial bee defensin-1 has very recently been identified in Revamil and it substantially contributes to the antibacterial activity of honey [START_REF] Kwakman | How honey kills bacteria[END_REF]. It remains to establish whether methylglyoxal and antibacterial bee peptide are also present in other natural honeys.

Along with methylglyoxal and defensin-1, the phenolic compounds in honey have been Further detailed studies are needed to identification of components responsible for antibacterial properties of honeydew honey.

In conclusion, our results document that Slovak honeydew honey has exceptional antibacterial activity against multi-drug resistant S. maltophilia isolates from cancer patients. In addition, in the most cases it exhibited considerably stronger antibacterial activity than manuka honey and it could potentially be used as an alternative therapeutic agent against S. maltophilia infections.

  modern medicine directs attention to natural products with antimicrobial activity and their use in clinical practice. The major arguments for implementing natural products such as honey are the low cost and absence of antimicrobial resistance risk. Honey can inhibit the growth wide range o bacteria, fungi, protozoa and viruses (Blair and Carter, 2005; Molan, 1992). Manuka honey derived from the floral source of Leptospermum scoparium in New Zealand has been claimed to have particularly high antimicrobial activity against various bacterial species (Allen et al., 1991; Lin et al., 2009; Lusby et al., 2005; Molan, 2006).

5 S

 5 . maltophilia clinical isolates (n = 20) from cancer patients were received from Department of Clinical Microbiology of National Institute for Oncology (Bratislava, Slovakia). These clinical isolates were collected during the period from December 2003 to March 2004.

  determined by the disk diffusion method with 20 antibiotic disks according to the criteria of the Clinical and Laboratory Standards Institute (CLSI) (Clinical and Laboratory Standards Institute, 2005) on Mueller-Hinton (MH) agar (Oxoid, Hampshire, UK). The following antimicrobial agents (disk content indicated in parentheses) were tested: amoxicillin-clavulanic acid (20/10 µg), ampicillin (10 µg), aztreonam (30 µg), cefepime (30 µg), cefotaxime (30 µg), cefoxitin (30 µg), cefpodoxime (10 µg), ceftazidime (30 µg), ceftriaxone (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), cloxacillin (5 µg), gentamicin (10 µg), nalidixic acid (30 µg), piperacillin-tazobactam (100/10 µg), streptomycin (10 µg), tetracycline (30 µg), trimethoprim (5 µg), sulfisoxazole (250 µg) trimethoprim-sulfamethoxazole (1.25/23.75 µg), E. coli ATCC 25922 was used as the control.Minimum inhibitory concentration assayDetermination of MIC was conducted according to the recommendation of the CLSI (Clinical and Laboratory Standards Institute, 2003) with minor modification of using TSB instead of MH broth. Briefly, one bacterial colony was suspended in PBS buffer, pH 7.2 and the turbidity of suspension was adjusted to 10 8 colony forming unit (cfu)/mL and diluted with medium to final concentration of 10 7 cfu/mL. Ten µl aliquots of suspension were inoculated to each well of sterile 96-well polystyrene plates (Sarstetd, Germany). The final volume in each well was 100 µL, consisting of 90 µL of sterile medium or diluted honey and 10 µL of bacterial suspension.After 18 h of incubation at 37°C, bacterial growth inhibition was determined by monitoring the 490 nm. The MIC was defined as the lowest concentration of antibiotic inhibiting bacterial growth. All tests were performed in triplicate and were repeated three times.

  maltophilia isolates. However, Tan et al.(2009) demonstrated that Malaysian tualang honey together with manuka honey (UMF 10+) with MIC of 10% (w/v) showed good antibacterial activity against S. maltophilia clinical strain with unknown antibiotic susceptibility profile.Both Medihoney, a medical grade manuka honey and Revamil, a medical grade buckwheat honey are well-characterized honeys with pronounced healing properties. It has been documented that antibacterial activity of manuka honey directly originates from the

  considered as potential antimicrobial agents (Gomez-Caravaca et al., 2006; Taormina et al., 2001). The honey phenolic compounds composition and consequent antibacterial and antioxidant capacity of honey depends on their floral sources. In present study, dark honeydew honey derived from the floral source of Abies spp. was used to investigate its bactericidal activity against multi-drug resistant S. maltophilia isolates. According to recent studies (Estevinho et al., 2008; Kwakman et al., 2010), we speculate that several non-peroxide factors, namely defensin-1 and phenolic compounds of honeydew honey participate in its strong antibacterial properties.

  EFRD). Grant: ITMS 26240220020 -"Establishment of biotherapeutic facility and technology proposal for production and development of bio-drugs". HT, Rahman RA, Gan SH, Halim AS, Hassan SA, Sulaiman SA, Kirnpal-Kaur B. 2009. The antibacterial properties of Malaysian tualang honey against wound and enteric microorganisms in comparison to manuka honey. BMC Complement Altern Med 9: 34. 26. Taormina PJ, Niemera BA, Beuchat LR. 2001. Inhibitory activity of honey against foodborne pathogens as influenced by the presence of hydrogen peroxide adn level of antioxidant power. Int J Food Microbiol 69: 217-225. 27. Vartivarian SE, Anaissie EJ, Bodey GP, Sprigg H, Rolston KV. 1994. A changing pattern of susceptibility of Xanthomonas maltophilia to antimicrobial agents: implications for therapy.

Table 1 .

 1 Antimicrob Agents Chemother 38: 624-627. 28. Wahdan HA. 1998. Causes of the antimicrobial activity of honey. Infection 26: 26-31. 29. Zhang L, Li XZ, Poole K. 2000. Multiple antibiotic resistance in Stenotrophomonas maltophilia: involvement of a multidrug efflux system. Antimicrob Agents Chemother 44: Incidence of antibiotic resistance in 20 clinical isolates of S. maltophilia AMC ATM CPD CRO CTX FEP FOX OB TMP TZP A, ampicillin; AMC, amoxicillin-clavulanic acid; ATM; aztreonam; C, chloramphenicol; CAZ, ceftazidime; CIP, ciprofloxacin; CPD, cefpodoxime; CRO, ceftriaxone; CTX, cefotaxime; FEP, cefepime; FOX, cefoxitin; G, gentamicin; OB, cloxacillin; NAL, nalidixic acid; S, streptomycin;

	287-293.

Table 2 .

 2 Minimum inhibitory concentration (%) of different honeys towards S. maltophilia

	clinical isolates
	S. maltophilia strain		Type of honey
	(n=20)	Honeydew	Manuka (UMF 15+)	Artificial
	57/03	7.5	15	50

Page 17 of 17 http://mc.manuscriptcentral.com/ptr Phytotherapy Research

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

http://mc.manuscriptcentral.com/ptr

Acknowledgements

We thank Mrs. Janka Zatkova for her technical assistance. This work was funded by the Operational Program Research and Development and co-financed with European Fund for


Kurt Appel

Thorsten Rose

Bernd Fiebich

Thomas ; Kammler

Christine ; Hoffmann

Dr Gabriele Weiss
email: gabriele.weiss@pascoe.de

Pascoe

Gabriele ; Weiß

Modulation of the γ-aminobutric acid (GABA) system by Passiflora incarnata L

Keywords: GABA, anxiety, insomnia, passion flower, Passiflora incarnata GABA, anxiety, insomnia, passion flower, Passiflora incarnata L., Pascoflair

Introduction

The genus Passiflora consists of 500 species that are mostly found in warm and tropical regions. The genus was first discovered by Spanish invaders in 1529. This plant has been used widely in herbal medicine in West India, Mexico, The Netherlands, South America, Italy and Argentina. Passiflora incarnata is the official species and monographed in several pharmacopoeias (e.g. Passiflorae Herba Pharm. Eur.).

