2014 Extraction, puri_cation and characterization of polysaccharides from Hawk tea

Talanta46(1998)449–455Extraction procedures for the determination of heavy metals incontaminated soil and sedimentGemma RauretDept.Quı´mica Analı´tica,Uni6ersitat de Barcelona,Barcelona,SpainReceived25May1997;accepted14October1997AbstractExtraction tests are commonly used to study the mobility of metals in soils and sediments by mimicking different environmental conditions or dramatic changes on them.The results obtained by determining the extractable elements are dependent on the extraction procedure applied.The paper summarises state of the art extraction procedures used for heavy metal determination in contaminated soil and sediments.Two types of extraction are considered:single and sequential.Special attention is paid to the Standard,Measurement and Testing projects from the European Commission which focused on the harmonisation of the extraction procedures and on preparing soil and sediment certified reference materials for extractable heavy metal contents.©1998Elsevier Science B.V.All rights reserved. Keywords:Extraction procedures;Heavy metals;Contaminated soil;Sediment;Certified reference materials1.IntroductionTrace metals in soils and sediments may exist in different chemical forms or ways of binding.In unpolluted soils or sediments trace metals are mainly bound to silicates and primary minerals forming relatively immobile species,whereas in polluted ones trace metals are generally more mobile and bound to other soil or sediments phases.In environmental studies the determina-tion of the different ways of binding gives more information on trace metal mobility,as well as on their availability or toxicity,in comparison with the total element content.However,the determi-nation of the different ways of binding is difficult and often impossible.Different approaches are used for soil and sediment analysis,many of them focused on pollutant desorption from the solid phase;others are focused on the pollutant adsorp-tion from a solution by the solid phase.Among those approaches based on desorption,leaching procedures are the most widely accepted and used.Extraction procedures by means of a single extractant are widely used in soil science.These procedures are designed to dissolve a phase whose element content is correlated with the availability of the element to the plants.This approach is well established for major elements and nutrients and it is commonly applied in studies of fertility and quality of crops,for predicting the uptake of essential elements,for diagnosis of deficiency or excess of one element in a soil,in studies of the physical-chemical behaviour of elements in soils0039-9140/98/$19.00©1998Elsevier Science B.V.All rights reserved. PII S0039-9140(97)00406-2G.Rauret/Talanta46(1998)449–455 450and for survey purposes.To a lesser extent they are applied to elements considered as pollutants such as heavy metals.The application of extrac-tion procedures to polluted or naturally contami-nated soils is mainly focused to ascertain the potential availability and mobility of pollutants which is related to soil-plant transfer of pollutants and to study its migration in a soil profile which is usually connected with groundwater problems[1]. For sediment analysis,extraction is used to asses long term emission potential of pollutants and to study the distribution of pollutants among the geochemical phases.As far as heavy metals are concerned sediments are usually a sink but may also become a source under certain condi-tions,especially in heavily contaminated areas or in drastically changing environments.Chemical extraction of sediments has proven to be adequate for determining the metal associated with source constituents in sedimentary deposits[2],but the general aim of many studies involving chemical extraction is the determination of element distri-bution among different phases of a sediment. Single extractants are usually chosen to evaluate a particular release controlling mechanism such as desorption by increasing salinity or complexing by competing organic agents.Generally,fractions can be isolated more specifically by using sequen-tial extraction schemes.For sediments these pro-cedures are frequently used and are designed in relation to the problems arising from disposal of dredged materials.Extraction tests,either in soils and sediments, are always restricted to a reduced group of ele-ments and as far as soil is concerned they are applied to a particular type of soil;silicious,car-bonated or organic.In a regulatory context,two applications for leaching tests can be recognised: the assessment or prediction of the environmental effects of a pollutant concentration in the environ-ment and the promulgation of guidelines or objec-tives for soil quality as for example for land application of sewage sludge or dredge sediments. The data obtained when applying these tests are used for decision makers in topics such as land use of soil or in countermeasures monly used extraction procedures in soils During the last decades several extraction pro-cedures for extractable heavy metals