双膜理论-邓修第二版

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERINGEffects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell culturesJiang-Lin Zhao &Li-Gang Zhou &Jian-Yong WuReceived:7September 2009/Revised:6January 2010/Accepted:6January 2010/Published online:2March 2010#Springer-Verlag 2010Abstract This study examined the effects of biotic and abiotic elicitors on the production of diterpenoid tanshi-nones in Salvia miltiorrhiza cell culture.Four classes of elicitors were tested,heavy metal ions (Co 2+,Ag +,Cd 2+),polysaccharides (yeast extract and chitosan),plant response-signaling compounds (salicylic acid and methyl jasmonate),and hyperosmotic stress (with sorbitol).Of these,Ag (silver nitrate),Cd (cadmium chloride),and polysaccharide from yeast extract (YE)were most effective to stimulate the tanshinone production,increasing the total tanshinone content of cell by more than ten-fold (2.3mg g -1versus 0.2mg g -1in control).The stimulating effect was concentration-dependent,most significant at 25μM of Ag and Cd and 100mg l -1(carbohydrate content)of YE.Of the three tanshinones detected,cryptotanshinone was stimulat-ed most dramatically by about 30-fold and tanshinones I and IIA by no more than 5-fold.Meanwhile,most of the elicitors suppressed cell growth,decreasing the biomass yield by about 50%(5.1–5.5g l -1versus 8.9g l -1in control).The elicitors also stimulated the phenylalanine ammonia lyase activity of cells and transient increases in the medium pH and conductivity.The results suggest that the elicitor-stimulated tanshinone accumulation was a stress response of the cells.Keywords Salvia miltiorrhiza .Cell culture .Tanshinones .Elicitors .Stress responseIntroductionSalvia miltiorrhiza Bunge (Lamiaceae),called Danshen in Chinese,is a well-known and important medicinal plant because its root is an effective herb for treatment of menstrual disorders and cardiovascular diseases and for the prevention of inflammation (Tang and Eisenbrand 1992).As its Chinese name refers,Danshen root is characterized by the abundance of red pigments which are mainly ascribed to numerous diterpene quinones generally known as tanshinones,e.g.,tanshinone I (T-I),tanshinone-IIA (T-IIA),and T-IIB,isotanshinone I and II,and cryptotanshinone (CT).Tanshinones constitute a major class of bioactive compounds in S .miltiorrhiza roots with proven therapeutic effects and pharmacological activities (Wang et al.2007).Danshen in combination with a few other Chinese herbs is an effective medicine widely used for the treatment of cardiovascular diseases and used as an emergency remedy for coronary artery disease and acute ischemic stroke.According to WHO statistics,cardiovas-cular diseases are and will continue to be the number one cause of death in the world (www.who.int/cardiovascular_diseases ).It is of significance to develop more efficient means for the production of Danshen and its active constituents.Although field cultivation is currently the major produc-tion means for Danshen and most other plant herbs,plant tissue cultures provide more well-controlled and sustainable systems for efficient production of desired bioactive compounds of the herb.Plant tissue cultures are the most useful and convenient experimental systems for examiningJ.-L.Zhao :L.-G.Zhou (*)Department of Plant Pathology,China Agricultural University,Beijing 100193,China email:lgzhou@J.-Y .Wu (*)Department of Applied Biology and Chemical Technology,The Hong Kong Polytechnic University,Hung Hom,Kowloon,Hong Kong email:bcjywu@.hkAppl Microbiol Biotechnol (2010)87:137–144DOI 10.1007/s00253-010-2443-4various factors on the biosynthesis of desired products and for exploring effective measures to enhance their produc-tion.The importance of Danshen for traditional and modern medicines has promoted the long-lasting research interest in the development of tiorrhiza tissue cultures for production of bioactive compounds for more than two decades.In an early study,Nakanishi et al.(1983)induced several cell lines from plant seedlings and screened out a cell line capable of producing significant amounts of CT and another diterpene,ferruginol.In later studies,the group performed a fuller evaluation and optimization of the medium for cell growth and CT production and,eventually,derived an effective production medium with a simpler composition(ten components)than the original Murashige and Skoog(MS) medium(about20components),achieving a high CT yield of 110mg l-1(Miyasaka et al.1987).Many recent studies have been focused on hairy root cultures of tiorrhiza transformed by Agrobacterium rhizogenes(Hu and Alfermann1993;Chen et al.2001)and by our group (Zhang et al.2004;Ge and Wu2005;Shi et al.2007).Most of the bioactive compounds in medicinal plants belong to secondary metabolites which are usually less abundant than primary metabolites in the plants.Since the accumulation of secondary metabolites in plants is a common response of plants to biotic and abiotic stresses, their accumulation can be stimulated by biotic and abiotic elicitors.Therefore,elicitation,treatment of plant tissue cultures with elicitors,is one of the most effective strategies for improving secondary metabolite production in plant tissue cultures(Chong et al.2005;Smetanska2008).The most common and effective elicitors used in previous studies include the components of microbial cells especially poly-and oligosaccharides(biotic)and heavy metal ions, hyperosmotic stress,and UV radiation(abiotic),and the signaling compounds in plant defense responses such as salicylic acid(SA)and methyl jasmonate(MJ;Zhou and Wu2006;Smetanska2008).Some of these elicitors,yeast extract(mainly the polysaccharide fraction),silver ion Ag+, and hyperosmotic stress(by an osmoticum)have also been applied and shown effective to enhance the production of tanshinones in tiorrhiza hairy root cultures(Chen et al.2001;Zhang et al.2004;Shi et al.2007).To the best of our knowledge,only a few studies have been documented on the effects of elicitors,YE,SA,and MJ,on the secondary metabolite production in Agro-bacterium tumefaciens transformed tiorrhiza cell cultures from one research group(Chen and Chen1999, 2000)but not any study in normal cell cultures.The present study focuses on the effects of common biotic and abiotic elicitors including polysaccharides,heavy metal ions, SA and MJ,and osmotic stress(with sorbitol)on the growth and accumulation of three major tanshinones T-I, T-IIA,and CT in suspension culture of normal tior-rhiza cells.In addition to the effects of various elicitors on the total tanshinone content of cells,the study will examine the effects on different tanshinone species and the potential relationship to plant stress response.Material and methodsCallus induction and cell suspension cultureYoung stem explants of tiorrhiza Bunge were collected from the botanical garden at the Institute of Medicinal Plant Development,Chinese Academy of Med-ical Sciences,Beijing,China,in May2005.The fresh explants were washed with tap water,surface-sterilized with 75%ethanol for1min,and then soaked in0.1%mercuric chloride for10min and rinsed thoroughly with sterilized water.The clean and sterilized explants were cut into∼0.5-cm segments and placed on solid MS medium(Murashige and Skoog1962)supplemented with sucrose(30g l-1),2,4-D(2mg l-1)and6-BA(2mg l-1)to induce callus formation. The callus culture of tiorrhiza was maintained on a solid,hormone-free MS medium with8g l-1agar and 30g l-1sucrose at25°C in the dark and subcultured every 4weeks.The culture was deposited in Lab Y1210at The Hong Kong Polytechnic University with a collection number of Danshen cell-1.All experiments in this study were performed in suspension culture of tiorrhiza cells in a liquid medium of the same composition as for the solid culture but excluding agar.The cell suspension culture was maintained in shake-flasks,i.e.,125-ml Erlenmeyer flasks on an orbital shaker operated at110–120rpm,at 25°C in the dark.Each of the flasks was filled with25ml medium and inoculated with0.3g fresh cells from18–21-day-old shake–flask culture.Elicitor preparation and administrationEight elicitors were tested,each at three concentrations,in the initial elicitation experiments(Table1).These are representative of the four major classes of elicitors for the induction of plant responses and the stimulation of secondary metabolite production in plant tissue cultures (Zhou and Wu2006;Smetanska2008).All elicitors except MJ were prepared as a concentrated stock solution in water and autoclaved at121°C for15min,and stored at4°C in a refrigerator prior to use.Yeast elicitor(YE)was the polysaccharide fraction of yeast extract(Y4250,Sigma, St.Louis,MO,USA)prepared by ethanol precipitation as described previously(Hahn and Albersheim1978;Ge and Wu2005).In brief,yeast extract was dissolved in distilled water(20g/100ml)and then mixed with400ml of ethanol and allowed to precipitate for4days at4°C in arefrigerator.The precipitate was redissolved in100ml of distilled water and subjected to another round of ethanol precipitation.The final gummy precipitate was dissolved in 50ml of distilled water and stored at4°C before use.The concentration of YE was represented by total carbohydrate content which was determined by the Anthrone test using sucrose as a reference.Chitosan solution was prepared by dissolving0.5g crab shell chitosan(C3646,Sigma)in1ml glacial acetic acid at55–60°C for15min,and then the final volume was adjusted to50ml with distilled water and the pH adjusted to5.8with NaOH(Prakash and Srivastava 2008).MJ(Cat.39,270-7,Sigma-Aldrich)was dissolved in 95%ethanol and sterilized by filtering through a microfilter (0.2µm).SA(10,591-0,Sigma-Aldrich),sorbitol(S3755, Sigma),and the salts of heavy metals including cobalt chloride(C8661,Sigma-Aldrich),silver nitrate(S7276, Sigma-Aldrich),and cadmium chloride(C5081,Sigma-Aldrich)were dissolved in distilled water to the desired concentrations and adjusted to pH5.8.Elicitor treatment was administered to the shake–flask culture of tiorrhiza cell on day18,which was about 2–3days before reaching the stationary phase.This time point is usually favorable for elicitation when the biomass concentration is high(compared with earlier days of growth),and the cell metabolism is still active(compared with that during or after stationary phase;Buitelaar et al. 1992;Cheng et al.2006).Each of the elicitor solutions was added into the culture medium with a micropipette at the desired concentration.After the elicitor addition,the shake–flask culture