Passiflora incarnata contains C-glycosyl flavones such as vitexin, isovitexin, schaftoside, isoschaftoside and isovitexin-2-Oglucoside phenols, glycosyl flavonoids and cyanogenic compounds [START_REF] Wohlmuth | Pharmacognosy and chemotypes of passionflower (Passiflora incarnata L.)[END_REF]).

Only a few human studies are available focusing on its potential role as an anxiolytic (Akhondzadeh et al. 2001[START_REF] Miyasaka | Passiflora for anxiety disorder[END_REF][START_REF] Movafegh | Preoperative oral Passiflora incarnata reduces anxiety in ambulatory surgery patients: a double-blind, placebo-controlled study[END_REF]. In some experiments, it has potential effects for treatment of some diseases like anxiety, opiates withdrawal, insomnia, ADHD (attention-deficit hyperactivity disorder) and cancer (Sarris et al. 2001[START_REF] Dhawan | Passiflora: a review update[END_REF][START_REF] Patel | Passiflora incarnata Linn: A phytopharmacological review[END_REF].

Very few pharmacological studies have been undertaken on the anxiolytic/sedative activity of Passiflora incarnata; most of these investigations have been carried out with different Passiflora species, such as P. edulis [START_REF] Deng | Anxiolytic and sedative activities of Passiflora edulis f. flavicarpa[END_REF][START_REF] Barbosa | The aqueous extracts of Passiflora alata and Passiflora edulis reduce anxiety-related behaviors without affecting memory process in rats[END_REF], P. alata [START_REF] Barbosa | The aqueous extracts of Passiflora alata and Passiflora edulis reduce anxiety-related behaviors without affecting memory process in rats[END_REF], P. coerulea [START_REF] Reginatto | Evaluation of anxiolytic activity of spray dried powders of two South Brazilian Passiflora species[END_REF], or P. quadrangularis [START_REF] De Castro | Possible anxiolytic effect of two extracts of Passiflora quadrangularis L. in experimental models[END_REF]) and with insufficient phytochemical characterization of the extracts.

The anxiolytic activity of an extract from Passiflora actinia has been shown to be exerted via the GABA receptor [START_REF] Lolli | Possible involvement of GABA A-benzodiazepine receptor in the anxiolytic-like effect induced by Passiflora actinia extracts in mice[END_REF]), but the much more widely -used Passiflora incarnata has not been investigated in this context. Although there are some reports speculating regarding the effects of Passiflora incarnata on the GABA system, investigations concerning the mechanism are still lacking.

Therefore, we hypothesized that the mode of action of the dry extract prepared from the flowers of Passiflora incarnata which is the sole active ingredient of the proprietary herbal drug Pascoflair ® 425mg might be modulation via the GABA-system.

Overall, we show that this dry extract of Passiflora incarnata inhibited the binding of

Material and Methods

Botanical drug preparation

Passiflora incarnata dry extract (Passiflorae Herba Ph. Eur., DER = 5-7:1, extraction solvent: 50% ethanol (V/V)) was provided by Pascoe pharmazeutische Präparate GmbH (Giessen, Germany). This dry extract is the sole active ingredient of the proprietary herbal drug Pascoflair ® 425 mg.

Dried extract was used in the pharmacological studies and was dissolved in a measured small amount of dimethlsulfoxide (DMSO) and diluted in the application solution immediately prior to testing. All test doses contained the same quantity of DMSO as did all control test solutions.

Chemicals used

[Butyryl-2,3-3 H]-SR 95531, [N-Methyl-3 H]-Ro-15-1788, [7,9-3 H]-Ro-15-4513 and GTP[γ-35 S] were from PerkinElmer (Massachusetts, USA). [ 3 H]CGP 54626 and SR 95531 hydrobromide were purchased from Biotrend Chemikalien GmbH (Cologne, Germany). Diazepam-ratiopharm  was from Ratiopharm GmbH (Ulm, Germany).

GABA (γ-amino-n-butyric acid), (±) Baclofen, GABase were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Vigabatrin was from Sanofi-Aventis (Frankfurt, Germany). Binding experiments (GABA-/Benzodiazepine-/Ethanol-site, GABA B -receptor) were performed as described [START_REF] Heaulme | Characterization of the binding of [3H]SR 95531, a GABAA antagonist, to rat brain membranes[END_REF][START_REF] Mehta | Characterization of a benzodiazepine receptor site with exceptionally high affinity for Ro 15-4513 in the rat CNS[END_REF][START_REF] Asay | Characterization of the binding of [3H]CGP54626 to GABAB receptors in the male bullfrog (Rana catesbeiana)[END_REF].

The assay was terminated by transfer of the samples on GF/C filter plates, presoaked with 0.1% polyethyleneimine. Filters were washed four times with 200 µl of ice-cold 50 mM Tris-HCl pH 7.4 and filter-bound radioactivity was determined by a microplate reader (Microbeta, Wallac, Finnland).

Test compound data are presented specific ligand binding to the receptor. Specific binding is defined as the difference between the total binding and the non-specific binding determined in the presence of reference compound.

The IC 50 values (concentration causing half-maximal inhibition of control specific binding) were determined by non-linear regression analysis of the competitive curves using the algorithm "sigmoidal dose-response" (GraphPadPrism, San Diego, USA). In case of ill-defined curves or algorithm-generated minima below the defined non-specific binding the IC 50 value was determined by graphical extrapolation.

GABA uptake experiments

Freshly dissected, rat cortex dissected from male Sprague-Dawley rats was immersed immediately in 10 volumes of ice-cold 0.32 M sucrose buffered with 10 mM HEPES pH 7.4 and homogenized. The resulting preparation was centrifuged at 900 x g for 10 minutes at 4 °C. The pellet was discarded and the supernatant was centrifuged again at 4 °C at 10.000 x g. The supernatant was discarded and the pellet was stored with 0.32 M sucrose/HEPES on ice until needed. The slices (n=4) were transferred into nylon mesh baskets and placed in 12 well plates with 3 ml buffer containing the test compounds and 1 mM nipecotic acid (GABA uptake inhibitor) per well. The plates were incubated for 10 min at room temperature.

These wells were the basal fractions. The slices were than transferred into new plates with 3 ml stimulation buffer per well (buffer with 50 mM K + , corresponding equimolar reduction of Na + to maintain osmolarity) containing the test substances and 1 mM nipecotic acid and the plates were incubated for 7,5 min. These wells were the stimulation fractions. The slices were removed and their wet weight was determined.

The buffer samples were purified from the tritated metabolites of [³H]-GABA by anionexchange chromatography. The pH of the samples was adjusted to 4,0 with acetic acid and the diluted samples were applied to a DOWEX column equilibrated with sodium acetate. After washing the colums with 0,1% Triton X-100, GABA was eluted with 0,4 M Tris pH 7,5. GABA containing fractions were used for liquid scintillation counting.