in soils have been developed and modified.In this respect,two groups of tests must be considered:the single reagent extraction test,one extraction solution and one soil sample,and in the sequential extrac-tion procedures,several extraction solutions are used sequentially to the same sample although this last type of extraction is still in development for soils.Both types of extraction are applied using not only different extracting schemes but also different laboratory conditions.This leads to the use of a great deal of extraction procedures. In Table1a summary of the most common leaching test are given.Table1Most common single extraction testsType and solution strength Reference Group[3]HNO30.43–2mol l−1Acid extractionAqua regia[4]HCl0.1–1mol l−1[3]CH3COOH0.1mol l−1[5]Melich1:[6]HCl0.05mol l−1+H2SO40.0125mol l−1EDTA0.01–0.05mols l−1[3] Chelatingagents at different pH[7]DTPA0.005mol l−1+TEA0.1mol l−1CaCl20.01mol l−1Melich3:[8]CH3COOH0.02mol l−1NH4F0.015mol l−1HNO30.013mol l−1EDTA0.001mol l−1NH4–acetate,acetic acidBuffered salt[9]buffer pH=7;1mol l−1solution[3]NH4–acetate,acetic acidbuffer pH=4.8;1mol l−1Unbuffered salt CaCl20.1mol l−1[3]solutionCaCl20.05mol l−1[3][3]CaCl20.01mol l−1NaNO30.1mol l−1[10]NH4NO31mol l−1[3]AlCl30.3mol l−1[11]BaCl20.1mol l−1[12]G.Rauret/Talanta46(1998)449–455451 Table2Extraction methods proposed for standardisation or standardised in some European countriesMethod MethodCountry Reference[15]Mobile trace element determination1mol l−1NH4NO3Germany[16]Available Cu,Zn and Mn evaluation for fer-France0.01mol l−1Na2–EDTA+1mol l−1tilisation purposesCH3COONH4at pH=7DTPA0.005mol l−1+TEA0.1mol l−1+CaCl20.01mol l−1at pH=7.3Available Cu,Zn,Fe and Mn evaluation inItaly0.02mol l−1EDTA+0.5mol l−1[17]acidic soilsCH3COONH4at pH=4.6DTPA0.005mol l−1+TEA0.1mol l−1+CaCl20.01mol l−1at pH=7.3[18]Availability and mobility of heavy metals inCaCl20.1mol l−1Netherlandspolluted soils evaluationSoluble heavy metal(Cu,Zn,Cd,Pb and[19] Switzerland NaNO30.1mol l−1Ni)determination and ecotoxicity risk evalu-ationUnited Kingdom EDTA0.05mol l−1at pH=4[20]Cu availability evaluationFrom Table1it can be observed that a single extraction including a large spectra of extractants are used.It ranges from very strong acids,such as aqua regia,nitric acid or hydrochloric acid,to neutral unbuffered salt solutions,mainly CaCl2or NaNO3.Other extractants such as buffered salt solutions or complexing agents are frequently ap-plied,because of their ability to form very stable water soluble complexes with a wide range of cations.Hot water is also used for the extraction of boron.Basic extraction by using sodium hy-droxide is used to assess the influence of the dissolved organic carbon in the release of heavy metals from soils.A large number of extractants are reviewed by Pickering[13]and Lebourg[14]. The increasing performance of the analytical techniques used for element determination in an extract,together with the increasing evidence that exchangeable metals better correlate with plant uptake,has lead extraction methods to evolve towards the use of less and less aggressive solu-tions[10].These solutions are sometimes called soft extractants and are based on non buffered salt solutions although diluted acids and complex-ant agents are also included in the group.Neutral salts dissolve mainly the cation exchangeable frac-tion although in some cases the complexing ability of the anion can play a certain role.Diluted acids dissolve partially trace elements associated to dif-ferent fractions such as exchangeable,carbonates, iron and manganese oxides and organic matter. Complexing agents dissolve not only exchange-able element fraction but also the element fraction forming organic matter complexes and the ele-ment fractionfixed on the soil hydroxides.Nowa-days it is generally accepted that extractants are not selective and that minor variations in analyti-cal procedures have significant effects on the re-sults.Some leaching procedures for soils have been adopted officially or its adoption is under study in different countries with different objectives[14]. An account of these methods are given on Table 2.monly used extraction procedures in sedimentsAs for soils,exchangeable metal in sediments are selectively displaced by soft extractants.Other extractants used are less selective and they co-ex-tract the exchangeable fraction together with metals bound to different sediment phases moreG.Rauret/Talanta46(1998)449–455 452or less extensively.The phases considered relevant in heavy metals adsorption in sediments are ox-ides,sulphides and organic matter.Fractionation is usually performed by using sequential extrac-tion schemes.The fractions obtained,when apply-ing these schemes,are related to exchangeable metals,metals mainly bound to carbonates, metals released in reducible conditions such as those bound to hydrous oxides of Fe and Mn, metals bonded to oxidable components such as organic matter and sulphides and residual frac-tion.The extractants more commonly used in sequential extraction schemes are generally ap-plied according to the following order:unbuffered salts,weak acids,reducing