of cells was maintained for another7days and then harvested for analysis.All treatments were performed in triplicate,and the results were averaged.After the initial experiments on the eight elicitors,the three most effective ones,Ag(25µM),Cd(25µM),and YE(100mg l-1)were applied in the following experiments on the time courses of elicitor-treated cell growth and tanshinone accumulation in the tiorrhiza cell culture.Measurement of cell weight,sucrose concentration, medium pH,and conductivityThe cells were separated from the liquid medium by filtration.The cell mass on the filter paper was rinsed thoroughly with water and filtered again,and blotted dry by paper towels and then dried at50°C in an oven to attain the dry weight.Sucrose concentration in the liquid medium was determined by the Anthrone test using sucrose as a reference(Ebell1969),and the medium pH and conduc-tivity were measured with the respective electrodes on an Orion720A+pH meter(Thermo Fisher Scientific,Inc., Beverly,MA,USA)and a CD-4303conductivity meter (Lutron,Taiwan),respectively.Measurement of PAL activityPhenylalanine ammonia lyase(PAL)was extracted from fresh tiorrhiza cells with borate buffer(pH8.8).The cells were ground in the buffer(0.15g ml-1)for2min with a pestle and mortar on ice,and then centrifuged at10,000rpm and4°C for20min to obtain a solid-free extract.The PAL activity was determined based on the conversion of L-phenylalanine to cinnamic acid as described by Wu and Lin(2002).Analysis of tanshinone contentsThe cell mass from culture was dried and ground into powder and extracted with methanol/dichloromethane(4:1, v/v,10mg ml-1)under sonication for60min.After removal of the solid,the liquid extract was evaporated to dryness and redissolved in methanol/dichloromethane(9:1,v/v). Tanshinone content was determined by high performance liquid chromatography(HPLC)on a HP1100system using C18column,acetonitrile/water(55:45,v/v)as the mobile phase,and UV detection at275nm as described previously (Shi et al.2007).Three tanshinone species CT,T-I,and T-IIA were detected and quantified with authentic standards obtained from the Institute for Identification of Pharmaceu-tical and Biological Products(Beijing,China).Total tanshinone content is the total content of the three tanshinones in the cells.Tanshinone content in the culture medium was negligible and not determined.ResultsCell growth and tanshinone accumulation in tiorrhiza cell cultureThe time course of tiorrhiza cell growth exhibited a lag phase or slow growth period in the first3–6days, a rapid,linear growth period between day9–18,and aTable1Elicitors and concentrations tested in the initial experiments Elicitors Unit ConcentrationC1C2C3Cobalt chloride(Co)µM 5.02550 Silver nitrate(Ag)µM 5.02550 Cadmium chloride(Cd)µM 5.02550 Salicylic acid(SA)µM1050100 Methyl jasmonate(MJ)µM1050100 Yeast elicitor(YE)mg l-150100200 Chitosan(CH)mg l-150100200 Sorbitol(SO)g l-152550stationary or declining phase in the later days,reaching the maximum biomass concentration (8.1g l -1)around day 21.The total tanshinone content of cells remained at a very low level from days 1–12and then increased steadily from days 12–27to a maximum of 0.16mg g -1.A significant portion (65%)of the tanshinone accumulation or content increase occurred during the stationary phase from days 21–27(Fig.1a ),which is characteristic of secondary metabolite production in a batch culture process.The time course of sugar (sucrose)concentration (Fig.1b )was nearly sym-metrical to that of cell growth,indicating a direct correlation of the cell growth (or biomass production)to sugar consumption.As the major carbon source,sugar was required for the S .miltiorrhiza cell growth,and when it was depleted (around day 21),the cell growth stopped,and the biomass concentration began to drop.As seen from Fig.1b ,the medium pH showed a notable drop in the first 3days (due to consumption of NH 4+and release of protons)and a gradual increase after day 6(due to consumption of nitrate NO 3-)(Morard et al.1998).Effects of various elicitors on cell growth and tanshinone productionFigure 2shows the effects of elicitor treatments on the cell growth and tanshinone accumulation in S .miltiorrhiza cell cultures,which were dependent both on the elicitor species and elicitor dose.As seen from Fig.2a ,most of the elicitor treatments except Co 2+and sorbitol at lower concentrations suppressed the cell growth to a lower biomass concentra-tion than that of the untreated control culture,and the growth suppression was more severe at a high elicitor dose.On the other hand,most of the elicitor treatments except Co 2+,sorbitol,SA,and MJ at lower concentrations increased the total tanshinone content of cell to a higher level than in the control (Fig.2b ).Overall the results indicated that the enhancement of tanshinone accumulation by an elicitor treatment concurred with a notable suppres-sion of cell growth or biomass production.Nevertheless,some of the elicitors had a much stronger stimulating effect on the tanshinone accumulation than the suppressing effect on the cell growth.In particular,Ag and Cd both at 25μM,and YE at 100mg l -1increased the total tanshinone content to 2.30mg g -1,about 11.5-fold versus that of the control (0.20mg g -1),but decreased the biomass production by no more than 50%(5.1–5.5g l -1versus 8.9g l -1).Another three elicitors,SA,MJ (both at 50μM),and sorbitol (50g l -1)increased the total tanshinone content by 2–3-fold but decreased the biomass by 30–45%compared with the control.The stimulating effect of chitosan on tanshinone accumulation (about 6-fold)was stronger than SA,MJ,and sorbitol but much weaker than Ag,Cd,and YE,while its suppressing effect on the cell growth was as severe as Ag,Cd,and YE.In summary,the results indicate that Ag,Cd,YE are the most favorable elicitors for the tanshinone production in S .miltiorrhiza cell culture and were used in the following experiments.Figure 3shows the time courses of cell growth and tanshinone production after treatment with the three most effective elicitors Ag (25μM),Cd (25μM),and YE (100mg l -1)and the control culture.All three elicitor treatments caused a steady decline of biomass concentration from initially 8.5g l -1to 5.3g l -1on day 6while biomass in00.040.080.120.160.20246810TT content (mg g -1)C e l l b i o m a s s (g d w l -1)dw TTa4.85.1 5.45.76.001020304036912151821242730p HS u c r o s e (g l -1)Culture time (d)bSucrosepHFig.1Time courses of biomass and total tanshinone content (a ),residue sugar (sucrose)and medium pH (b )in S .miltiorrhiza cell cultures (error bars for standard deviations,n =3)246810C e l l b i o m a s s (g l -1)0.00.51.01.52.02.5Control AgCdSAMJYECH SOT T c o n t e n t (m g g -1)Elicitor treatmentCo Fig.2Effects of various elicitors on biomass growth (a )andtanshinone production (b )in S .miltiorrhiza cell cultures (elicitors added to cultures on day 18at three concentrations C1,C2,and C3as shown in Table 1,and cultures harvested on day 25;error bars for standard deviations,n =3)the control culture was increased during this period (Fig.3a ).In the meantime,the tanshinone content of cells in the three elicitor-treated cultures increased sharply and most rapidly by Ag (from 0.14to 1.98mg g -1),while that of control increased slightly (from 0.14to 0.21mg g -1;Fig.3b ).The volumetric total tanshinone yields (the products of total tanshinone content and cell dry weight)were 1.9mg l -1in the control,and 9.2mg l -1,10.7mg l -1and 11.7mg l -1in cultures treated with 100mg l -1YE,25μM Cd,and Ag,respectively (on day 6).Another test was performed on the effects of two and three elicitors in combinations in the S .miltiorrhiza cell culture.As shown in Fig.4,the tanshinone content was increased about 20%with either two elicitors and about 40%with all three elicitors in combination compared with that with a single elicitor.The results suggest an additive or synergistic effect of these elicitors on the tanshinone accumulation in the cells.However,the combined use of two or three elicitors also suppressed the cell growth (biomass)more severely than with a single elicitor.Effects of elicitor treatments on different tanshinone species Of the three tanshinone species detected,CT was stimulated most significantly by all elicitors without exception;T-IIA was stimulated by most elicitors,and T-I was stimulated significantly only by chitosan but slightly stimulated or suppressed by other elicitors (Table 2).The highest CT content was about 2mg g -1(1,854–2,011μg g -1)in cellcultures treated with 25μM Ag and Cd,and 100mg l -1YE,about 31–34fold of the control level (60μg g -1),the highest T-I content 0.27mg g -1with 100mg l -1chitosan (3.4-fold of the control 80μg g -1)and the highest T-IIA content 0.37mg g -1with 25μM Cd (6-fold of the control 60μg g -1).As seen from the HPLC chromatograms (Fig.5),the cultures treated with the three different elicitors exhibited a similar profile with virtually identical major peaks.The experimental results do not suggest any specificity of particular tanshinone species to the type of elicitors,YE and chitosan as biotic polysaccharides,Cd and Ag as abiotic heavy metals,or SA and MJ as plant stress signaling pared with that of control,the HPLC profiles of elicitor-treated cultures also had three new unknown peaks appearing before the CT peak,between 10.0–11.5min and a high peak at 11.1min,which0.00.51.01.52.02.5123456T T c o n t e n t (m g g -1)Time after treatment (d)b4681012C e l l b i o m a s s (g l -1)Control Ag 25Cd 25YE 100aFig.3Time courses of biomass (a )and total tanshinone content (b )in S .miltiorrhiza cell cultures after treatment with Ag (25µM),Cd (25µM),and YE (100mg l -1;error bars for standard deviations,n =3)24681012345Cell dry weight (g l -1)T T c o n t e n t (m g g -1)Elicitor treatmentTTdwFig.4Effects of single and combined elicitors on S .miltiorrhiza cell growth and tanshinone accumulation (elicitors added to cell cultures on day 18at the same concentrations as in Fig.3,and cultures harvested on day 25;error bars for standard deviations,n =3)Table 2Effects of various elicitors on the accumulation of three tanshinones in S .miltiorrhiza cells Treatment aContent,μg/g (fold of content control)CTT-IT-IIA Control 59.9(1)81.6(1)57.6(1)Co-50263.7(4.4)67.5(0.83)55.5(0.96)Ag-251,817.5(30)71.0(0.87)225.8(3.9)Cd-251,854.0(31)80.3(0.98)369.0(6.4)SA-100390.0(6.5)78.5(0.96)72.8(1.3)MJ-100299.8(5.0)109.5(1.3)82.6(1.4)YE-1002,011.4(34)90.3(1.1)190.3(3.3)CH-100597.2(10)276.0(3.4)98.8(1.7)SO-50584.6(9.8)56.9(0.70)83.0(1.4)CT