Determination of GABA transaminase (GABA-T) activity

For the determination of GABA transaminase activity the rate of the reaction is determined by measuring the NADPH production at 340 nm at room temperature for 15 min within the linear range with a spectrophotometer in disposible PMMA cuvettes.

Results

Binding of Passiflora incarnata extract to GABA A receptor

The specific GABA A -receptor antagonist SR95531 competes for the binding of [ 3 H]-SR95531 to rat brain membranes in a concentration dependent manner (data not shown). The bound radioactivity in the presence of 100 µM SR95531 was defined as non-specific binding of the radioligand to rat brain membranes.

Passiflora incarnata extract competes for the binding of [ 3 H]-SR95531 to rat brain membranes in a concentration dependent manner (Fig. 1).

The IC 50 value of Passiflora incarnata extract at this site was 101 µg/ml. Therefore, binding to the GABA-site of the GABA A receptor is a likely mode of action of Passiflora incarnata extract. The IC 50 values for competitive binding of Passiflora incarnata extract at the benzodiazepine site are very high (944 µg/ml). In addition, the binding to this site is not modulated by the presence of GABA as observed for the reference compound (Figure 2). Therefore, it is very unlikely that Passiflora incarnata extract acts via this binding site.

Binding of Passiflora incarnata extract to GABA B receptor

Baclofen -a derivative of GABA that is a specific agonist of GABA B receptorscompetes with the binding of [ 3 H]-CGP 54626 -a selective and potent GABA B receptor antagonist-to rat brain and rat hippocampus GABA B receptors in a concentrationdependent manner. The bound radioactivity in the presence of 1 mM Baclofen was defined as non-specific binding of the radioligand to the membranes.

Passiflora incarnata extract competes with the binding of [ 3 H]-CGP 54626 to rat brain and rat hippocampus GABA B receptors in a concentration-dependent manner the IC 50 was 120 µg/ml (data not shown).

Binding of Passiflora incarnata extract to GABA B receptor ([ 35 S]-GTPγ γ γ γS-binding)

Baclofen -a derivative of GABA that is a specific agonist of GABA B receptorsstimulated the [ 35 S]-GTPγS binding to rat hippocampal membranes in a concentrationdependent manner (agonist mode). The selective and potent GABA B receptor antagonist CGP 54626 reduced the Baclofen-evoked signal in a concentration-dependent manner to the level of the basal signal (no effector added, antagonist mode). Passiflora incarnata extract has a lower IC 50 value (31 mg/ml) in the antagonist mode than in the agonist mode (115 mg/ml). Therefore Passiflora incarnata extract is an antagonist of the GABA B receptor (Figure 3).

GABA uptake and GABA release experiments

Up to 1000 µg/ml the Passiflora incarnata extract investigated in this study had no effect on potassium-evoked release of [³H]-GABA synthesized from [³H]-glutamine from rat cortical slices (data not shown).

The extract inhibited [³H]-GABA uptake into rat cortical synaptosomes with an EC 50 of 95.7 µg/ml (Figure 4).

Discussion

Extracts from passion flower (Passiflora incarnata) have been used to reduce anxiety and insomnia and there are numerous studies in mice and rats which demonstrate a reduced anxiety and stress with passion flower treatment [START_REF] Dhawan | Anti-anxiety studies on extracts of Passiflora incarnata Linneaus[END_REF][START_REF] Miyasaka | Passiflora for anxiety disorder[END_REF]). In addition there is evidence that extracts from Passiflora incarnata may be helpful in the treatment of substance addictions as amphetamine, nicotine, marihuana and alcohol (Capasso und Sorrentino, 2005, Dhawan et al., 2002and Dhawan and Sharma, 2003).

Although the anxiolytic activity of Passiflora species has been repeatedly evaluated in the last years, there is only limited information on the mechanism of action.

This study provides a comprehensive insight into the mode of action of a dry extract prepared from the flowers of Passiflora incarnata which is the sole active ingredient of the proprietary herbal drug Pascoflair ® 425mg on the GABAergic system.

Preclinical studies have suggested that GABA levels may be decreased in animal models of depression, and clinical studies reported low plasma and CSF GABA levels in mood disorder patients. The Passiflora incarnata extract investigated in this study had no effect on potassium-evoked release of [³H]-GABA synthesized from [³H]glutamine from rat cortical slices but the extract inhibited [³H]-GABA uptake into rat cortical synaptosomes with a EC 50 of 95,7 µg/ml. GABA transaminase (GABA-T), an enzyme target in the therapy of anxiety, epilepsy and related neurological disorders was not affected by Passiflora incarnata which is in accordance with already published results [START_REF] Awad | Effects of traditionally used anxiolytic botanicals on enzymes of the gamma-aminobutyric acid (GABA) system[END_REF]. Because preadministration of Flumazenil (Ro 15-1788), an antagonist of the benzodiazepine binding site of the GABA A receptor, attenuates the effects of Passiflora incarnata in vivo [START_REF] Grundmann | Anxiolytic activity of a phytochemically characterized Passiflora incarnata extract is mediated via the GABAergic system[END_REF][START_REF] Medina | Chrysin (5,7-di-OH-flavone), a naturally-occurring ligand for benzodiazepine receptors, with anticonvulsant properties[END_REF]) it was assumed that Passiflora incarnata and Diazepam share the same pharmacology.

Our study showed that the IC 50 values for competitive binding of Passiflora incarnata extract at the benzodiazepine site are very high moreover the binding to this site is not modulated by the presence of GABA so it seems very unlikely that the mode of action of Passiflora incarnata extract includes binding to the benzodiazepine site. Similarly it is unlikely that it acts via the ethanol site. However, it is very likely that binding to the GABA-site of the GABA A receptor is one of the clinically relevant modes of action of Passiflora incarnata extract.

Another possible target might be the binding of Passiflora incarnata extract to the GABA B receptor. There is accumulating evidence that modulators of the GABA B receptor (a GPCR -G protein coupled receptor) might act as anxiolytic [START_REF] Frankowska | Effects of GABAB receptor ligands in animal tests of depression and anxiety[END_REF] and might be helpful in the treatment of substance addictions [START_REF] Martin | GABA Receptors[END_REF].

The Passiflora incarnata extract investigated in this study inhibited the binding of [ 3 H]-CGP 54626 to GABA B -receptors in a concentration dependent manner. This could be verified using the [ 35 S]-GTPγS binding assay. It was found that Passiflora incarnata has a lower IC 50 value in the antagonist mode than in the agonist mode. Therefore

Passiflora incarnata needs to be classified as antagonist of the GABA B receptor. This opens possibilities for further investigations because GABA B antagonists may provide a pharmacological therapy for cognitive impairment [START_REF] Helm | GABAB receptor antagonist SGS742 improves spatial memory and reduces protein binding to the cAMP response element (CRE) in the hippocampus[END_REF].

Although the compounds responsible for the therapeutic activity of Passiflora incarnata are yet to be identified, this study provides novel evidence of the mechanism of action of a dry extract of Passiflora incarnata with respect to the GABAergic system.

  Grundmann et al. 2008, who investigated the same dry extract (sole active ingredient in Pascoflair ® 425mg) which was used in this study, and Zhong et al. 2008 showed that Passiflora incarnata increased the time mice spent in the open arm of the elevated plus maze and they postulated that the effects were mediated via GABA receptors.