agents,oxidising agents and strong acids.In Table3the extractants most commonly used to isolate each fraction are given[21].The water soluble fraction may be obtained by two ways,by sampling sediment pore solution using in situ filtration,dialysis tubes or bags,or by a leaching procedure in the laboratory.When this procedure is used the pH may be indeterminate because of the low buffering capacity of the extractant and problems with readsorption occurs.Exchangeable fraction uses an electrolyte such as salts of strong acids and bases or salts of weak acids and bases at pH7to prevent oxyhydroxy phases precipitation. The carbonate bound fraction generally uses an acid such as acetic or a buffer solution acetic acid-sodium acetate at pH5.These reagents are not able to attack all the carbonate content,as for example dolomitic carbonates,neither to attack carbonate selectively as they also remove partially organically bound trace metals.The fraction ob-tained when a reducing solution is used as extrac-tant is mainly related to metals bound to iron and manganese oxides.Hydroxylamine in acid solu-tion is the reducing agent most widely used to solubilise these oxides although iron oxide is not completely dissolved.Ammonium oxalate seems to be most effective when used in the dark,al-though some problems in heavy metals oxalate phase precipitation may occur even at low pH. The sodium dithionite/citrate/carbonate reagent dissolves the oxide and hydroxyoxides but can attack iron rich silicates.So reducing extractants are neither selective nor completely effective for iron and manganese oxides.Other group of ex-tractants used sequentially includes oxidising reagents which destroy organic matter and also oxidises sulphides to sulphates.The extractants most widely used in this group are H2O2and NaOCl.Hydrogen peroxide seems to be more efficient if used after the oxide extraction step. The most widely used extraction scheme is the one proposed by Tessier[22]which has been modified by several authors[23–25].Many of these modifications make more specific the isola-tion of the iron and manganese oxide and hydrox-ide phases.The Tessier procedure is schematised in Table4together with the modified procedures of Fo¨rstner[26]and of Meguelatti[24].4.Harmonisation and method validationOwing to the need of establishing common schemes in Europe for extractable trace metals in soils and sediments the EC Standards,Measure-ment and Testing Programme,formerly BCR(Bu-Table3Most common extractants used in sequential extraction schemesType and solution strenght GroupH2OWater soluble fractionExchangeable and weakly NaNO30.1mol l−1 adsorbed fractionKNO30.1mol l−1MgCl21mol l−1CaCl20.05mol l−1Ca(NO3)20.1mol l−1NH4OAc1mol l−1pH=7 Carbonate bound fraction HOAc0.5mol l−1HOAc/NaOAc1mol l−1pH=5Fractions bound to hydrous NH2OH.HCl0.04mol l−1 oxides of Fe and Mn in acetic or nitric acidNH4OxSodium ditionite,sodiumcitrate,sodium bicarbonate(DCB)Organically bound fraction H2O2NaOClG.Rauret/Talanta46(1998)449–455453 Table4Sequential extraction schemes2345Method1HF/HClO4NaOAc1mol l−1H2O28.8mol l−1Tessier et al.NH2OH.HCl0.04molMgCl2mol l−1l−1HNO3/NH4OAc residual pH7pH525%HOAcsilicate phaseorganic matter+sul-exchangeable carbonate Fe/Mn oxidesphideHNO3NH4Ox/HOx0.1mol H2O28.8mol l−1NH2OH.HCl0.1molFo¨rstner NaOAc1moll−1l−1l−1residualpH7NH4OAcpH5easily reducible pH3in darksilicate phasemoderately reducible organic matter+sul-exchan+carbphideNaOAc1mol l−1NH2OH.HCl0.1molMeguellati BaCl21mol l−1H2O28.8mol l−1+ashingl−1HNO3+HF/HClresidualpH525%HOAcorganic matter+sul-pH7phidesilicate phaseFe/Mn oxidesexchangeable carbonatereau Community of Reference),has sponsored from1987several projects focused on single ex-traction for soils and sequential extraction for soils and sediments.The project started with the intercomparison of existing procedures tested in an interlaboratory exercise[27].The next step was to adopt common procedures for single extraction of trace metals from mineral soils.The second step was to adopt a common procedure for se-quential extraction of sediment.As a conclusion of thefirst step,single extraction procedures using acetic acid,0.43mol l−1,and EDTA,0.005mol l−1for mineral soils and a mixture of DTPA, 0.005mol l−1diethylenetriamine pentaacetic acid, 0.01mol l−1CaCl2and0.1mol l−1tri-ethanolamine for calcareous soils were adopted for extractable Cd,Cr,Cu,Ni,Pb and Zn.In order to improve the quality of the determination of extractable metal content in different types of soil using the procedures previously adopted,the extraction procedures were validated by means of intercomparison exercises[28,29].Moreover the lack of suitable certified reference materials for this type of studies did not enable the quality of the measurements to be controlled.With the pur-pose to overcome this problem three certified reference materials:a terra rossa soil,a sewage amended soil and a calcareous soil have been prepared and their extractable trace metal con-tents were certified(CRM483,CRM484and CRM600)[30,31].The second step of the EC,Standards,Mea-surement and Testing was focused on a feasibility study on the adoption