cryptotanshinone,T-I tanshinone I,T-IIA tanshinone-IIAaNumber after each elicitor symbol represents the elicitor concentra-tion as shown in Table 1may be ascribed to tanshinone relatives of higher polarity than CT induced by the elicitors.PAL activity,pH,and conductivity changes induced by elicitorsFigure 6shows the changes of intracellular PAL activity and medium pH and conductivity in the S .miltiorrhiza cell culture after the treatment by Ag (25μM),Cd (25μM),and YE (100mg l -1).The PAL activity of cells was stimulated by all three elicitors to the similar level,from 1.4-to 1.9-fold of the control level over 6days (Fig.6a ).PAL is a key enzyme at the entrance step in the phenylpropanoid pathway in plants,and its activity increase stimulated by the elicitors is suggestive of an enhanced secondary metabolism in the plant cells (Taiz and Zeiger 2006).The pH and conductivity of culture medium were also increased (to higher levels than those of the control)by all three elicitors but more significantly by YE (Fig.6b,c ).Most significant increases (differences from the control level)in the medium pH and conductivity were shown in the very early period from day 0–1.The increase in medium conductivity in the early period was most probably attributed to the release of potassium K +ion from the cells or K +efflux across the cell membrane (Zhang et al.2004).Transient medium pH increase (alkalinization)and K +efflux across the cell membrane are early and important events in the elicitation of plant responses and phytoalexin production (Ebel and Mithöer 1994;Roos et al.1998).The conductivity decline in the later period after day 1of Ag +and Cd 2+-treated cultures and the control cultures can be attributed to the consumption of inorganic and mineral nutrients in the culture medium (Kinooka et al.1991).Overall,the results here provide further evidence forthe01234R e l a t i v e P A L a c t i v i t yControl Ag CdYEa5.05.45.86.26.6M e d i u m p H b2.03.04.05.06.00246M e d i u m c o n d u c t i v i t y (m S )Time after treatment (d)cFig.6Time courses of PAL activity (a ),medium pH (b ),and conductivity (c )of S .miltiorrhiza cell cultures after elicitor treatments in comparison with the control (error bars for standard deviation,n =3)elicitor activities of Ag,Cd,and YE in stimulating the stress responses and secondary metabolism of the S. miltiorrhiza cells.DiscussionThe effects of various elicitors on tanshinone accumulation found here in the normal tiorrhiza cell cultures are in general agreement with those found in transformed cell and hairy root cultures of tiorrhiza.In transformed cell cultures(Chen and Chen1999),the CT accumulation was also stimulated significantly by YE but not by SA or MJ alone,and YE also inhibited the cell growth.The tanshinone(mainly CT)production in hairy root cultures was also enhanced significantly(3–4fold)by Ag(Zhang et al.2004)and YE(Shi et al.2007).In all these culture systems,CT was the major tanshinone species stimulated by various elicitor treatments.CT has been identified as a phytoalexin in tiorrhiza plant which plays a defense role against pathogen invasion of the plant(Chen and Chen 2000).In this connection,the stimulated CT accumulation by the elicitors may be a defense or stress response of the cells.CT was also the major diterpenoid produced by a normal tiorrhiza cell line which was initially grown in the MS medium and then transferred to a production medium containing only about half of the nutrient compo-nents of the MS medium(Miyasaka et al.1987).It is very possible that the improvement of CT yield in this production medium was also attributed,at least partially, to the stress imposed by the nutrient deficiency which suppressed growth but stimulated secondary metabolite accumulation.MJ or its relative jasmonic acid has been shown effective for stimulating a variety of secondary metab-olites in plant tissue cultures such as hypericin in Hypericum perforatum L.(St.John’s Wort)cell cultures (Walker et al.2002),paclitaxol(diterpenoid)and related taxanes in various Taxus spp.and ginsenosides in Panax spp.(Zhong and Yue2005),and bilobalide and ginkgo-lides in Ginkgo biloba cell cultures(Kang et al.2006). However,MJ showed only a moderate or insignificant stimulating effect on tanshinone accumulation in normal and transformed tiorrhiza cell cultures.The discrep-ancy suggests that the effects of various elicitors on secondary metabolite production in plant tissue cultures are dependent on the specific secondary metabolites.This argument is also supported by the much stronger stimu-lation of CT than T-I and T-IIA by most elicitors found in our tiorrhiza cell cultures.In addition,the hairy roots appeared more tolerant to the elicitor stress,and the growth was less inhibited by the elicitors or even enhanced in some cases,e.g.,by YE(Chen et al.2001)and sorbitol(Shi et al.2007).Moreover,sorbitol as an osmotic agent significantly stimulated the tanshinone accumulation(3–4folds)in tiorrhiza hairy root cultures,but not so significantly in the cell cultures.This shows that the elicitor activities for the same metabolites can vary with the tissue culture systems.In conclusion,the polysaccharide fraction of yeast extract and two heavy metal ions Ag+and Cd2+were potent elicitors for stimulating the tanshinone production in tiorrhiza cell culture.The stimulated tanshinone production by most elicitors was associated with notable growth suppression.CT was more responsive to the elicitors and enhanced more dramatically than another two tanshinones,T-I and IIA.The results from this study in the tiorrhiza cell cultures and from previous studies in hairy root cultures suggest that the cell and hairy root cultures may be effective systems for CT production, provided with the elicitors.As most of the elicitor chemicals are commercially available or can be readily prepared in the laboratory and easily administered to the cell and root cultures,they are suitable for practical applications in the laboratory or large-scale production. Acknowledgements This work was supported by grants from The Hong Kong Polytechnic University(G-U502and1-BB80)and the China Hi-Tech Research and Development Program(2006AA10A209).ReferencesBuitelaar RM,Cesário MT,Tramper J(1992)Elicitation of thiophene production by hairy roots of Tagetes patula.Enzyme Microb Technol14:2–7Chen H,Chen F(1999)Effects of methyl jasmonate and salicylic acid on cell growth and cryptotanshinone formation in Ti transformed Salvia miltiorrhiza cell suspension cultures.Biotechnol Lett 21:803–807Chen H,Chen F(2000)Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures.Plant Cell Rep19:710–717Chen H,Chen F,Chiu FCK,Lo CMY(2001)The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza.Enzyme Microb Technol28:100–105Cheng XY,Zhou HY,Cui X,Ni W,Liu CZ(2006)Improvement of phenylethanoid glycosides biosynthesis in Cistanche deserticola cell suspension cultures by chitosan elicitor.J Biotechnol 121:253–260Chong TM,Abdullah MA,Lai QM,Nor’Aini FM,Lajis NH(2005) Effective elicitation factors in Morinda elliptica cell suspension culture.Process Biochem40:3397–3405Ebel J,Mithöer A(1994)Early events in the elicitation of plant defence.Planta206:335–348Ebell LF(1969)Variation in total soluble sugars of conifer tissues with method of analysis.Phytochemistry8:227–233Ge XC,Wu JY(2005)Tanshinone production and isoprenoid pathways in Salvia miltiorrhiza hairy roots induced by Ag+and yeast elicitor.Plant Sci168:487–491。

Solvothermal in situ synthesis of Fe3O4-multi-walled carbon nanotubes with enhanced heterogeneous Fenton-like activityJingheng Deng a,1,Xianghua Wen a,*,Qinian Wang ba State Joint Key Laboratory of Environment Simulation and Pollution Control,Department of Environmental Science and Technology,Tsinghua University,Beijing100084,PR Chinab College of Environment and Chemical Engineering,China University of Mining and Technology,Beijing100083,PR China1.IntroductionRecent development of high quality monodispersion ofmagnetite nanoparticles has allowed for the reinvestigation ofnew applications,such as being used as advanced magneticmaterials,ferrofluid,rechargeable conversion electrode material,in biological imaging and environmental remediation[1–5].Forpractical application,the magnetic nanoparticles must havecontrollable sizes,uniform morphologies,good crystallinity,andhigh dispersity since all of these affect their magnetic propertiesand the catalytic activities[6,7].However,magnetic nanoparticlesare very easy to aggregate into larger clusters owing to anistropicdipolar attraction,losing the dispersibility and specific properties[8,9].Therefore,a large number of studies on stabilizing magneticnanoparticles have been carried out.One way is to modify thenanoparticles with organic chemicals,such as surfactants orpolymers,or to coat the nanoparticles with inorganic chemicals,such as silica or carbon[10–13].In recent years,carbon nanotubes(CNTs)have been consideredas high-performance supports due to their intriguing nanoscaledimensions,high specific surface area,electrical properties,mechanical strength,and chemical stability[14,15].The utilizationof CNTs as a support for Fe3O4nanoparticles has drawn favorableattention because it prevents agglomeration and thus enhancestheir performances[16].More importantly,CNTs can serve notonly as supports but also as components with synergistic andhybrid properties[17–19].Successful loading of Fe3O4nanopar-ticles onto the outer surface of CNTs have been performed throughthe following techniques:high-temperature decomposition meth-od[20],hydrothermal or solvothermal process[21,22],polymerwrapping and the layer-by-layer assembly method[23],electro-static interaction[24–26],and wet chemistry[27,28].Thesetechniques have demonstrated to be useful to synthesize Fe3O4nanoparticles and CNTs hybrid materials.However,high-temper-ature method(5008C)was harsh and energy-consuming.Self-assembly method was operationally tedious and complicated.Wetchemistry method attained relatively poor crystallinity andmagnetic properties,and it was difficult to control Fe3O4nanoparticles size and size distribution[29].Among thesemethods,in situ hydrothermal or solvothermal synthesis providean promising approach,which possess some advantages over theother techniques for controlling particle size,morphology,and sizedistribution[30].For example,Wang et al.[20]successfullyprepared10–25nm magnetite nanoparticles deposited on theCNTs in a benzene medium.Zhang et al.