[

  ³H]-SR95531 to GABA A -receptors and of [ 3 H]-CGP 54626 to GABA B -receptors in a concentration dependent manner. Using the [ 35 S]-GTPγS binding assay Passiflora incarnata could be classified as an antagonist of the GABA B receptor. The Passiflora incarnata extract inhibited [³H]-GABA uptake into rat cortical synaptosomes and showed no effect on GABA transaminase and GABA release.

  rats were sacrificed by decapitation. Rat brain (minus cerebellumand medulla oblongata, i.e. the hippocampus and cerebellum) tissue was homogenized at 4°C in 10 ml 10 mM Tris-HCl pH 7.4, 1 mM EDTA, and centrifuged for 15 min at 25,000xg. The pellet was finally resupended in 50 mM Tris-HCl pH 7.4, 4 mM MgCl 2 and 1 mM EDTA, frozen in liquid nitrogen and stored at -80°C until usage.

  ]-GTPγS-binding assays were carried out as described(Bidlack und Parkhill, 2004):Membranes were preincubated with the respective effectors for 15 min, the incubation was initiated by the addition of 200 pM [ 35 S]-GTPγS (60 min incubation at 30°C in a final volume of 100 µl assay buffer).The assay was terminated by transfer of the samples on GF/C filter plates. Filters were washed four times with 200 µl of ice-cold 50 mM Tris-HCl pH7.4 and bound radioactivity was determined by a microplate reader (Microbeta, Wallac, Finnland).From every data point the non-specific binding (binding of [ 35 S]-GTPγS in the presence of 10 µM GTPγS) was subtracted and the value normalized to the respective control signal of the respective plate.EC 50 -and IC 50 values (concentration causing half-maximal stimulation/inhibition of control specific binding) were determined by non-linear regression analysis of the competitive curves using the algorithm "sigmoidal dose-response" (GraphPadPrism, San Diego, USA). In case of ill-defined curves the IC 50 was determined by graphical exploration of the algorithm-generated curve.

Figure 1 :

 1 Figure 1: Competitive binding of Passiflora incarnata extract at the GABA-site of

Figure 2 :

 2 Figure 2: Competitive binding of Passiflora incarnata extract to the

Figure 3 :

 3 Figure 3:Effect of Passiflora incarnata extract on rat hippocampal GABA B -

Figure 4 :

 4 Figure 4: Effects of a Passiflora incarnata extract on the GABA uptake

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

http://mc.manuscriptcentral.com/ptr

Conflict of interest

Financial support for the analysis was provided by PASCOE pharmazeutische Präparate GmbH, the manufacturer of PASCOFLAIR ® 425 mg. The sponsor had no influence on the conduct of the analysis.


Nicola Robinson

Ava Lorenc
email: ava.lorenc@tvu.ac.uk

Responding to patient demand: community pharmacists and herbal and nutritional products for children

Keywords: herbal, pharmacists, children, nutrition supplements and other complementary and alternative medicine (CAM)

Responding to patient demand: community pharmacists and herbal and nutritional products for children

Ava Lorenc, Nicola

Introduction

Complementary and alternative medicine may be defined as healthcare outside of conventional medicine. It is used by 38% of adults and 12% of children, in particular overthe-counter products (homeopathy, herbal, vitamins/minerals and other food supplements) [START_REF] Nahin | Costs of complementary and alternative medicine (CAM) and frequency of visits to CAM practitioners: United States, 2007[END_REF]. Almost all pharmacies in the UK (99%) stock herbal and nutritional products (HNPs) and most (81%) receive CAM queries [START_REF] Barnes | Experiences with complementary remedies: a survey of community pharmacists[END_REF]. Whether pharmacists should stock HNPs is debatable, given limited evidence of efficacy and safety [START_REF] List | An ethical dilemma[END_REF]. However, such HNP provision may be a natural extension of the pharmacist's role (Boon 2005), particularly in supporting self-care (DH 2008). Patients and pharmacists believe pharmacists have some responsibility for HNP (Boon 2005) but may lack knowledge, especially for herbal medicine (Cramer et al 2010). The decision to stock these products may be a balance between profit, ethics and patient demand [START_REF] Naidu | Attitudes of Australian pharmacists toward complementary and alternative medicines[END_REF]. Few studies have explored in-depth the issues of HNP for pharmacy practice, including pharmacists' attitudes to HNP and the tension between patient demand and evidence-based practice.

Methods

As part of a larger study on complementary and traditional medicines for children (Robinson Quantitative diary data is displayed as frequency tables. Framework Analysis, a highly structured and systematic method, was used for qualitative interview data [START_REF] Ritchie | Qualitative data analysis for applied policy research[END_REF]

Results

Table one provides details on the four pharmacies and interviewees. Twenty-nine HNP diary events were recorded. Of these, 86% were product purchases and 79% were advice requests, mainly (45%) for under 4 year olds. Ethnicity of children was varied (24% White, 24% Asian, 14% Black, 38% missing). Many interviewees perceived ethnic variation in HNP use; White populations using more HNPs and Afro-Caribbeans more conventional medicine.

Most children (69%) were currently not taking medication and 83% were not having prescriptions filled. Pharmacists generally perceived HNP were used to avoid conventional medicine (e.g. antidepressants or steroids), and cited that this would also be when they would recommend it. Similarly, from the diaries, HNPs were used predominantly for health maintenance or minor conditions e.g. teething, colds and colic (see table 3). Interviewees also identified that HNPs were used for child development, wellbeing, eczema, dermatitis, asthma and nocturnal enuresis. Pharmacists perceived increasing demand, and reported that they would like to stock more products to meet this demand.

"There is clearly a patient demand for alternative treatments; people are now looking to perhaps try alternative before conventional" "for children a lot of them prefer using like the, the homeopathic teether, which is a common one, um, I really like the rescue remedy for calming people down" Nutritional (vitamin/mineral) and herbal dietary supplements and aromatherapy were especially popular (table 2), average cost per event £4.26. Pharmacists cited stocking around 100 HNPs, mainly vitamins, minerals, herbs and homeopathy; with some specific foods, massage oils, and Bach flower remedies. Supplier information and local demand informed stock choice. Location of the pharmacy was important in determining which HNPs were stocked due to variations in customer demand, with less busy pharmacies stocking less.

"if we get more than a couple of requests we usually maybe order a couple in and see how that sells" Diary forms were completed by pharmacy assistants (15 events) or pharmacists (13 events).

For all recorded events staff felt competent giving advice, although in 31% of events staff reported needing more knowledge. In 41% of events staff used information to give advice.

HNP use was encouraged in 59%, with only 10% recommending another type of HNP and only 3% advising against HNP use, with no concerns over the child's safety. No interviewees spontaneously mentioned safety issues and none cited direct experience of adverse events.

During the interviews only one pharmacist reported that they would routinely ask customers about HNP, although all felt they 'should' ask about and record HNP use, but that it was not 'natural' to do so; customer-initiated discussion was seen as much more common.

Recommendations were dependent on knowledge and stocked products.

"they do often ask 'well you know what I take so is it alright to take it with my medication', yes, it's a common request that patients ask before they purchase it" "I don't think it's something we would necessarily suggest straight away" "we are meant to record people using supplements, particularly if they're on other medication"

Three pharmacists were open to HNPs; the other was 'not a big fan'. Pharmacists appeared to separate their personal and professional beliefs/experience, which created tension. Ethnic background did not appear to affect HNP beliefs or knowledge; professional experience of conventional medicine harm/overuse appeared more influential.