and validation of a sequen-tial extraction scheme for sediment samples.In a workshop held in1992in Sitges(Spain)a sequen-tial extraction scheme was proposed which in-cludes three steps:acetic acid,hydroxylamine hydrochloride or a reducing reagent and hydrogen peroxide or an oxidising reagent.This procedure is schematised in Table5.Moreover in this work-shop the main analytical limitations in sequential extraction of trace metals in sediments were thor-oughly discussed and practical recommendations were given[32,33].These recommendations deal with sampling and sample pre-treatment,practical experiences with reagents and matrices and ana-lytical problems after extraction.Once the scheme was designed,it was tested through two round robin exercises using two dif-ferent type of sediment,silicious and calcareous [34].In these exercises some critical parameters in the protocol were identified such as the type and the speed of the shaking and the need of an optimal separation of the liquid–solid phases af-ter the extraction.It was stated that the sedimentG.Rauret/Talanta46(1998)449–455 454should be continually in suspension during the extraction.In these intercomparison exercises an important decrease was noted on the acceptable set of values for concentration in the extract lower than10m g l−1,which illustrates the difficulties experienced by a number of laboratories in the determination of such concentration levels in these matrices.It was concluded that when elec-trothermal atomic absorption spectrometry is used for thefinal determination,the method of standard additions is strongly recommended for calibration.The results obtained in the round robin exercises encouraged to proceed with the organisation of a certification campaign in order to produce a sediment reference material follow-ing the sequential extraction scheme adopted.So the next step of the project was the preparation of a sediment certified reference material for the extractable contents of Cd,Cr,Cu,Ni.Pb and Zn,following the three-step sequential extraction procedure.A silicious type sediment with rather high trace metal content was chosen for this pur-pose.This material has been recently certified for five metals,Cd,Cr,Ni,Pb and Zn in thefirst step,Cd,Ni and Zn in the second step and Cd,Ni and Pb in the third step[35].Not all the elements were certified because the lack of reproducibility atributable to non adherence to the protocol,in the acceptance of too large tolerances in the con-ditions specified in it or in the existence of critical aspects in the procedure referred mainly to the second step.These aspects were mainly pH,redox conditions and possible losses of sediment in the transfer.The results obtained in the certification exercise recommended to continue the develop-ment of the extraction protocol in order to in-crease reproducibility.Consequently the causes of non reproducibility are now under study in a new SMT project.5.ConclusionsThe advantages of a differential analysis over investigations of total metal contents and about the usefulness of single and sequential chemical extraction for predicting long-term adverse effects of heavy metals from polluted solid material,soils and sediments,is beyond any doubt.The ad-vances in thisfield,especially to make available soil and sediment certified reference materials for extractable element contents by using harmonised procedures,is going to increase the quality of the results due to the possibility of verifying the ana-lytical quality control.Nevertheless some problems need to be solved with these procedures for example:(1)reactions are not selective and are influenced by the experi-mental conditions so it is necessary to identify the main variables which involves a lack of reproduci-bility when applying a procedure,to write very well defined protocols and to validate them;(2) labile fractions could be transformed during sam-ple preparation and during sequential extraction schemes application so problems encountered when preparing certified reference materials are not representing all the problems to be found when working with environmental samples such as wet sediments,some work in this area is needed;(3)analytical problems due to the low level of metals to be measured in the different fractions especially when using soft extractants; and(4)the procedures need to be optimised and validated for different type of soils,including organic soils and sediments.Table5EC Standard,Measurements and Testing procedureConditionsStep10.11mol l−1HOAc,V m−140ml g−1temp.20o C,shaking overnight20.1mol l−1NH2OH.HCl(pH=2with HNO3)V m−140ml.g−1temp.20o Cshaking overnight8.8mol l−1H2O2(pH=2–3with HNO3)3V m−1=10ml g−1room temperature1h.New addition10ml g−185o C for1h.reduce volume to few ml.1mol l−1NH4Oac(pH=2with HNO3)V m−1=50ml g−120o Cshaking overnightG.Rauret/Talanta46(1998)449–455455References[1]H.A.van der Sloot,L.Heasman,Ph.Quevauviller(Eds.),Harmonization of leaching/extraction test,Chap.3, 1997,41–56.[2]H.A.van der Sloot,L.Heasman,Ph.Quevauviller(Eds.),Harmonization of leaching/extraction test,Chap.5, 1997,pp.75–99.[3]I.Novozamski,Th.M.Lexmon,V.J.G.Houba,Int.J.Environ.Anal.Chem.51(1993)47–58.