[31]coated20–60nmFe3O4nanoparticles onto CNTs using hydrazine hydrate.However,in both cases the iron oxide nanoparticles were not small enoughand the particle size distribution was not narrow enough.As thecatalytic and magnetic properties of magnetic nanoparticlesdepend on the size,small enough nanoparticles are desired.Materials Research Bulletin47(2012)3369–3376A R T I C L E I N F OArticle history:Received16November2011Received in revised form23June2012Accepted28July2012Available online3August2012Keywords:A.Magnetic materialsC.X-ray diffractionC.Photoelectron spectroscopyD.Catalytic propertiesA B S T R A C TFe3O4-multi-walled carbon nanotubes(Fe3O4-MWCNTs)hybrid materials were synthesized by asolvothermal process using acid treated MWCNTs and iron acetylacetonate in a mixed solution ofethylene glycol and ultrapure water.The materials were characterized using X-ray powder diffraction,scanning and transmission electron microscopy,X-ray photoelectron spectroscopy,and vibratingsample magnetometry.The results showed that a small amount of water in the synthesis system playeda role in controlling crystal phase formation,size of Fe3O4,and the homogeneous distribution of theFe3O4nanoparticles deposited on the MWCNTs.The Fe3O4nanoparticles had diameters in the range of4.2–10.0nm.They displayed good superparamagnetism at room temperature and their magnetizationwas influenced by the reaction conditions.They were used as a Fenton-like catalyst to decompose AcidOrange II and displayed a higher activity than nanometer-size Fe3O4.ß2012Elsevier Ltd.All rights reserved.*Corresponding author.Tel.:+861062772837;fax:+861062772837.E-mail address:xhwen@(X.Wen).1Current address:Changsha Research Institute of Mining and Metallurgy,Changsha410012,PR China.Contents lists available at SciVerse ScienceDirectMaterials Research Bulletinj o u rn a l h om e p a ge:w w w.e l s e v i e r.c o m/l o c a t e/m a t r e s b u0025-5408/$–see front matterß2012Elsevier Ltd.All rights reserved./10.1016/j.materresbull.2012.07.021Additionally,they used either benzene or hydrazine hydrate as solvent that is poisonous.Nowadays,safe and environmentally friendly solvents are favored to attain desired target products.In this context,the main purposes of this paper are to present an environmentally friendly,easily-controlled approach to coat monodisperse Fe3O4nanoparticles on multi-walled carbon nano-tubes(MWCNTs).We present in situ solvothermal approach to fabrication Fe3O4-MWCNTs.Iron acetylacetonate(Fe(acac)3)is used as the iron source in the solvothermal synthesis of Fe3O4-MWCNTs because of its low cost,easy preparation,and mild decomposition[32].Ethylene glycol is environmentally friendly solvent and reducing ing ethylene glycol as a solvent and a reducing agent has been tried to produce many metal and bimetallic nanoparticles[33–35].As a weak reducing agent, ethylene glycol can reduce Fe(III)to Fe(II),but not further reduce Fe(II)to metal iron which is a merit in the Fe3O4fabrication. Previous studies suggested that the presence of water in non-aqueous played an important role in controlling oxides nanopar-ticles particle size and size distribution[36,37].Water is such a solvent that is nontoxic,nonflammable,and cheap.The physico-chemical properties of water,such as the dielectric constant,ion product,and dynamic viscosity,change considerably under near-critical condition(temperature between150and2508C)[38,39]. The near-critical water can even play the role of acid or base catalyst in organic synthesis.Accordingly,this paper focuses primarily on the small particle size Fe3O4nanoparticles uniformly coating on MWCNTs in water with ethylene glycol as the reducing agent and solvent,without exertion of any additional protective agents.The effects of synthesis temperature,reaction time,and water amount on the catalyst properties were analyzed.The probable mechanisms of Fe3O4nanoparticles in situ decoration on MWCNTs are discussed.In addition,the catalytic activity of the Fe3O4-MWCNTs in the decomposition Orange II was explored.2.Experimental2.1.MaterialsThe as-received MWCNTs(diameter,30–50nm;length, $20m m)used in this work were purchased from Chengdu Institute of Organic Chemicals,Chinese Academy of Science.All the other chemicals were analytical grade and used without further purification.Ethylene glycol and H2O2were purchased from the Beijing Chemical Reagents Company.Fe(acac)3was purchased from Alfa Aesar Co.Powder Fe3O4was obtained from Aladdin(99%,Shanghai,China).The Acid Orange II was purchased from Hengye Jingxi Chemical Co.,Ltd.2.2.Purification of MWCNTsFor purification and oxidation the MWCNTs surface to facilitate a uniform Fe3O4deposition on their outer walls, 2.0g of the pristine MWCNTs were dispersed in200mL68%HNO3by exerting ultrasonic dispersion(Kunshan,KQ-250DE,50kHz,100W)and refluxed at1408C with constant stirring for14h.After cooling to room temperature,the black suspension was washed through a vacuumfilter with adequate ultrapure water until pH neutral,and then dried.The dried MWCNTs were milled into powder for use.2.3.Preparation of Fe3O4-MWCNTsIn a typical experiment,MWCNTs(0.1g)and Fe(acac)3(0.2g) were added to a solvent mixture of ethylene glycol(10mL)and various amount of ultrapure water(0–7mL).The suspension was poured into80mL stainless steel autoclave and sealed after the suspension bubbled with purified N2.And then the autoclave was placed in oil bath and heated to2008C for30min,and to2608C for another30min.The autoclave was taken out of the oil bath and cooled to room temperature naturally.The sample wasfiltrated, washed with ethanol several times and vacuum dried at808C for 24h.For comparison,the same preparation processes were applied to fabrication of nanometer-size Fe3O4.2.4.CharacterizationThe as-prepared products were characterized using X-ray powder diffraction(XRD)(Rigaku D/max TTRIII,Cu K a, l=0.15406nm),scanning electron microscopy(SEM)(JSM-7401,Japan),transmission electron microscopy(TEM)(JEM-2010,Japan),and high-resolution transmission electron microsco-py(HRTEM)(JEM-2010,Japan)equipped with selected-area electron diffraction(SAED)and energy-dispersive X-ray spectrom-eter(EDX).The particles size of Fe3O4nanoparticles was obtained from TEM image using Nano Measure software(Fudan University, China).Chemical characterization of the samples surface was recorded using X-ray photoelectron spectroscopy(XPS)(PHI Quantera,ULVAC-PHI,Inc.),with Al K a(h n=1486.7eV)X-ray as the excitation source.The magnetic properties were measured at room temperature using a vibrating sample magnetometer(VSM) (730T,Lake Shore,USA).2.5.Catalyst activity testsThe heterogeneous catalytic activities of the Fe3O4-MWCNTs were evaluated based on the results of the degradation of Acid Orange II(Orange II).The reaction suspension was prepared by adding30mg Fe3O4-MWCNTs powder into60mL Orange II solutions with its initial concentration of0.25mmol LÀ1and pH of3.5as adjusted by H2SO4or NaOH.The reactions were initiated by adding1.0mL0.9mol LÀ1H2O2and stirred continuously in a temperature controlled condition.After30min,the samples were filtered through a0.45m m cellulosefilter paper and analyzed for Orange II concentration by using Hach DR5000UV-vis spectro-photometer.3.Results and discussion3.1.Effects of water/ethylene glycol ratio on the Fe3O4nanoparticles’formation on MWCNTsFig.1illustrates XRD patterns of the purified MWCNTs and Fe3O4-MWCNTs using various amount of water at the same reaction temperature(2608C)and holding time(30min,the holding time means the time between the reaction systems reach 2608C and start to take out of oil bath).In Fig.1a,the diffraction peaks at2u=26.08can be indexed to the(002)reflection of MWCNTs.It showed acid-oxidation pretreatment of MWCNTs did not cause significantly structural damage to the MWCNTs.From the XRD patterns of the coated MWCNTs(Fig.1b–h),we can see that the characteristic peaks of MWCNTs still exist and the other peaks at2u values about18.38,30.18,35.48,43.18,53.38,56.98,62.58 can be easily indexed as the cubic spinel phase of Fe3O4(JCPDF:19-0629).However,In Fig.1i,new diffraction peaks came out at2u values about24.18,33.08,40.88,49.58in addition to the above mentioned peaks.It indicated that the third phase existed.Judging from the pattern,the corresponding peaks results can be indexed to a-Fe2O3(JCPDF33-0664).The result demonstrated that the purity of the product Fe3O4depended on the volume ratio of water/ ethylene glycol.Previous studies on the synthesis of Fe3O4in organic solvent have suggested that a suitable amount of water was significant for the crystallization.Excessive water might lead to further oxidation of Fe3O4and transformation to Fe2O3J.Deng et al./Materials Research Bulletin47(2012)3369–3376 3370[30,40,41].Therefore,the detection of Fe 2O 3might be ascribed to the oxidation of Fe 3O 4to a -Fe 2O 3by excessive water.Thus,in order to prepare pure Fe 3O 4,the volume ratio of water/ethylene glycol should be maintained at no more than 7:10in the fabrication system.The results based on XRD data of the products prepared with various ratios of water/ethylene glycol (while other condi-tions were kept constant)were listed in Table 1.It should be noted that g -Fe 2O 3has the same inverse spinel cubic structure and an almost identical lattice constant as Fe 3O 4[42].Therefore,the results of XRD cannot confirm the formation of Fe 3O 4.XPS is a surface chemical analysis technique that can beused to analyze the chemical environment and oxidation state of the atom.The as-obtained sample was then further analyzed using XPS technique to verify if the nanoparticles on MWCNTs were Fe 3O 4or g -Fe 2O 3.Fig.2reveals wide scan of sample 3and the Fe 2p spectrum of sample 3(inset of Fig.2).As seen from Fig.2,the wide scan exhibits Fe,O,and C elements existence.The core level spectrum peaks is at about 711and 724eV,corresponding to Fe 2p 3/2and 2p 1/2levels,respectively.The absence of satellite peak at the 716–720eV regions excluded the existence of g -Fe 2O 3phase in the Fe 3O 4-MWCNTs,which indicated that the sample was pure Fe 3O 4[43,44].3.2.Effects of water/ethylene glycol ratio on the Fe 3O 4nanoparticles’monodispersion on MWCNTsThe water content in solvent not only influenced the crystal phase of Fe 3O 4but also its size and dispersion on the MWCNTs.Fig.3depicts TEM (a)and HRTEM (b)micrographs of sample 1.It can be seen from Fig.3a that the Fe 3O 4nanoparticles were not dispersed adequately and instead aggregates were formed.The diameters of the aggregates ranged from 55to 110nm.Fig.3b shows HRTEM image of Fe 3O 4nanoparticles on the MWCNTs.The clear lattice fringes having a distance of 0.299nm reveals that the growth of Fe 3O 4nanoparticles occurs preferentially on the (220)plane.The inset of Fig.3b shows the SEAD patterns.The bright circular rings indicated Fe 3O 4nanoparticles were polycrystalline.Fig.4a shows the typical SEM and Fig.4b the TEM micrographs of sample 2,from which one can see that the Fe 3O 4clusters gradually dispersed and petal-like structure formed.The diameters of the Fe 3O 4clusters ranged from 14to 40nm,and the average diameter was 29.5nm.While the volume ratio of water/ethylene glycol increased to 2:10,as shown in Fig.5a,the Fe 3O 4clusters were thoroughly dispersed forming Fe 3O 4nanoparticles that were uniformly distributed without aggregation on the MWCNTs.Fig.5b presents the histogram of the particle size distribution and Gaussian fitting curve of sample 3.The diameters had a narrow size distribution and ranged from 4.2to 10.0nm.The average diameter of Fe 3O 4particles was 7.4nm which was slightly bigger than the data analyzed by the XRD (6.8nm).In addition,the iron content in the Fe 3O 4-MWCNTs was determined by EDX being 15.1wt%.Fig.6illustrates the SEM micrographs of sample 4,sample 5,sample 6,and sample 7.As seen from Fig.6,the Fe 3O4Fig.1.XRD patterns.