Only one pharmacist reported professional training on HNPs, none had detailed knowledge.

Pharmacists did feel (P3 particularly) that more knowledge would mean they could stock and recommend more HNPs, improve patient choice and help patients.

Only a few sources of HNP product information were cited; mainly the same as conventional medicine -through suppliers, internet, customers, and occasionally reading. A need for more evidence and information was expressed.

"you read about it…, from time to time, and mainly through conversing with patients you pick up these stories" "we have a vitamin, a brand, natures aid, and they're really good, they provide you with a whole file of every single item and what it's used for, …a reference, which is quite a good idea really"

Discussion

This study identified that parents often approach pharmacies concerning herbal remedies/topical products and nutritional supplements for their children, independent of This study has shown that pharmacists are uniquely placed as conventional healthcare providers and are influential, responsive and empathetic to patient choice, including HNP.

Expanding the range of services provided by community pharmacies and improving patient choice are part of the new role of pharmacies in the UK [START_REF] Richardson | Community pharmacy: moving from dispensing to diagnosis and treatment[END_REF]. The new community pharmacy contract in the UK emphasises the role of pharmacist to support self-care, which may include the use of herbal, vitamin and nutritional supplements (NHS

2005):

"To enhance access and choice for people who wish to care for themselves or their families"

In this study pharmacists were, generally, open to HNPs and understood that parents often wish to seek an alternative to conventional medication for their children and themselves, which many empathised with, due to experience of the overuse and harm of medicines.

Belief in overuse/harm of medicines may be related to the Asian ethnic background of all the pharmacists [START_REF] Horne | Medicine in a multi-cultural society: the effect of cultural background on beliefs about medications[END_REF]. Although no pharmacists mentioned their ethnic background influencing their attitudes or behaviour related to HNPs, the influence of the pharmacist's personal background was debatable. One pharmacist's strong belief in HNPs despite being brought up with and used to orthodox medicine was of particular interest.

Although staff felt competent, they felt more HNP product knowledge would benefit their business and their customers. Pharmacists want to maintain professionalism and are strongly influenced by professional experience and factors, for example, believing in HNPs despite being brought up with and familiar with orthodox medicine. The emphasis on professional factors, rather than personal background, may be due to a desire to appear 'professional', highlighting the tension between personal and professional roles [START_REF] Bourgeault | CAM in inter-professional context: Canada[END_REF] Cohen (2002) suggests that if pharmacists continue to stock CAM products, they must improve their training. Three pharmacists were trained over 10 years ago, which may account for their lack of HNP education. The literature shows that many pharmacists believe CAM education should be mandatory [START_REF] Chang | Pharmacists' knowledge and attitudes toward herbal medicine[END_REF], part of pharmacy training [START_REF] Koh | Pharmacists' Patterns of Use, Knowledge, and Attitudes Toward Complementary and Alternative Medicine[END_REF] or continuing education (Charrois et al 2007). Continuing professional development (CPD) may therefore be needed to ensure unbiased advice is available to help parents make informed choices (Cramer et al 2010).

Due to limited sample size and specific location, findings from this study may not be generalizable, but does provide in-depth insight into issues which warrant further exploration.

Given the prevalent use of CAM in the UK and that most pharmacies do stock CAM products and receive queries about CAM products [START_REF] Barnes | Experiences with complementary remedies: a survey of community pharmacists[END_REF], there may be a need for further research both on the efficacy and safety of HNPs, but also on the relationship between pharmacists, pharmacies and the public.

Conclusions

Pharmacists appear to understand and empathise with customer demand for herbal and nutritional products for children and are in a unique position to provide advice and products.

However, in order to maintain professionalism, which is important to them, pharmacists may need information and CPD.

  et al 2009), interviews and diary recording took place in four community pharmacies in a highly multi-ethnic community in Northwest London, between March -June 2008. A diary sheet was completed by pharmacists or assistants for each purchase or enquiry of a HNP for a child for two separate one week periods. Information included child demographics, conventional healthcare, product enquiries, purchases and pharmacist response. Semistructured interviews of up to one hour were subsequently carried out (by AL) with one pharmacist from each pharmacy. Pharmacists completed consent forms, and questionnaires about themselves, the pharmacy and HNP stock. Interviews discussed beliefs, knowledge and education on HNPs and their use for children, reason for stocking HNPs, use of HNP information, perception of HNP use in the community and safety issues.

  "Q: Do you think it[HNPs] should be something that you learn about or? P: Yeah definitely, yeah, I think people are going more for alternative medicines, and Ayurvedic"

  One in particular had very positive beliefs, due to seeing HNPs work for others, particularly for avoiding conventional medicine wherever possible. This contrasted with her personal background:"my Dad's a pharmacist and we grew up in that …we have two doctors [in the family] so they're obviously more geared to [conventional medicine]…. Q: And do you think that affects your attitudes to it[HNPs] now? P: No, because I'm much more open to it, I think I would personally prefer it, and I'm not one to take tablets at all"

  . The previous phases of our study demonstrated that many parents actively choose HNPs to avoid overuse of conventional medicine, which pharmacists recognised(Robinson et al 2009).

  Lorenc A, Blair M. 2009 Developing a decision-making model on traditional and complementary medicine use for children.

  and is described elsewhere (Robinson et al 2009). Harrow Research Ethics Committee approved the study in February 2007, ref 06/Q0405/92.

Table One : Pharmacies Interviewee

 One

										Phytotherapy Research			Page 12 of 14
	Pharmacy	No. Pharmacists	No. other staff	Number of diary events	Estimated no. of	prescriptions per day	CAM Products in stock	(estimated by	pharmacist)	Age	Sex	Ethnicity	Full/part time	Time in current	position	When trained
	1	1	5	4	200	100	35 -44	M	Asian	Full	5yrs	1998
							approx			British
	2	1	5	10 280	150	25 -34	F	Asian	Full	4yrs,	1997
												British	5months
	3	1	3	0 Missi	missing	45 -54	M	Asian	Full	4yrs	1984
					ng	data			British	5months
	4	2	4	data 15 100	60 -70 F o 25 -34 r	F	Asian British	Part	3yrs	2001
										P
										e
											e r
												R
												e
													v i e
														w

http://mc.manuscriptcentral.com/ptr

Table 2 : CAM purchases from diary data

 2

	Page 13 of 14			Phytotherapy Research
	Group of CAM	Products	Number Details/names of products
	Nutritional	Vitamins/minerals	3	Zincovit ® (vitamin C)
	supplements (n=7)	Tonics	2	Multivitamins Abidec ® multivitamins Minadex ® tonic
		Essential fatty acids	2	Seven seas	® cod liver oil liquid
				Omega 3 syrup
	Herbal/ aromatherapy	Aromatherapy oils	4	Clove oil Olbas ® for children
	topical products			Citronella oil
	(n=9)			Menthol and eucalyptus
		Creams	2	Arnica cream
				Tea tree cream
		Oils	2	Olive oil
		Balm	1	Happinose ® (decongestant)
	Herbal oral	Teething powder	2	Ashton and Parsons Powder	®
	products (n=3)	Echinacea	1	Echinacea for children
	Other (n=6)	Constipation remedy	1	Califig ® (syrup of figs)
		Colic mixtures Flower remedies	2 1	® Woodwards gripe mixture Colief ® Bach flowers Rescue Remedy ®
		Teething granules	2	Nelson	® teething granules

http://mc.manuscriptcentral.com/ptr

Table 3 : Conditions treated with CAM from diary data

 3

		Page 14 of 14
	Health maintenance	6
	Teething	4
	Respiratory Tract	4
	infection (including colds)
	GI problems (including	4
	colic)
	Immune stimulation	2
	Skin problems	2
	Allergy	2
	ENT	1
	Cuts/injury	1
	Stress	1
	Toothache	1