[4]E.Colinet,H.Gonska,B.Griepink,H.Muntau,EURReport8833EN,1983,p57.[5]A.M.Ure,Ph.Quevauviller,H.Muntau,B.Griepink,Int.J.Environ.Anal.Chem.51(1993)135–151.[6]C.L.Mulchi,C.A.Adamu,P.F.Bell,R.L.Chaney,Com-mon.Soil Sci.Plant Anal.23(1992)1053–1059.[7]W.L.Lindsay,W.A.Norvell,Soil Sci.Soc.Am.J.42(1978)421–428.[8]A.Melich,Common.Soil Sci.Plant Anal.15(1984)1409–1416.[9]A.M.Ure,R.Thomas, D.Litlejohn,Int.J.Environ.Anal.Chem.51(1993)65–84.[10]S.K.Gupta,C Aten,Int.J.Environ.Anal.Chem.51(1993)25–46.[11]J.C.Hughes,A.D.Noble,Common.Soil Sci.Plant Anal.22(1991)1753–1766.[12]C.Juste,P.Solda,Agronomie8(1988)897–904.[13]W.P.Pickering,Ore Geol.Rev.1(1986)83–146.[14]A.Lebourg,T.Sterckeman,H.Cielsielki,N.Proix,Agronomie16(1996)201–215.[15]DIN(Deutches Institut fu¨r Normung)(Ed.)Bo-denbeschaffenheit.Vornorm DIN V19730,in:Boden -Chemische Bodenuntersuchungsverfahren,DIN,Berlin, 1993,p.4.[16]AFNOR(Association Francaisede Normalization),AFNOR,Paris,1994,p.250.[17]UNICHIM(Ente Nazionale Italiano di Unificazione),UNICHIM,Milan1991.[18]V.J.G.Houba,I.Novozamski,T.X.Lexmon,J.J.van derLee,Common.Soil Sci.Plant Anal.21(1990)2281–2291.[19]VSBo(Veordnung u¨ber Schadstoffgehalt im Boden)Nr.814.12,Publ.eidg.Drucksachen und Materialzentrale, Bern,1986,pp.1–4.[20]MAFF(Ministry of Agriculture,Fisheries and Food),Reference Book427MAFF,London1981.[21]A.Ure,Ph.Quevauviller,H.Muntau,B.Griepink,Re-port EUR14763EN.1993.[22]A.Tessier,P.G.X.Campbell,M.Bisson,Anal.Chem.51(1979)844.[23]W.Salomons,U.Fo¨rstner,Environ.Lett1(1980)506.[24]M.Meguellati,D.Robbe,P.Marchandise,M.Astruc,Proc.Int.Conf.on Heavy Metals in the Environment, Heidelberg,CEP Consultants,Edinburgh,1983,p.1090.[25]G.Rauret,R.Rubio,J.F.Lopez-Sanchez,Int.J.Envi-ron.Anal.Chem.36(1989)69–83.[26]U.Fo¨rstner,in:R.Lechsber,R.A.Davis,P.L’Hermitte(Eds.),Chemical Methods for Assessing Bioavailable Metals in Sludges,Elsevier,London,1985.[27]A Ure,Ph.Quevauviller,H.Muntau,B.Griepink,Int.J.Environ.Anal.Chem.51(1993)135–151.[28]Ph.Quevauviller,chica,E.Barahona,G.Rauret,A.Ure,A Gomez,H.Muntau,Sci.Total Environ.178(1996)127–132.[29]Ph.Quevauviller,G.Rauret, A.Ure,R.Rubio,J-FLo´pez-Sa´nchez,H.Fiedler,H.Muntau,Mikrochim.Acta 120(1995)289–300.[30]Ph.Quevauviller,G.Rauret,A.Ure,J.Bacon,H.Mun-tau,Report EUR17127EN,1997.[31]Ph.Quevauviller,chica,E.Barahona,G.Rauret,A.Ure,A.Gomez,H.Muntau,Report EUR17555EN,1997.[32]Ph.Quevauviller,G.Rauret,B.Griepink,Int.J.Environ.Anal.Chem.51(1993)231–235.[33]B.Griepink,Int.J.Environ.Anal.Chem.51(1993)123–128.[34]Ph.Quevauviller,G.Rauret,H.Muntau,A.M.Ure,R.Rubio,J-F Lo´pez-Sanchez,H.D.Fiedler, B.Griepink, Fres.J.Anal.Chem.349(1994)808–814.[35]Ph.Quevauviller,G.Rauret,J-F.Lo´pez-Sanchez,R.Rubio, A.Ure,H.Muntau,Report EUR17554EN, 1997.. .。

马齿苋中抗炎活性物质的提取、分离及结构鉴定张会敏1，邢岩2，仇润慷1，张丽梅2，倪贺3，赵雷1*（1.华南农业大学食品学院，广东广州 510642）（2.国珍健康科技（北京）有限公司，北京 100000)（3.华南师范大学生命科学学院，广东广州 510640）摘要：以活性物质示踪为导向，建立脂多糖诱导的RAW264.7巨噬细胞炎症模型对马齿苋中的抗炎物质进行跟踪，采用柱层析提取法、硅胶柱色谱分离法、制备液相色谱法及气相色谱-质谱联用技术对抗炎物质进行提取分离和结构鉴定。

结果表明，石油醚-乙醇、无水乙醇和纯水溶剂依次对马齿苋样品进行提取，三种粗提物将细胞中一氧化氮（Nitric Oxide，NO）的分泌量分别减少至33.13、25.83和20.53 μmol/L，其中石油醚相粗提物的抑制效果最强（P<0.05）。

对石油醚相进一步分离得到四个组分，Fr.1、Fr.2和Fr.3组分具有较强的抗炎效果，但Fr.1和Fr.2组分含有潜在的毒性成分，选择Fr.3组分继续分离。

Fr.3组分经硅胶柱分离得到三个组分，Fr.3.1组分表现出最强的抑制NO的分泌量效果（11.80 μmol/L）。

经制备液相色谱进一步纯化及气质分析，确定Fr.3.1组分的主要成分为硬脂酸（47.09%）、邻苯二甲酸二(2-乙基己)酯（13.21%）和其他成分。

该研究建立了一种从马齿苋中分离纯化出抗炎物质方法，为马齿苋的开发利用提供理论参考。

关键词：马齿苋；抗炎活性；提取分离；鉴定文章编号：1673-9078(2024)03-191-199 DOI: 10.13982/j.mfst.1673-9078.2024.3.0324Extraction, Separation and Structural Identification of Anti-inflammatory Active Substances from Purslane (Portulaca oleracea L.)ZHANG Huimin1, XING Y an2, QIU Runkang1, ZHAGN Limei2, NI He3, ZHAO Lei1*(1.College of Food Science, South China Agricultural University, Guangzhou 510642, China)(2.Guozhen Health Technology (Beijing) Co. Ltd., Beijing 100000, China)(3.College of Life Sciences, South China Normal University, Guangzhou 510640, China)Abstract: To track the anti-inflammatory substances in purslane, the lipopolysaccharide-induced RAW264.7 macrophage inflammation model was established, which was guided by the tracer of active substances. The extraction, separation and structural identification of anti-inflammatory substances in purslane were performed by column chromatography (for extraction), silica gel column chromatography (for separation), and preparative high performance liquid chromatography and gas chromatography-mass spectrometry (for analyses). The results showed that the three crude extracts obtained from purslane through sequential extractions with petroleum ether-ethanol, anhydrous ethanol and pure引文格式：张会敏,邢岩,仇润慷,等.马齿苋中抗炎活性物质的提取、分离及结构鉴定[J] .现代食品科技,2024,40(3):191-199.ZHANG Huimin, XING Yan, QIU Runkang, et al. Extraction, separation and structural identification of anti-inflammatory active substances from purslane (Portulaca oleracea L.) [J] . Modern Food Science and Technology, 2024, 40(3): 191-199.