(a)Purified MWCNTs;(b–e)Fe 3O 4-MWCNTs prepared using volume ratios of water/ethylene glycol (v/v,mL)(b)0:10,(c)1:10,(d)2:10,(e)3:10,(f)4:10,(g)5:10,(h)6:10,(i)7:10.Symbols (&),(*)and (5)represent peaks of MWCNTs,Fe 3O 4,a -Fe 2O 3,respectively.Table 1Product phases and crystal size derived from the XRD patterns under various reaction conditions.Sample Volume ratio(water:ethylene glycol)Temperature (8C)Time (min)Phase by XRD Size (nm)a10:1026030Fe 3O 4 6.821:1026030Fe 3O 47.432:1026030Fe 3O 48.343:1026030Fe 3O 49.754:1026030Fe 3O 411.065:1026030Fe 3O 411.876:1026030Fe 3O 414.287:1026030Fe 3O 4+a -Fe 2O 321.192:1020030Fe 3O 4 2.2102:1021030Fe 3O 4 5.5112:1022030Fe 3O 4 6.0122:1023030Fe 3O 4 6.2132:1024030Fe 3O 4 6.7142:1025030Fe 3O 47.8152:1027030Fe 3O 48.7162:1026020Fe 3O 48.3172:1026040Fe 3O 48.6182:1026050Fe 3O 49.1192:1026060Fe 3O 412.6aCrystal size calculated based on the (311)diffraction peaks of Fe 3O 4according to Scherrer’s equation,D =(0.89l )/(b cos u).Fig.2.XPS wide scan spectrum of sample 3,the inset is Fe 2p spectrum.J.Deng et al./Materials Research Bulletin 47(2012)3369–33763371nanoparticles displayed uniform sizes and a narrow size distribu-tion,homogeneously decorated on the MWCNTs.The average crystal size of the Fe 3O 4calculated based on XRD pattern slightly increased with the increase of the volume ratio water/ethylene glycol from 3:10to 6:10(Table 1).This may be attributed to the increase of the autogeneous pressure while water content increases,while high pressure system plays part in forming larger size formation [45].Small nanoparticles are of important for futureapplications for their size-dependent properties.Therefore,the optimal ratio of water/ethylene glycol was 2:10in the present system.In addition to water content in solvent,MWCNTs could influence on Fe 3O 4nanoparticles’homogeneous dispersion.MWCNTs in bulk solution acted as seeds for heterogeneous nucleation.Defects and organic groups on MWCNTs surface are in general preferable nucleation sites.A lot of small nuclei couldformFig.4.SEM (a)and TEM micrographs (b)of the sample2.Fig.5.TEM micrograph of sample 3(a)and the histogram of Fe 3O 4particle size distribution(b).Fig.3.TEM (a)and HRTEM (b)micrographs of sample 1,the inset of (b)is the corresponding SAED pattern.J.Deng et al./Materials Research Bulletin 47(2012)3369–33763372within a short time and tend to deposit on the MWCNTs because heterogeneous nucleation has lower free energy barriers in comparison with homogeneous nucleation.3.3.The effect of temperature on crystallizationFixing the volume ratio of water/ethylene glycol at 2:10and holding reaction time,the effect of temperatures on the crystallization was explored.Fig.7displays XRD patterns of samples obtained at various temperatures.As seen from Fig.7,all samples exhibited diffraction peaks at 2u values around 18.38,30.18,35.48,43.18,53.38,56.98,62.58.This ascribed to Fe 3O 4characteristic diffraction peaks.The broad diffraction peaks indicated that the crystalline size of Fe 3O 4particles was quite small.The average crystal size was summarized in Table 1.It was noticeable that the intensities of diffraction peaks gradually increased with increasing solvothermal temperature from 200to 2708C.The low intensities between 2008C and 2308C explicated Fe 3O 4poor crystalline.It was worth pointing out that in the hydrothermal processing,large amounts of Fe(acac)3would remain in the solution when the reaction temperature was lower than the decomposition temperature of 2508C (t =30min).It was reported that Fe(acac)3did not thoroughly decompose and there was about 8.8%residual mass even at temperature below 2628C [46].Accordingly,the optimal temperature was 2608C in the present system.3.4.The effect of holding time on the crystallization and crystal sizeFor further investigating the effects of holding time on crystallization and size of Fe 3O 4at the determined temperature and ratio of water/ethylene glycol,holding time was tested from 20min to 60min.The XRD patterns in Fig.8revealed that diffraction peaks intensities gradually increased with prolonging holding time,especially for 60min.It suggests that the crystallini-ty of nanoparticles Fe 3O 4increased with reaction time.The average crystal size was summarized in Table 1.The results showed little change in the mean crystal size throughout different reaction times from 20min to 50min.However,the mean crystal size increased remarkably to 12.6nm at 60min.This may be attributed to Ostwald ripening.As a result,in order to gain small nanoparticles Fe 3O 4,the holding temperature time should be controlled at 30min for this experiment.Therefore,the optimumexperimentalFig.6.SEM micrographs of sample 4(a),sample 5(b),sample 6(c),and sample 7(d).Fig.7.XRD patterns of Fe 3O 4-MWCNTs prepared at various temperatures.Symbols (&)and (*)represent peaks of MWCNTs,Fe 3O 4.J.Deng et al./Materials Research Bulletin 47(2012)3369–33763373conditions for improved dispersion of Fe 3O 4nanoparticles on MWCNTs were:0.1g MWCNTs,0.2g Fe(acac)3,2mL water and 10mL ethylene glycol,2608C temperature,30min holding temperature time.3.5.Magnetic properties of Fe 3O 4-MWCNTsThe magnetic properties of Fe 3O 4-MWCNTs samples prepared at various temperatures were also investigated.Magnetization curves at room-temperature are shown in Fig.9.The saturation magnetization values of samples prepared at 200,210,220,230,240,250,260,2708C were 5.07,13.06,14.10,16.02,18.07,18.72,20.0,22.06emu/g,respectively,which was lower than that of the pure Fe 3O 4.The decrease in the saturation magnetization mainly resulted from the existence of MWCNTs [21].With the elevation of temperature,the saturation magnetization increased.This was attributed to the increase in the crystallinity of Fe 3O 4nanopar-ticles.The coercivity and the remnant magnetization of all the samples were close to nil.This suggests superparamagnetism of Fe 3O 4nanoparticles.Magnetic material has the advantage of external magnetic field separation in practical applications.The inset in Fig.9illustrates the ease separation of the sample using a magnet near the vial wall.This shows the high magnetic sensitivity of Fe 3O 4-MWCNTs.3.6.Mechanism for the formation of Fe 3O 4-MWCNTs hybrid materialsSince there are few details on the mechanism behind Fe 3O 4-MWCNTs hybrid formation,the following mechanism was proposed based on experimental results.The formation of Fe 3O 4-MWCNTs hybrids involved basically two steps:Step (1):Ethylene glycol reduces the Fe (III)salt to an Fe (II)intermediate before forming into Fe 3O 4through decomposition at high temperature [47].The reaction process probably involves the following chemical reactions:HOCH 2CH 2OH !CH 3CHO þH 2O(1)CH 3CHO þ6Fe ðacac Þ3þ9H 2O !2Fe 3O 4þCH 3COOHþ18Hacac(2)Step (2):To some extent,the MWCNTs serves as a nuclei for Fe 3O 4epitaxial growth via heterogeneous nucleation because free energy for heterogeneous nucleation is lower than that for homogeneous nucleation.Defects and organic groups on MWCNTs surface facilitate the binding of metal atoms.Moreover,solvent properties such as polarity,viscosity,and softness would strongly influence the solubility and transport behavior of the precursors [48].In the present system,the addition of water changes the chemical properties of the solvent such as viscosity,surface tension,dielectric constant,interionic attraction,and the solute–solvent interaction because of solubility differences [49].This addition of water also changes the pH value of the local solution,which could have a significant effect on heterogeneous nucleation,crystal growth,and phase formation in the present study [50].Additionally,heterogeneous nucleation depends on foreign particles size,curvature,and its surface properties.However,we do not yet know enough about which factor plays a key role in the homogeneous growth of Fe 3O 4on MWCNTs.Better understanding of the formation mechanism is needed.3.7.Enhanced activity of Fe 3O 4-MWCNTs as heterogeneous Fenton-like catalystFe 3O 4nanoparticles could be used as heterogeneous Fenton-like catalysts for catalytic oxidation of organic compounds,such as phenol and aniline [51,52].In order to illustrate the significantly enhanced catalytic activity of Fe 3O 4-MWCNTs,Fe 3O 4-MWCNTs,powder Fe 3O 4and nanometer-size Fe 3O 4were used to catalyze the H 2O 2-driven degradation of Orange II under identical conditions.In order to test the enhanced catalytic activity of Fe 3O 4-MWCNTs,powder Fe 3O 4,nano-Fe 3O 4,Fe 3O 4-MWCNTs and MWCNTs were respectively employed to catalyze the degradation of Orange II by adding H 2O 2to start the reaction (catalysts:30mg,Orange II:60mL,0.25mmol L À1,pH:3.5,H 2O 2:15mmol L À1).Fig.10shows that powder Fe 3O 4was a poor catalysis for H 2O 2-driven degradation of Orange II (only 15.8%).Even for nanometer-size Fe 3O 4,only a degradation efficiency of 37.1%was observed.The result was consistent with Zhu’s report on Fe 3O 4nanoparticles exhibiting low catalytic activity for degrading stable organic compounds [53].In contrast,the degradation efficiency of Fe 3O 4-MWCNTs reached 94.0%,which was two times more than that of nanometer-size Fe 3O 4.When using MWCNTs,it represents its own adsorption efficiency (21.6%adsorption of anionic dye Orange II;data not shown)because it does not catalyze H 2O 2to degrade Orange II in the system.These comparisons were made based on equal weight of catalysts.If the comparison was based on the amount of Fe 3O 4used,the degradation efficiency of Fe 3O 4-MWCNTs would be as high as six times that ofnanometer-sizeFig.8.XRD patterns of Fe 3O 4-MWCNTs prepared at various time.Symbols (&)and (*)represent peaks of MWCNTs,Fe 3O 4.Fig.9.Room temperature magnetization curves of samples prepared at various temperatures.Inset:photograph of magnetic separation of the Fe 3O 4-MWCNTs from solution.J.Deng et al./Materials Research Bulletin 47(2012)3369–33763374Fe 3O 4due to Fe 3O 4accounting for 19.5%(the 15.1wt%Fe is equivalent to 19.5%Fe 3O 4)of Fe 3O 4-MWCNTs.The significant activity enhancement can be attributed to homogeneous dispersion of the Fe 3O 4nanoparticles,which increases the active sites for substrate access.Other reasons for high activity can be explained as following.Fe 3O 4demonstrates Fenton-like behavior because it can decompose H 2O 2and produce hydroxyl free radical.In the meantime,the valence states of the surface iron are interconverted between 2+and 3+during the reaction with H 2O 2[54].It is well known that the oxidation of the ferrous iron by H 2O 2is much faster than the reduction of the ferric iron by H 2O 2.Thus,in order to speed up the redox cycle between ferrous and ferric species,it should improve the reduction of the ferric iron by H 2O 2.It is reported by Lim et al.