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

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

http://mc.manuscriptcentral.com/ptr

Acknowledgements and funding

We are very grateful to all the pharmacy staff who took part. This work was supported by a King's Fund Partners for Health grant Deleted: CAM Deleted: these products Deleted: CAM such as Deleted: products Deleted: supplements Deleted: CAM


Hans-Juergen Meyer

Mariusz Kubus

Jochen Glaser

Andrzej Kłonkowski

H.-Jürgen Meyer

Professor Rüdiger Kniep

Dr H.-J Meyer

Rare Earth Carbodiimide Silicates: RE 2 (CN 2 )(SiO 4 )

Keywords: rare earth, carbodiimide, silicate Wiley-VCH ZAAC Rare earth, Carbodiimide, Silicate, Metathesis, Structure elucidation, X-ray diffraction

Rare earth carbodiimide silicates RE 2 (CN 2 )(SiO 4 ) with RE = Y, La, and Pr where synthesized via solid state metathesis reactions of RECl 3 , Li 2 (CN 2 ), and SiO 2 or Li 2 SiO 4 , respectively, in silica tubes at 550 °C. All three compounds crystallize with different structures, although all of them represent distorted derivatives of the sodium chloride type structure. The structure of Y 2 (CN 2 )(SiO 4 ) was refined monoclinically (C2/m, Z = 2, a = 1301.382(5) pm, b = 377.630(1) pm, c = 527.656(2) pm, β = 93.9816(2) °) from X-ray powder data. The crystal structure of La 2 (CN 2 )(SiO 4 ) was refined in a different monoclinic space group (P2 1 /c, Z = 4, a = 660.3(1) pm, b = 1282.01(2) pm, c = 656.2(1) pm, β = 105.23(2) °), and the structure of Pr 2 (CN 2 )(SiO 4 ) was refined triclinically (P1, Z = 2, a = 646.7(2) pm, b = 669.2(2) pm, c = 671.8(2) pm, α = 86.18(3) °, β = 73.22(3) °, γ = 74.08(3) °) from X-ray single crystal data.

Introduction

A great deal of rare earth carbodiimide compounds was synthesized following solid state metathesis (SSM) reactions between rare earth trihalides and lithium carbodiiimide. On heating these mixtures, metathesis reactions reveal their characteristics, with a soft ignition coming about when heating up reaction mixtures into a temperature region within 450 to 550 °C. The series of quasi-binary carbodiiimide RE 2 (CN 2 ) 3 compounds [START_REF] Neukirch | [END_REF]2] with trivalent rare earth ions (RE = Y -Lu, except Pm, La) are fundamental examples of rare earth carbodiimide chemistry. But there are also multinary carbodiimide (or in some cases cyanamide) compounds represented by the formulas REX(CN 2 ) (X = Cl, F) [3,4], RE 2 X(CN 2 )N (X = Cl, Br, I) [5,6], LiRE 2 X 3 (CN 2 ) 2 (X = F) [4], as well as very recently discovered compounds LiRE(CN 2 ) 2 (RE = La, Ce) and Ce 3 (CN 2 ) 3 N [7]. All these compounds being obtained from SSM reactions, departing from different stoichiometries of REX 3 and Li 2 (CN 2 ) mixtures, account for the rapid growth of this new family of rare earth carbodiimide compounds, and the thereon resting development of tetracyanamidosilicates [8].

An extension of this preparative concept to higher complexity comes about when another reaction partner is being introduced into a given SSM reaction. The introduction of oxide into a SSM reaction was previously explored with various oxygen sources in order to develop a high yield synthesis of Y 2 O 2 (CN 2 ):Eu [9]. During reactions performed in silica tubes an unknown side phase appeared which later turned out to be Y 2 (CN 2 )(SiO 4 ), which was the starting point of this work.

Mixed anion compounds like RE 2 (CN 2 )(SiO 4 ) are known for various examples of rare earth orthosilicates containing yet another anion. The doubly charged carbodiimide ion and its assignment as a pseudo-chalcogenide [9,10,11] leads to compounds having the general formula RE 2 Ch(SiO 4 ) being known for Ch = O [12], S [13], Se [14] and Te [15]. From a structural point of view, the compounds RE 2 O 3 , RE 2 O 2 S and RE 2 O 2 (CN 2 ) have been shown to crystallize in closely related structures [9].

The luminescence behaviour of lanthanide-doped carbodiimide oxide compounds having the general formula RE 2 O 2 (CN 2 ):Ln [16,17,18] provides an interesting potential of carbodiimide compounds. Moreover, the luminescence behaviour of Y 2 O 2 (CN 2 ):Eu appears to be similar with that of the well-known red light emitter Y 2 O 2 S:Eu [9]. In addition, the rare earth silicates Y 3 SiO 5 :Ln (Ln = Ce or Tb) are phosphors of current interest, being used in field emission displays (FEDs) [19].

In this work we present the novel RE 2 (CN 2 )(SiO 4 ) compounds, originally obtained from a reaction of rare earth carbodiimide with silica glass, and we describe the selective SSM preparations of these compounds, including their crystal structures.

Experimental Section

Page 2 of 13

Wiley-VCH

ZAAC

Synthesis of Li 4 (SiO 4 )

Lithium orthosilicate was prepared by solid state reaction on heating amorphous silica (Ventron, 99.9%, -325 mesh) and lithium carbonate (Li 2 CO 3 , Alfa, ultra pure). Amorphous silica and Li 2 CO 3 were carefully mixed in an agate mortar in 1:2 molar ratio and heated at 800 °C for 4 h in air [20].

SiO 2 + 2 Li 2 CO 3 → Li 4 SiO 4 + 2 CO 2 ↑
Lithium orthosilcate obtained from the reaction was X-ray pure.

Synthesis of RE 2 (SiO 4 )(CN 2 )

Method 1: Reaction mixtures were prepared in an argon filled glove box starting from carefully homogenized mixtures of RECl 3 (ABCR 99.9 %), Li 2 (CN 2 ) (prepared as previously reported [7]) and SiO 2 (Ventron, 99.9%, ≈ 325 mesh). Reaction mixtures of 61.3 mg LaCl 3 , 61.8 mg PrCl 3 and 48.8 mg YCl 3 , respectively, were loaded with 54 mg Li 2 CN 2 , 15 mg SiO 2 and 20 mg KCl (Merck, p.a.) being carefully mixed and ground in a agate mortar. Each mixture was placed into a dry silica ampoule. Ampoules were evacuated, sealed under vacuum and heated up to 550 °C (5 °C/min), held at this temperature for 3 days, and then cooled down to room temperature. The reaction products were washed with water several times, rinsed with ethanol, and dried at 85 °C in air. Results of powder XRD measurement revealed that the title compounds were the main crystalline products with estimated yields >90 %.