收稿日期：2023-03-16基金项目：国家自然科学基金资助项目（31771980）；广东省自然科学基金（2023A1515012599）作者简介：张会敏（1996-），女，硕士研究生，研究方向：活性物质分离提取，E-mail：；共同第一作者：邢岩（1981-），女，博士，助理研究员，研究方向：抗氧化与抗衰老，E-mail：通讯作者：赵雷（1982-），男，博士，教授，研究方向：天然产物绿色修饰及热带水果加工，E-mail：191water solvents reduced the secretion of nitric oxide (NO) in the cells to 33.13, 25.83 and 20.53 μmol/L, respectively, with the crude petroleum ether extract exhibiting the strongest inhibitory effect (P<0.05). The petroleum ether phase was further separated into four fractions, with the Fr.1, Fr.2 and Fr.3 fractions had stronger anti-inflammatory effects, though the Fr.1 and Fr.2 fractions contained potential toxic components. Therefore, the Fr.3 fraction was selected for further separation. The Fr.3 fraction was separated through a silica gel column to obtain three fractions. The Fr.3.1 subfraction exhibited the strongest inhibitory effect against the NO secretion (11.80 μmol/L). The Fr.3.1 subfraction was further purified by the preparative liquid chromatography and GC-MS analysis, and the main components of the Fr.3.1 subfraction were identified as stearic acid (47.09%), di(2-ethylhexyl)phthalate (13.21%) and other components. This study established a method for separating and purifying anti-inflammatory substances from purslane, and provides a theoretical reference for the development and utilization of purslane.Key words: Portulaca oleracea L.; anti-inflammatory activity; extraction and isolation; identification炎症是机体受到外部刺激时做出的一种保护性生理反应，能够及时清除体内受损或死亡的细胞，帮助机体恢复内部平衡[1] 。

T.A.L.,vol.41,n o2,pp.1–25EXTRANS,AN ANSWER EXTRACTION SYSTEMDiego MOLLÁRolf SCHWITTER Michael HESSRachel FOURNIER*Résumé-AbstractLes systèmes d’Extraction de Réponses(ER)récupèrent dans des documents des expressions qui répondent directementàdes questions du langage cou-rant.L’ER pour des manuels techniques exige un haut niveau de rappel et de précision;pourtant,ce sont de petites unités de texte qui doiventêtre ré-cupérées.C’est pourquoi il est important d’effectuer une analyse linguistique détaillée.Nous présentons ici ExtrAns,un système d’ER pour des manuels Unix qui utilise une analyse syntaxique complète,une désambiguïsation par-tielle et un module de résolution d’anaphores pour générer les formes logiques minimales correspondant aux documents etàla requê procédure de re-cherche se sert d’un algorithme de démonstration de la requête sur la repré-sentation en clauses de Horn des formes logiques minimales.Les ambiguïtés non résolues sont traitéesàl’aide d’une mise enévidence graduelle.Answer Extraction(AE)systems retrieve phrases in textual documents that dir-ectly answer natural language questions.AE over technical manuals requires very high recall and precision,and yet small text units must be retrieved.It is therefore important to perform linguistic analysis in detail.We present ExtrAns, an AE system over Unix manuals that uses full parsing,partial disambiguation, and anaphora resolution to generate the minimal logical forms of the docu-ments and the query.The search procedure uses a proof algorithm of the user query over the Horn clause representation of the minimal logical forms.Re-maining ambiguities in the retrieved sentences are dealt with by graded high-lighting.Mots Clefs-KeywordsComposants linguistiques en RI,extraction de réponses,formes logiques mi-nimales.Linguistic components in IR,answer extraction,minimal logical forms.*Computational Linguistics Group,University of Zurich.E-mail:{molla,schwitt,hess, fournier}@ifi.unizh.ch.cDiego MOLLÁ,Rolf SCHWITTER,Michael HESS,Rachel FOURNIER INTRODUCTIONThe need for systems capable of retrieving precise information from tex-tual documents in an efficient way is becoming more obvious by the day.The fact that the known methods,such as document retrieval and information ex-traction,are increasingly inadequate or insufficiently powerful in the task of loc-alising very precise information,has been recognised by the TREC programme committee with the formation of the Question Answering T rack(Voorhees E.M. &Harman D.1999).Since fully-fledged text-based question answering is still too ambitious for practical purposes,realistic compromise solutions are clearly needed.We suggest answer extraction as one such solution and introduce ExtrAns,an implementation of an answer extraction system1.Document Retrieval(DR)techniques are the prototypical form of inform-ation retrieval methods,to the point that textbooks about information retrieval typically cover almost exclusively DR(van Rijsbergen C.1979;Salton G.& McGill M.J.1983).They have the advantage of allowing arbitrary queries over very large document collections(many gigabytes in size)covering arbit-rary domains.One of the disadvantages of DR is the very fact that such sys-tems retrieve entire documents,which is unhelpful if documents are dozens, or hundreds,of pages long.T ypically,DR systems(be they based on Boolean, vector space,or probabilistic principles)are keyword based,i.e.they take into consideration only the content words of documents and queries,discarding all the morphological and syntactic information including all function words. This is why these systems cannot distinguish the command copiesfiles from the commandfiles copies(lost ordering information),or export from Germany to the UK from export to Germany from the UK(lost function word informa-tion).T rue,some DR systems can use phrasal search terms(such as com-puter design),to be found as a whole in the documents,but then a number of relevant documents(such as those containing design of computers)will no longer be retrieved.Statistical methods are used occasionally tofind inherent relations between words,such as synonymy(Deerwester S.et al.1990),or to determine simple dependencies between the words in a sentence(Strza-lkowski T.et al.1997).Some DR systems use partial linguistic information and even ontologies to create more accurate indexing terms(Woods W.A.1997; Strzalkowski T.et al.1998).However,to the best of our knowledge,no DR system produces full syntactic parses of either documents or queries.In fact, the common belief holds that it does not pay off to use deep linguistic analysis in DR(Lewis D.D.