[55]that the electron-poor groups which is associated with iron,could promote the reduction of the ferric ion by H 2O 2to enhance catalytic activity.In the case of Fe 3O 4-MWCNTs,MWCNTs have nonplanar sp 2-hybridized framework.Iron is bound to MWCNTs which leads to a partial electron transfer to the graphite due to the d orbital of Fe hybridizing strongly with the p z orbital of the graphitic carbon [56–58].Accordingly,the Fe (III)facilitates the reduction by H 2O 2to accelerate the cycle between Fe (III)and Fe (II)because the electron density of iron transfers from the iron center.The significantly enhanced catalytic activity is attributed to synergetic effect originated from Fe 3O 4and MWCNTs hybrid structure [59].This hypothesis needs further experimental and theoretical studies to confirm.4.ConclusionsIn this work,we have devised a simple solvothermal approach for the preparation of Fe 3O 4-MWCNTs hybrid materials using various water volumes to control the phase,size and narrow distributions of Fe 3O 4nanoparticles.The effects of temperature and time during synthesis on the properties of the catalyst were analyzed.The results showed that,through the addition of water at various amount into the reaction system,pure Fe 3O 4nanoparticles with uniform size and good dispersion coated on the MWCNT surface can be obtained.The Fe 3O 4nanoparticles had diameters in the range of 4.2–10.0nm,average grain size of 7.4nm with narrow size distribution.The possible mechanism for the formation ofFe 3O 4-MWCNTs was explained according to the MWCNTs serves as Fe 3O 4heterogeneous nucleation.Typical superparamagnetism at room temperature for Fe 3O 4-MWCNTs was demonstrated using magnetization measurements.Moreover,the saturation magnetizations of these materials change regularly with the size and crystallinity of Fe 3O 4.The Fe 3O 4-MWCNTs were used as a Fenton-like catalyst to decompose Orange II and displayed a higher activity than nanometer-size Fe 3O 4.The high activity could be attributed to Fe 3O 4nanoparticles’narrow size distribution,good dispersion on MWCNTs and the iron electron transfer to MWCNTs,Fe (III)facilitating reduction by H 2O 2.AcknowledgmentsThis work was supported by the National Natural Science Foundation of China (No.20677033)and the Special Fund of State Key Joint Laboratory of Environment Simulation and Pollution Control (08Z01ESPCT)and China Postdoctoral Science Foundation (No.20090450379).References[1]H.T.Hai,H.Kura,M.Takahashi,T.Ogawa,J.Colloid Interface Sci.341(2010)194–199.[2]Z.Liu,J.Wang,D.H.Xie,G.Chen,Small 4(2008)462–466.[3]H.T.Pu,F.J.Jiang,Nanotechnology 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Special ArticleCOMPLIANCE AMONG PHARMACIES IN CALIFORNIAWITH A PRESCRIPTION-DRUG DISCOUNT PROGRAMFOR MEDICARE BENEFICIARIESJ OY H. L EWIS, D.O., M ATTHIAS S CHONLAU, P H.D., J ORGE A. M UÑOZ, M.S., M.P HIL., S TEVEN M. A SCH, M.D., M.P.H., M AYDE R. R OSEN, R.N., B.S.N., H ANNAH Y ANG, B.S.P.H., AND J OSÉ J. E SCARCE, M.D., P H.D.A BSTRACTBackground Several states have developed pre-scription-drug discount programs for Medicare bene-ficiaries. In California, Senate Bill 393, enacted in 1999,requires pharmacies participating in the state Med-icaid program (Medi-Cal) to charge customers whopresent a Medicare card amounts based on Medi-Calrates. Because Medicare beneficiaries may not be ac-customed to presenting their Medicare cards at phar-macies, we assessed the compliance of pharmacieswith Senate Bill 393.Methods Fifteen Medicare beneficiaries who re-ceived special training and acted as “standardized pa-tients” visited a random sample of pharmacies in theSan Francisco Bay area and Los Angeles County inApril and May 2001. According to a script, they askedfor the prices of three commonly prescribed drugs:rofecoxib, sertraline, and atorvastatin. The script en-abled us to determine whether and when, during theirinteractions with pharmacists or salespeople, the dis-counts specified in Senate Bill 393 were offered. Phar-macies at which the appropriate discounts were of-fered were considered compliant.Results The patients completed visits to 494 phar-macies. Seventy-five percent of the pharmacies com-plied with the prescription-drug discount program; atonly 45 percent, however, was the discount offered be-fore it was specifically requested. The discount wasoffered at 91 percent of pharmacies that were part ofa chain, as compared with 58 percent of independentpharmacies (P<0.001). Compliance was higher in theSan Francisco Bay area than in Los Angeles County(84 percent vs. 72 percent, P=0.004) and was higherin high-income than low-income neighborhoods (81percent vs. 69 percent, P=0.002). A Medicare bene-ficiary taking all three drugs would have saved an av-erage of $55.70 per month as compared with retailprices (a savings of 20 percent).Conclusions Discounts required under California’sprescription-drug discount program for Medicarebeneficiaries offer substantial savings. Many patients,however, especially those who use independent phar-macies or who live in low-income neighborhoods, maynot receive the discounts. (N Engl J Med 2002;346:830-5.)Copyright © 2002 Massachusetts Medical Society.From RAND Health, Santa Monica, Calif. (J.H.L., M.S., J.A.M., S.M.A., M.R.R., H.Y., J.J.E.); and the Greater Los Angeles Veterans Af-fairs Health Care System, Los Angeles (J.H.L., S.M.A.). Address reprint requests to Dr. Lewis at RAND, 1700 Main St., Santa Monica, CA 90407-2138, or at joys@.XPENDITURES for drugs have grown inparallel with their increasing importance intreating and preventing disease.1In 1998,prescription-drug spending in the United States was estimated to be $91 billion, more than twice the amount spent in 1990.2 Elderly Medicare beneficiaries are particularly vulnerable to the high costs of prescription drugs and to the adverse health consequences of missed drug treatments. Thirty-five percent of people over the age of 65 years have three or more chronic conditions; people over 65 account for 34 percent of pharmaceutical expenditures3 but make up 13 percent of the population.4 However, one third of Medicare beneficiaries have no prescription-drug coverage,2,5 and many of those who do have in-termittent coverage.6 Medicare beneficiaries who lack prescription-drug coverage, especially those with low incomes, use fewer drugs and have higher out-of-pock-et costs than covered beneficiaries.7-9The federal government is examining ways to make prescription drugs more affordable for Medicare ben-eficiaries. However, many states have already developed pharmaceutical-assistance programs for persons over the age of 65. Four states (California, Florida, Maine, and Vermont10) have passed laws to provide Medicare beneficiaries with discounted prescription-drug prices based on prices in the Medicaid program. Eighteen states introduced bills in 2001 that would establish similar policies.11California Senate Bill 393 was enacted in 1999. This law, which applies to all participating pharmacies in the California Medicaid program (Medi-Cal), states that “the pharmacy, upon presentation of a valid prescrip-tion for the patient and the patient’s Medicare card, shall charge Medicare beneficiaries a price that does not exceed the Medi-Cal reimbursement rate for pre-scription medicines, and an amount, as set by the California Department of Health Services, to cover electronic transmission charges.”12 The California De-EPHARMACY COMPLIANCE WITH A PRESCRIPTION-DRUG DISCOUNT PROGRAMpartment of Health Services sent three notices and a Medi-Cal bulletin alerting pharmacies to the enact-ment of this law, and the sponsoring legislator con-ducted press conferences about it.However, the law may not be fully achieving its goal. Because Medicare itself does not cover the cost of pre-scription drugs, Medicare beneficiaries may be unac-customed to presenting Medicare cards to pharmacies. The new law included no provisions for educating beneficiaries or monitoring the compliance of phar-macies. This study assessed the compliance of phar-macies with California Senate Bill 393.METHODSWe trained a group of volunteers, all Medicare beneficiaries, to represent “standardized patients”13,14 and visit a sample of phar-macies participating in the Medi-Cal program in April and May 2001. As standardized patients, they would follow a consistent script that enabled us to determine whether and when, during their interactions with pharmacists or salespeople, the discounts required by Senate Bill 393 were offered. The study protocol was approved by the RAND institutional review board.PharmaciesWe obtained a list of all the licensed pharmacies in Los Angeles County and in the San Francisco Bay area counties of Alameda, Contra Costa, Marin, San Francisco, Sonoma, and San Mateo from the Board of Pharmacy of the California Department of Consumer Affairs. We contacted each pharmacy by telephone to determine whether it was independent or part of a chain (defined as three or more outlets with common ownership) and whether it participated in the Medi-Cal program. We used the 2001 Claritas