Method 2: Reaction mixtures were prepared in an argon filled glove box starting from carefully homogenized mixtures of RECl 3 , Li 2 (CN 2 ) and Li 4 SiO 4 being carefully mixed and ground in a agate mortar. Each mixture was placed into a dry silica ampoule. Ampoules were evacuated, sealed under vacuum and heated up to 550 °C (5 °C/min), held at this temperature for 3 days, and then cooled down to room temperature. The reaction products were washed with water several times, rinsed with ethanol, and dried at 85 °C in air. Results of powder XRD measurement revealed reflections of the title compounds as the only crystalline products with estimated yields >95 %.

Powder X-Ray Diffraction

The powder XRD patterns of all reaction products were collected with a Stadi-P (Stoe, Darmstadt) powder diffractometer using Cu-K α1 (λ = 154.06 pm) radiation (germanium monochromator). Most samples occurred to be X-ray pure, except for some samples being prepared following method 1 which were slightly contaminated with Li 2 SiO 3 .

The patterns were indexed by using the program system WinXPow [START_REF] Winxpow | Version 1.10: Diffractometer Software[END_REF]. The structure of Y 2 (CN 2 )(SiO 4 ) was indexed monoclinically with a = 1303. The crystal structure Y 2 (CN 2 )(SiO 4 ) was solved and refined in the monoclinic space group C2/m from powder XRD data (a = 1301.38 (7) pm, b = 377.63(2) pm, c = 527.66(3) pm and β = 93.982(4) °, Z = 2) using the program packages EXPO [START_REF] A: Altomare | Extra: a Program for extracting Structure-Factor Amplitudes from Powder Diffraction Data[END_REF] and Fullprof [START_REF] Rodriguez-Carvajal | WinPLOTR: a Windows tool for powder diffraction patterns analysis[END_REF] based on 258 independent reflections and 40 refined parameters, yielding R Bragg = 6.42.

Positions of (CN 2 ) 2-and (SiO 4 ) 4-were refined in a disordered fashion on similar positions. No sign of a superstructure could be detected. Atom positions are listed in Table 2 and selected bond lengths are given in Table 3.

Single-Crystal X-Ray Diffraction

Colorless transparent crystals of La 2 (CN 2 )(SiO 4 ) and light green crystals of Pr 2 (CN 2 )(SiO 4 ) were selected from the reaction product and mounted on the tip of a glass fiber for a crystal structure determination. Intensity data were collected on a Stoe IPDS diffractometer using Mo-K α radiation. The data sets were corrected for Lorentz and polarization effects. Absorption corrections were carried out using crystal-shape optimization (X-Shape, X-RED). The program package SHELX97 [START_REF] Sheldrick | SHELX-97: Program Package for Crystal Structure Determination[END_REF] was used for crystal structure solutions and anisotropic refinements of all atoms. Some selected crystallographic data and some details of the structure refinements are listed in Table 1. Atomic positions and equivalent isotropic displacement parameters are given in Table 2, and a summary of selected bond lengths is given in Table 3.

Further details of the crystal-structure investigation may be obtained from the Fachinformationszentrum Karlsruhe, D-73644 Eggenstein-Leopoldshafen, Germany (fax: (+49)7247-808-666; e-mail: crysdata@fiz-karlsruhe.de), on quoting the depository number CSD-420943 for La 2 (CN 2 )(SiO 4 ) and CSD-420944 for Pr 2 (CN 2 )(SiO 4 ).

Results and Discussion

Solid state metathesis reactions were used for the synthesis of three selected examples of hitherto unknown new rare earth carbodiimide silicates. Powders of Y 2 (CN 2 )(SiO 4 ) were originally obtained as a side phase from reactions of YCl 3 and Li 2 (CN 2 ) with the wall of the silica container. This reaction was successfully reproduced in our synthesis described as method 1 in the experimental section by using small sized SiO 2 as a reactant. Subsequent reactions described in method 2 are straight forward SSM reactions to combine a rare earth element (as RECl 3 ) simultaneously with carbodiimide (as Li 2 (CN 2 )) and orthosilicate (as Li 4 (SiO 4 )) ions. Since RE 2 (CN 2 )(SiO 4 ) compounds behave stable in air and in water, the coproduced LiCl can be easily removed from the reaction products. The use of KCl in the reaction mixture was intended to allow better crystal growth, due to the formation of a LiCl/KCl flux in course of the reaction. The The three compounds Y 2 (CN 2 )(SiO 4 ), La 2 (CN 2 )(SiO 4 ), and Pr 2 (CN 2 )(SiO 4 ) crystallize with three different crystal structures, all being related to the motif of the arrangement of ions in the NaCl type structure, where the centers of gravities of (CN 2 ) 2-and (SiO 4 ) 4-ions occupy anion positions in an alternating fashion. However, the structure refined for Y 2 (CN 2 )(SiO 4 ) revealed disordering of these anions, resulting in a picture in which anions superimpose each other at the same lattice voids. Although the refinement from powder data may reveal an intrinsically higher degree of uncertainty of a given structure refinement, no evidence for the presence of a superstructure was detectable from powder X-ray patterns. With respect to the difficulty related with this structure, we will only discuss the structures of La 2 (CN 2 )(SiO 4 ) and Pr 2 (CN 2 )(SiO 4 ) in more detail, with their structures being projected in Figures 1 and2, in which the NaCl type related unit sections are emphasized as dotted lines. The alternating arrangements the two types of anions in the structures introduce a layered nature, being characterized by alternating [RE(CN 2 )] + and [RE(SiO 4 )] -sequences (with RE = La, Pr). As a consequence, the crystal structures of RE 2 (CN 2 )(SiO 4 ) contain two crystallographically distinct RE ions displayed in Figures 3 and4 4) pm for RE = Pr. In spite of their significant deviations from ideal D ∞h symmetry, the [N=C=N] 2-ions are considered as carbodiimide ions (rather than cyanamide) for reasons discussed previously [7].

  3(3) pm, b = 377.6(6) pm, c = 527.8(1) pm, β = 93.96(1) ° and V = 259.2(2) Å 3 based on 28 single indexed lines, La 2 (CN 2 )(SiO 4 ) was indexed monoclinically with a = 666.1(2) pm, b = 1282.0.(3) pm, c = 651.8(2) pm, β = 105.13(1) ° and V = 537.4(3) Å 3 based on 37 single indexed lines, and Pr 2 (CN 2 )(SiO 4 ) was indexed Page 3 of 13 Wiley-VCH ZAAC triclinically with a = 646.5(3) pm, b = 670.0(5) pm, c = 671.5(4) pm, α = 86.09(3) °, β = 73.29(2) °, γ = 74.09(3) ° and V = 267.9(3) Å 3 based on 22 single indexed lines.

  RE 2 (CN 2 )(SiO 4 ) synthesis provides a general concept for SSM reactions to obtain mixed anion compounds.