&Sparck Jones K.1996).Information Extraction(IE)techniques are similar to DR techniques in that they,too,are suitable for processing text collections of basically unlimited size(normally,messages in a stream)covering a potentially wide range of topics.However,IE systems differ from DR systems in that they identify those messages in a stream that fall into a(usually very small)number of specific 1This research is funded by the Swiss National Science Foundation,project No.12–53704.98.EXTRANS,AN ANSWER EXTRACTION SYSTEM topics,and extract from those a very limited amount of highly specific data. This information is placed into a frame-like database record with afixed number of predefined role slots,with one type of frame for each type of report.Such systems typically use some kind of very shallow syntactic analysis because of run-time requirements(Appelt D.E.et al.1993;Grisham R.&Sundheim B.1996;Chinchor N.A.1998).Clearly,the kind of information extracted by these systems is much more precise and specific than what is delivered by DR systems.On the other hand,IE systems do not allow for arbitrary questions.T ext-based Question Answering(QA)systems would be the ideal solution to the problem offinding precise and localised information.QA systems read texts,assimilate their content into knowledge bases,and generate answers to arbitrary questions phrased in unrestricted natural language.T wo well-known examples of QA systems are Unix Consultant(UC),which performs QA over a(hand-crafted)data base of facts about Unix(Wilensky R.et al.1994),and LILOG(Herzog O.&Rollinger C.-R.1991),which performs QA over a small number of travel guide texts in German.QA systems must integrate very deep syntactic and semantic analysis of both documents and questions,search in knowledge bases,inference over textual and world knowledge,and generation of answers in natural language.Some systems(such as UC)even include a user modelling component that keeps track of previous interactions with the user and makes sure that the overall dialogue between the user and the ma-chine sounds natural.Each of these tasks is in itself very difficult to implement with the current state of the art in technology.As a result,all past and existing fully-fledged QA systems work only over very narrow domains,for extremely small volumes of text,and with very high development costs.One may attempt to produce a reduced version of a QA system,for example,by using a very simple knowledge representation(Katz B.1997).T o our knowledge,however, it has not been possible to use such systems with real-world text in practical applications.Answer Extraction(AE)lies between text-based QA on the one hand,and DR and IE on the other.AE systems allow questions in arbitrarily phrased,un-restricted,natural language,over a collection of texts in equally unrestricted natural language,but they merely pinpoint the exact phrases in the documents of the collection which contain the explicit answers to the specific question.AE systems do not try to perform inferences over the content of the documents (or world knowledge),and they do not generate answers either.In this respect they are much more modest than fully-fledged QA systems.It is interesting to see that the systems which participated in the QA track of the TREC-8confer-ence(Voorhees E.M.&Harman D.1999)did not do QA in the classical sense but AE as defined in this paper.Other attempts to use DR techniques for AE are under way(Woods W.A.1997).There are also attempts to produce AE over FAQs(Burke R.D.et al.1997;Winiwarter W.1999)by trying to match the user’s query with an existing query in a FAQ,and returning the answer given in the FAQ.Diego MOLLÁ,Rolf SCHWITTER ,Michael HESS ,Rachel FOURNIER Tokeniser Link GrammarPruner Lemmatiser Disambiguator Anaphora Knowledge BaseNL QueryUNIX ManpagesDisplayAEMinimal Logical Forms Horn Clause Logic Information flowResources Figure 1:The architecture of ExtrAnsExamples for possible applications of AE methods are interfaces to ma-chine-readable technical manuals or on-line help systems for complex soft-ware.In these applications very high retrieval precision on the level of indi-vidual phrases is mandatory (queries in these domains tend to be very spe-cific),and high recall is equally vital (technical texts typically explain things only once).High recall and precision are best achieved if we determine