Demographic Update15 as a source of data on per capita income and population data according to age, sex, race, and Hispanic or non-Hispanic eth-nic background for calendar year 2000 within each ZIP Code in the study counties.Of the 1689 retail pharmacies that participated in Medi-Cal, we excluded 42 that were located in remote areas of Los Angeles Coun-ty (those in ZIP Codes 90265, 91350, 91354, 91355, 91381, 91384, 93534, 93535, 93536, 93550, 93551, and 93552) and grouped the remaining 1647 pharmacies into eight strata according to region (Los Angeles County vs. Bay area), type of store (chain vs. independent), and income level (high vs. low) within the neighbor-hood of the pharmacy, defined according to ZIP Code. We used the population-weighted median income in all the ZIP Codes in the study counties ($21,989) as a cutoff to categorize income levels as high or low.We drew a stratified, random sample of 500 of the 1647 phar-macies, allocated among the eight strata in proportion to the size of each stratum. The sample size was chosen to provide 90 percent statistical power to detect a 15 percent difference in compliance with Senate Bill 393 between two equal groups of pharmacies. Prescription DrugsWe selected three brand-name prescription drugs used to treat chronic conditions that frequently affect older persons. The drugs, and their legislated Medi-Cal prices for a 30-day supply, were rofe-coxib (Vioxx), $64.55; sertraline (Zoloft), $63.11; and atorvastatin (Lipitor), $90.79.16-18 They are among the 50 drugs most requested by Medicare beneficiaries, according to pharmacy-price inquiries since Senate Bill 393 was enacted.12Collection of DataForty-one Medicare beneficiaries with acting experience under-went a four-hour training session during which they learned a script to use when visiting pharmacies and learned to complete an en-counter form after each visit. The scripted encounter was designed to ascertain whether the legally required discounts were offered when older customers initially asked for prices, when they asked for a “senior discount,” when they requested a “Medicare discount,” or not at all. When speaking to the pharmacist or salesperson at each pharmacy, the patient was to begin by saying, “Hi. I left my pre-scription at home. I just called home and found out what I need. While I’m here, I would like to know how much my medications would cost. Can you help me?” The patient then presented a list of the three study drugs and their doses. After obtaining the pric-es, the patients asked whether the pharmacy offered a “senior dis-count” and, if it did, what the discounted prices would be. Finally, the patient presented a Medicare card and asked for a “Medicare discount.”Answers to common questions that might be asked of patients at a pharmacy were standardized. For example, in response to the question, “Do you have insurance?” the patient was to respond,“Y es, but it doesn’t cover my pills.” If pressed for more information or asked directly about Medicare, he or she would respond, “I just have Medicare” and show a Medicare card. The patients were also trained to look for printed signs that indicated the availability of “senior” or “Medicare” discounts.The training sessions concluded with a test in which two of the investigators acted out five different scenarios. Fifteen of the volun-teers (11 men and 4 women) who had previously demonstrated that they knew the script and its variants and who accurately filled out the encounter forms for all five of these scenarios were hired to act as patients and were assigned a set of pharmacies. Once the patients were in the field, the principal investigator reviewed the encounter forms daily for purposes of quality control.Statistical AnalysisThe principal investigator and one additional investigator reviewed all the encounter forms to assess whether and when each pharmacy had offered the required discount. Pharmacies were considered to have complied with Senate Bill 393 if the pharmacist had quoted the Medi-Cal prices for the study drugs or if he or she had stated that a Medicare discount was offered but could not provide prices without “checking the computer” (i.e., checking the state’s data base of Medi-Cal prices, a process that incurred a charge). Pharma-cies were also considered to have complied with the law if at any point during the interaction the pharmacist quoted prices lower than the Medi-Cal prices. Conversely, if the pharmacist claimed that a Medicare discount was offered but quoted prices higher than the Medi-Cal prices, the pharmacy was considered noncompliant. The principal investigator and the additional investigator agreed on all but four of the encounter forms, which were then adjudicated by the entire project team.We used chi-square tests19to assess the statistical significance of differences in compliance between pharmacies in Los Angeles County and those in the Bay area, pharmacies that were part of a chain and those that were independent, and pharmacies in high-income and low-income neighborhoods. We used multiple logistic-regression analysis20 to assess the independent effects of region, type of pharmacy, and characteristics of the population in the neighbor-hood on compliance with the law. Adjusted odds ratios obtained from the logistic-regression analysis were converted to relative risks.21 A P value of less than 0.05 (by two-tailed testing) was con-sidered to indicate statistical significance.22RESULTSThe distribution of the 500 study pharmacies among the eight sampling strata is shown in Table 1. Of these 500 pharmacies, 3 were closed at the time of the visit, and 2 did not have the study drugs; onevisit could not be completed. Thus, the 15 patients completed visits to 494 pharmacies. T able 1 also shows that overall, independent pharmacies were dispropor-tionately located in low-income neighborhoods. Compliance of PharmaciesOf the 494 study pharmacies to which visits were completed, 372 (75 percent) offered the required dis-count. Of the pharmacists at these stores, 22 quoted Medi-Cal prices, 5 quoted prices lower than Medi-Cal prices, and 345 stated that the discounted prices were available but did not provide them, stating that they could not check the prices in the computer with-out a prescription or sale.Of the 122 pharmacies where the discount was not offered, pharmacists at 86 stated that there was no Medicare discount; 24 said that their prices reflected the Medicare discount, when in fact their prices were higher than Medi-Cal prices; 8 said that their prices were lower than the Medicare-discount prices, when in fact their prices were higher; and 4 would not quote any prices. Only 71 of the 494 pharmacies (14 per-cent) had signs indicating the availability of a “sen-ior” or “Medicare” discount, and at only 67 of these stores was the discount offered.As Table 2 shows, compliance with Senate Bill 393 was higher in the San Francisco Bay area than in Los Angeles County (84 percent vs. 72 percent, P=0.004); higher among pharmacies that were part of a chain than among independent pharmacies (91 percent vs.58 percent, P<0.001); and higher among pharmacies located in high-income neighborhoods than pharma-cies located in low-income neighborhoods (81 percent vs. 69 percent, P=0.002). At 11 of the noncompliant independent pharmacies, the pharmacist told the pa-tient to go to a “big chain” store for discounts.At only 45 percent of the pharmacies did pharma-cists offer the mandated discount before being specif-ically asked about it. At these pharmacies the discount was offered either when the patient initially asked for prices (7 percent of the pharmacies) or when the pa-tient asked for a senior discount (38 percent). At 30percent of the pharmacies the discount was offered only after the patient presented a Medicare card and specifically asked for a Medicare discount.As Figure 1 illustrates, pharmacists at 63 percent of the pharmacies in the San Francisco Bay area, as com-pared with 36 percent of the pharmacies in Los An-geles County, offered the Medicare discount before being specifically asked about it (P<0.001). Most strikingly, pharmacists at 67 percent of chain-store pharmacies actively offered the Medicare discount be-fore being asked, as compared with pharmacists at 21 percent of independent pharmacies (P<0.001). Final-ly, the Medicare discount was actively offered at 52 percent of the pharmacies in high-income neighbor-hoods as compared with 36 percent of those in low-income neighborhoods (P<0.001).According to multiple logistic-regression analysis, the type of pharmacy (chain or independent) was the only significant independent predictor of compli-ance with Senate Bill 393. Pharmacists at chain phar-macies were 1.58 times (95 percent confidence inter-val, 1.49 to 1.64) as likely as those at independent pharmacies to offer the discount (P<0.001). The geo-graphic region (P=0.95), income level of the neigh-borhood (P=0.06), and proportions of blacks (P= 0.39), Hispanics (P=0.57), and Asians or Pacific Islanders (P=0.71) in the neighborhood were not as-sociated with compliance or noncompliance.*We defined neighborhoods according to ZIPCodes and income level using a cutoff value of$21,989 (the population-weighted median incomein the study regions).T ABLE 1. S TUDY S AMPLE OF 500 P HARMACIES.R EGION AND T YPE OF P HARMACYI NCOME L EVEL OFN EIGHBORHOOD*HIGH LOWno. of pharmaciesLos Angeles CountyChainIndependent737876122San Francisco Bay areaChainIndependent92291911*P values were calculated by the chi-square test.†We defined neighborhoods according to ZIP Codes and income level using a cutoff value of $21,989 (the population-weighted median income in the study regions).T ABLE 2. C OMPLIANCE OF 494 P HARMACIES WITH S ENATE B ILL 393, A CCORDING TO G EOGRAPHIC R EGION, T YPE OF P HARMACY, AND I NCOME L EVEL OF THE N EIGHBORHOOD.V ARIABLEN O. OFP HARMACIESM EDICARED ISCOUNT O FFEREDPV ALUE*no. (%)Geographic regionLos Angeles CountySan Francisco Bay area344150246 (72)126 (84)0.004Type of pharmacyChainIndependent257237235 (91)137 (58)<0.001Income level of neighborhood†HighLow270224218 (81)154 (69)0.002PHARMACY COMPLIANCE WITH A PRESCRIPTION-DRUG DISCOUNT PROGRAMTPHARMACY COMPLIANCE WITH A PRESCRIPTION-DRUG DISCOUNT PROGRAM13.14.15.16.17.278:1186-90.18./www./。