  . La1 is surrounded by four carbodiimide (La1-N: 265 -284 pm) and three silicate ions (La1-O: 242 -283 pm) and La2 by two carbodiimide (La2-N: 265 -269 pm) and four silicate ions (La2-O: 238 -288 pm), whereas silicate groups connect via one or two oxygen atoms with one and the same lanthanum atom. The coordination environment of Pr1 involves two carbodiimide (Pr1-N: 252 -257 pm) and four silicate ions (Pr1-O: 228 -275 pm) and Pr2 is surrounded by three carbodiimide (Pr2-N: 253 -270 pm) and three silicate ions (Pr2-O: 244 -264 pm). Each of the presented RE 2 (CN 2 )(SiO 4 ) structure contains one distinguishable [NCN] 2-ion, having N-C-N bond angles of 177(1) ° with C-N bond length of 121(2), 126(2) pm for RE = La, and N-C-N bond angles of 176.4(8) ° with C-N bond length of 125(1), 122(

Figure 1 .

 1 Figure 1. Crystal structure of La 2 (CN 2 )(SiO 4 ). The NaCl type related unit cell is indicated by the dotted line, with La1 at the corner.

Figure 2 .

 2 Figure 2. Crystal structure of Pr 2 (CN 2 )(SiO 4 ). The NaCl type related unit cell is indicated by the dotted line, with Pr2 at the corner.

Figure 3 .

 3 Figure 3. Coordination environment of lanthanum ions in the structure of La 2 (CN 2 )(SiO 4 ).

Figure 4 .Figure 1 .

 41 Figure 4. Coordination environment of praseodymium ions in the structure of Pr 2 (CN 2 )(SiO 4 ).

Figure 2 .Figure 3 .

 23 Figure 2. Crystal structure of Pr 2 (CN 2 )(SiO 4 ). The NaCl type related unit cell is indicated by the dotted line, with Pr2 at the corner.

Figure 4 .

 4 Figure 4. Coordination environment of praseodymium ions in the structure of Pr 2 (CN 2 )(SiO 4 ).

Table 1 .

 1 Single crystal data on structure refinements on RE 2 (CN 2 )(SiO 4 ) with RE = La, Pr. P1

	Empirical formula	La 2 (

CN 2 )(SiO 4 ) Pr 2 (CN 2 )(SiO 4 )

  ≤ h ≤ 7, -15 ≤ k ≤ 15, -7 ≤ l ≤ 7 -7 ≤ h ≤ 7, -7 ≤ k ≤ 7, -7 ≤ l ≤ 7

						ZAAC	Page 6 of 13
	Formula weight / g/mol		409.94	413.94
	Temperature / K					293(2)	293(2)
	Wavelength / Å					0.71073	0.71073
	Crystal system					monoclinic	triclinic
	Space group (No.)			P2 1 /c (14)	P1
	Unit cell dimensions / pm or °	a = 660.3(1)	a = 646.7(2), α= 86.18(3)
						b = 1282.0(2), ß= 105.23(2)	b = 669.2(2), β= 73.22(3)
						c = 656.2(1)	c = 671.8(2), γ = 74.08(3)
	Volume / 10 6 pm	3			535.9(2)	267.7(1)
	Z					4	2
	Density (calculated) / g/cm	3	5.081	5.136
	Absorption coefficient / mm	-1	15.891	18.150
	F(000)					720	368
	Crystal size / mm	3			0.14 x 0.13 x 0.04	0.12 x 0.10 x 0.06
	Theta range for data collection / ° 3.18 to 24.71	3.17 to 25.04
	Index ranges -7 Reflections collected 5318	2710
	Independent reflections		939 [R(int) = 0.0883]	884 [R(int) = 0.0271]
	Completeness to theta		100.0 %	93.3 %
	Refinement method			Full-matrix least-squares on F	2	Full-matrix least-squares on F	2
	Data / restraints / parameters	939 / 0 / 92	884 / 0 / 91
	Goodness-of-fit on F	2		1.114	1.293
	Final R indices [I > 2σ(I)]		R1 = 0.0375	R1 = 0.0244
						wR2 = 0.1057	wR2 = 0.0665
	R indices (all data)			R1 = 0.0379	R1 = 0.0257
						wR2 = 0.1064	wR2 = 0.0670
	6 Largest diff. peak and hole / e/10 pm 3	2.127 and -2.910	1.047 and -2.726
						Wiley-VCH

Table 2 .

 2 Atomic coordinates and equivalent isotropic displacement parameters (10 4 pm 2 ) for RE 2 (CN 2 )(SiO 4 ) (RE = La, Pr). U eq is defined as one third of the trace of the orthogonalized U ij tensor. Isotropic displacement parameters U iso (10 4 pm 2 ) are given for RE = Y.

	La 2 (CN 2 )(SiO 4 )
	Atom	x	y	z	U eq
	La1	0.4131(1)	0.09933(6) 0.2186(1)	0.0144(3)
	La2	0.0294(1)	-0.1454(1) 0.3295(1)	0.0151(3)
	Si	0.0924(6)	-0.0995(3) 0.8115(6)	0.0150(8)
	O1	0.105(2)	0.0532(8)	0.370(1)	0.017(2)
	O2	0.253(2)	-0.0075(8) -0.085(2)	0.024(2)
	O3	-0.026(2)	-0.1656(7) -0.042(2)	0.016(2)
	O4	0.222(2)	-0.1802(8) 0.705(2)	0.015(2)
	C	-0.474(2)	-0.152(1)	0.272(2)	0.017(3)
	N1	-0.574(2)	-0.0974(9) 0.360(2)	0.024(3)
	N2	-0.372(2)	-0.2047(9) 0.172(2)	0.019(3)

Pr 2 (CN 2 )(SiO 4 )

	Page 7 of 13				ZAAC ZAAC	Page 8 of 13
	Y 2 (CN 2 )(SiO 4 )
	Atom	x	y	z	U iso	Occ.
	Y	0.1291(1)	0	0.3749(3)	0.0063(5) 1
	Si	0.0930(6)	1/2	0.819(2)	0.003(3)	0.5
	O1	0.116(2)	1/2	0.108(5)	0.023(4)	0.5
	O2	0.203(2)	1/2	0.656(5)	0.016(4)	0.5
	O3	0.0352(6)	0.136(2)	0.730(1)	0.013(3)	0.5
	C	0.155(2)	1/2	0.865(6)	0.02(1)	0.5
	N1	0.152(2)	1/2	0.090(5)	0.023(4)	0.5
	N2	0.177(2)	1/2	0.643(6)	0.016(4)	0.5
	Atom	x	y	z	U eq
	Pr1	0.06189(5) 0.23652(5) 0.33127(5)	0.0068(2)
	Pr2	0.45537(5) 0.31934(6) -0.21243(5) 0.0084(2)
	Si	-0.0428(3) 0.2955(3)	-0.1844(3)	0.0072(4)
	O1	0.1118(8)	-0.1304(8) 0.3035(7)	0.010(1)
	O2	-0.1359(7) 0.6117(7)	0.3701(7)	0.0089(9)
	O3	0.0896(8)	0.2237(8)	-0.0145(7)	0.011(1)
	O4	0.2758(8)	0.5199(8)	0.1105(7)	0.011(1)
	C	0.563(1)	0.178(1)	0.303(1)	0.010(2)
	N1	0.478(1)	0.070(1)	0.236(1)	0.019(2)
	N2	0.6401(9)	0.299(1)	0.3758(9)	0.011(1)
					Wiley-VCH Wiley-VCH

Acknowledgement. The authors like to thank the Deutsche Forschungsgemeinschaft (Bonn) for support of this research.