a r X i v :c o n d -m a t /9610092v 5 23 J a n 1997The two dimensional antiferromagnetic Heisenberg model in the presence of anexternal fieldM.S.Yang and K.H.M¨u tterPhysics Department,University of Wuppertal,D-42097Wuppertal,Germany(February 6,2008)We present numerical results on the zero temperature mag-netization curve and the static structure factors of the two dimensional antiferromagnetic Heisenberg model in the pres-ence of an external field.The impact of frustration is alsostudied.PACS number:75.10-b,75.10.Jm,75.40.GbI.INTRODUCTIONThe discovery of high-T c superconductors 1has re-newed the interest in the two dimensional antiferromag-netic spin-12Heisenberg model in the presence of an externalfield.Our analysis is based on a numerical computation of the ground states at fixed magnetization M =S/N on the fol-lowing square lattices :N =4×4,N =6×6(1.5)with periodic boundary conditions and N =3×3+1,N =7×7+with helical boundary conditions.9In the latter case the Hamiltonian can be considered to be one dimensionalH=Nx=1 S(x)( S(x+1)+ S(x+k))+α N x=1 S(x)( S(x+k−1)+ S(x+k+1)) (1.7)with four types of couplings.Square lattices are realized for:N=k2+1,k=3,5,7(1.8)N=k2−1,k=3,5,7(1.9) as can be seen for k=5,N=26in Fig.1.The helical boundary conditions yield a quantization of the two dimensional momenta p=(p1,p2):p1=2πNkn,n=0,...,NN,N)atfixed magnetization M=S 2.Near saturation M→12,N)=α+12−12,N)=4,(2.2)withα≤12)−1(ǫ(α,M,N)−ǫ(α,M=12D1(α)Z−12D3(α)Z3(2.3)They can be absorbed in the definition of an”optimized”scaling variable:Z=(1−2M)−c(M)M)κ.(2.5)The choice for c(M)guarantees,that the expansion(2.3)satisfies the”initial”condition(2.2)for M=1Ni.e.Z=0independent of the exponentκ.Forκ≃5,weachieve the best scaling of the numerical data as can beseen from Fig.2.Afit of the numerical data to a Taylorexpansion in Z yields the coefficients D n(α),n=1,2,3listed in table1.We have repeated thefit with one ad-ditional term in the Taylor expansion(2.3).Such afityields only slight modifications of thefirst two coefficient.It is remarkable to note,that thefirst coefficient D1(α)decreases withαand vanishes at some valueα=α∗where0.49<α∗<0.5.This has important consequence for themagnetization curve M=M(B,α)which is obtained fromǫ(α,M,N=∞)by differentiation:∂ǫ2M=1D1(α)(2.7)Forα=α∗-where D1(α∗)=0-however,wefind asquare root behavior:M=14−BLet us go back to the discussion of the magnetization curves in the two dimensional model.The coefficients D n (α),n =1,2,3in the polynomial (2.3)were fitted bythe data points in the regime Z <13<M <13,which might indicate the emergence ofa ”plateau”.Unfortunately,we do not reach this shoul-der with our largest systems N =48,50shown in the upper insets of Fig.3b.The lower inset of Fig.3b contains the results for N =36with periodic boundary conditions.Here,we observe a shoulder around M =0.42which seems to be absent in the upper insets for the sys-tems with N =48,50.The variation of the magnetization curve with αon the4×4system has been computed by Lozovik and Notych.12These authors claim to see indi-cations for ”plateaus”at various M -values for α≃0.538.III.THE FIELD-DEPENDENCE OF THE STATICSTRUCTURE F ACTORS AT P=(π,π)In the one dimensional spin-13in the data forN =24,26and at M =0.42for N =36(inset Fig.5b).In both cases,this is just the position,where we found a shoulder in the corresponding magnetization curves.The dip at M =12antiferro-magnetic Heisenberg model develop singularities at field-dependent momenta (p 1(M )=2πM ,p 3(M )=π(1−2M )).We therefore address here the question,whether such sin-gularities can be found in the momentum dependence of the static structure factors for the two dimensional model as well.It will turn out,that the appearance of these sin-gularities crucially depends on the frustration parameter α.In this section we first treat the unfrustrated case α=0.0.The magnetization M =512,α=0,N ),N =24,36,48in thefirst Brillouin zone can be read offFig.6.These values appear to be constant along lines of constant :x =cos p 1+cos p 2(4.1)represented by the dashed curves in Fig.6.The behav-ior can be seen directly along the line p 1+p 2=π,x =0.Therefore,we expect the longitudinal structure factor todepend on the variable x only.In Fig.7we have plotted the longitudinal structure factor at M=18(N=24,48)and M=52)the magnetiza-tion curves show a linear behavior in two dimensions but a square root behavior in one dimension.As was pointed out in section2,this behavior changes,if the frustration parameter exceeds a critical valueα∗.If these features are correlated,we might hope tofind soft-modes in the two dimensional antiferromagnetic Heisenberg model as well,provided we weaken the an-tiferromagnetic order by frustrating the system.In order to test this hypothesis,we need a reliable criteria for the existence or nonexistence of soft-modes.In the one dimensional case5,we looked for singulari-ties(breaks,cusps etc)in the momentum distribution of the structure factors atfixed magnetization M.As is demonstrated in Figs.10a,b for the one dimensional case,pronounced structures are produced as well by the soft-modes in the M-dependence.The position of these singularities M1(p)=p2πdefine the soft-mode trajectories,which travel from p=0(ferro-magnetic order)to p=π(antiferromagnetic order)and vice versa.Keepingfixed the momentum,we have to meet the soft-mode at an appropriate value of the mag-netization.In this respect it is much easier tofind the soft-modes in the one dimensional case.In case of the frustrated two dimensional Heisenberg model,the points p=(π,π)and p=(π,0), p=(0,π) play a special role.Singularities in the structure factors at these points indicate antiferromagnetic and collinear antiferromagnetic order,respectively.Therefore,it is rather plausible to assume,that the soft-mode trajec-tories connect these points.On the system with N=5×5−1=24sites,the momentum closest to p=(π,π)and p=(0,π)are p=π(23)and p=π(16),respectively.Here,we have the best chance to observe soft-mode effects in the M-distributions of the static structure factors.The sen-sitivity of the M-distributions to a change of the frus-tration parameter can be seen in Figs.11a,b and12.In the unfrustrated caseα=0.0(open symbols),the M-distributions are smooth.Switching on the frustration parameterα(solid symbols),we observe the emergence of a peak at M=13,23in S3( p=π(16),M,α)(Fig.11b),re-spectively.The M-distribution of the transverse struc-ture factor at p=(23)πis shown in Fig.12.The data forα=0.5show a break at M=0.42,which reminds us to the structure observed in the one dimensional case shown in Fig.10b.VI.DISCUSSION AND CONCLUSIONIn this note,we have studied the zero temperature properties of the two dimensional spin-1by one variable,namely(4.1).There is no indication for the existence of singularities,which might give a hint to field-dependent soft-modes-i.e.zero frequency excita-tions.2.Frustration leads to new phenomena in the magne-tization curve and the static structure factors.The point α=0.5appears to be of special interest.Here,the mag-netization curve develops a square root singularity(2.8) near saturation(M→12in the frustration parameterα.Going be-yondα=12−1(called”one magnonstates”)have different momenta forα<12,respectively.Level crossings are”felt”by the Lanczos algorithm,which meets increasing problems tofind the ”true”ground state among two competing states.The problem can be solved,if these two states differ in their quantum numbers.One of the two competing states is filtered out,if the starting vector has the appropriate quantum numbers.Therefore,an extension of our anal-ysis to the strong frustration regimeα>12will generate interesting phe-nomena.E.g.they might be responsible for the plateau in the magnetization curve predicted by S.Gluzman.10ACKNOWLEDGMENTSWe got the numerical data for the static structure fac-tor on the6×6system-which enter in Fig.9-from H.J. Schulz and thefield-dependence of the one dimensional structure factors in Figs.10a,b from M.Schmidt.We thank H.J.Schulz and M.Schmidt for their support and S.Gluzman for discussions on the possible existence of plateaus in the magnetization curve.We are indebted to M.Karbach and G.M¨u ller for a critical reading of the manuscript and many useful comments.FIG.1.The square lattice5×5+1=26with helical bound-ary conditionFIG.2.Scaling behavior of the difference ratio(2.3)in the strongfield limit for the unfrustrated(α=0.0open symbols) and the frustrated case(α=0.5solid symbols).The curves represent polynomialfits in the scaling variable(2.4)FIG.3.The magnetization curves computed onfinite sys-tems with N=24,26,36,48and50with the method of Bonner and Fisher[10]a)the unfrustrated caseα=0.0b)the frus-trated case withα=0.5FIG.4.Thefield-dependence of the longitudinal structure factor S3( p=(π,π),M,α,N)forα=0.0(open symbols)and α=0.5(solid symbols).The curves are shown to guide the eyeFIG.5.Thefield-dependence of the transverse structure factor S1( p=(π,π),M,α,N)for a)α=0.0,b)α=0.5FIG.6.The momentum dependence of the longitudinal structure factor S3( p=(p1,p2),M=53,312FIG.8.Same as Fig.7for the transverse structure factor. The curves are shown to guide the eyeFIG.9.Same as Fig.7for M=0.0FIG.10.Field dependence of the one dimensional structurefactors atfixed momenta p=33π,13πa)the longitu-dinal case b)the transverse caseFIG.11.Field dependence of the two dimensional longi-tudinal structure factors atfixed momenta a) p=π(23)b) p=π(16)open symbolsα=0.0and solid symbolsα=0.5 FIG.12.Field dependence of the two dimensional trans-verse structure factor atfixed momentum p=π(23)TABLE I.The coefficients D n(α)of the expansion(2.3)α00.30.40.450.490.4950.5D2(α) 1.374 1.086 1.162 1.763 3.115 3.376 3.663FIG.10.00.10.20.30.40.50.60.7Z3.03.23.43.63.84.0F IG .20123B0.00.10.20.30.40.5F I G0123B 0.00.10.20.30.40.5F I GM012345S 3(,),M ,,N )F IG .40.00.10.20.30.40.5M 02468101214F IG .5a0.00.10.20.30.40.5M 02468101214F IG .5b000.10.20.30.40.50.60.70.80.91.0p 1/0.00.10.20.30.40.50.60.70.80.91.0S 3(p =(p 1,p 2),M =5/12,=0.0,N )0.2699510.3295380.3296770.2956390.3313660.3374620.3370760.3386620.0000000.2251160.3171350.3277210.2895890.3295250.3339160.3376590.3387920.3395330.2024780.3078790.3275900.3278400.3099330.2374780.2875750.3293130.3357090.3358050.3296980.3378710.3393210.3390340.339886F IG .6c o s (p 1)+c o s (p 2)0.00.10.20.30.40.50.60.70.8F I-2.0-1.5-1.0-0.50.00.51.01.5c o s (p 1)+c o s (p 2)0.00.10.20.30.4F I-2.0-1.5-1.0-0.50.00.51.01.5c o s (p 1)+c o s (p 2)0.00.51.01.52.0F I0.00.10.20.30.4M 0.00.51.01.52.02.5F IG .0.00.10.20.30.4M 0.00.20.40.60.81.01.21.4F IG .0.00.10.20.30.40.5M 0.00.20.40.60.81.01.21.41.61.82.0F IG .11a0.00.050.10.150.20.250.30.350.40.450.5M 0.00.20.40.60.81.01.2S 3p =(1/6,5/6),M ,,N )F IG .11b0.00.10.20.30.40.5M 0.00.20.40.60.81.01.21.41.61.82.0S 1p (2/3,2/3),M ,,N )-2MF IG .12。