part of the meaning of sentences (both of questions and texts)and locate relevant phrases in the documents on the basis of their meaning.This is computation-ally expensive but we can approach the final goal gradually.We can begin with a fairly simple yet useful system for technical manuals,since these are moder-ately sized document collections and they cover a very limited domain,and we can then refine the system in a stepwise manner to cover wider domains and larger volumes of data —for example,a recent follow-up of ExtrAns is Web-ExtrAns,an AE system over XML technical manuals that is using,for the time being,the maintenance manual of a commercial aircraft.Apart from extending the coverage,we may also attempt to gradually increase the depth of analysis,and ultimately we might even arrive at a fully-fledged text-based QA system.The main goal of this paper is to show that the current state of the art in NLP technologies makes it possible to implement useful AE systems over technical manuals.1.THE EXTRANS SYSTEM1.1.OverviewExtrAns finds those exact phrases in a collection of technical documents that directly answer a user query.Figure 1gives an overview of ExtrAns’gen-eral architecture.The current version of ExtrAns runs over 500unedited UnixEXTRANS,AN ANSWER EXTRACTION SYSTEM manual pages(manpages).These highly technical documents arefirst pre-processed by the tokeniser of ExtrAns that exploits all formatting information and domain-specific typographic conventions.For the syntactic analysis of document sentences and user queries,ExtrAns uses“Link Grammar”(LG), which consists of a very fast parser and a grammar of English written in the spirit of dependency grammars.The LG parser outputs all the alternative de-pendency structures for a sentence,showing the words that are linked and the types of the links.From the output of LG,obviously wrong structures are filtered out by a pruner that relies on a set of hand-crafted rules for the Unix domain.Since LG does not carry out any morphological analysis,ExtrAns uses a third-party lemmatiser that generates the lemmas of the inflected word forms(Humphreys K.et al.1996).In a next step,different forms of attachment ambiguities are resolved by a disambiguator trained with data from the man-pages.Following that,pronominal anaphors are resolved(on purely syntactic information,in contrast to the disambiguator that uses statistical knowledge). From these(partially disambiguated)dependency structures ExtrAns derives one or more Minimal Logical Forms(MLFs)as semantic representation for the core meaning of each sentence.MLFs have been designed to keep the balance between expressivity and processability for the AE task at hand.For processing reasons,MLFs are translated into Horn clause logic and asserted into the knowledge base.Unlike sentences in documents,which are processed off-line,MLFs of user queries are computed on-line and are proved by refuta-tion over the documents.ExtrAns’AE procedure alwaysfinds all proofs for a user query and assumes that the more often a part of an extracted sentence was used for the proof the more adequate it is.Adequacy is displayed by highlighting the pertinent parts of the sentences found,both individually(see Figure2in Section1.9)and in the context of the whole document.ExtrAns is particularly user-friendly not only because of this feature but also because it is very robust.It uses a keyword mechanism for unanalysable parts of sentences and a graceful fall-back strategy that relaxes the proof criteria in a stepwise manner if direct hits cannot be found.1.2.TokeniserSince ExtrAns has to be able to cope with unrestricted text,it needs a very reliable tokeniser.ExtrAns’tokeniser processes the NAME,SYNOPSIS, and DESCRIPTION section of the manpages.Apart from identifying regular word forms and sentence boundaries,the tokeniser has to recognise a set of domain-specific words.Therefore,the tokeniser uses information from the SYNOPSIS section together with a set of heuristics to recognise these spe-cial words and represent them as normalised tokens.The Unix manpages are encoded in T roff format where command names are printed in boldface and arguments in italics.As a consequence a considerable amount of formatting information can be exploited and added to the representation of the tokens.For example,command names are distinguished from homographic word formsDiego MOLLÁ,Rolf SCHWITTER,Michael HESS,Rachel FOURNIER (like“eject”in eject is used for those removable media devices that do not have a manual eject button)and are represented by adding an unambiguous sub-script to the token(e.g.eject is tagged and represented as ).Such subscripts are also introduced for words that are used as arguments(e.g.fi-lename1.arg,device.arg,nickname.arg),path names(e.g.</usr/5bin/ls>.path, </etc/hostname.le>.path)and command options(e.g.<-c>.opt,<-kbd>.opt, <-3>.opt).The tokeniser supplies a unique sentence identifier,the tokenised data and information about the offset position of every token with respect to the input sentence and the T roff sourcefile.This information is used later to highlight phrases in the retrieved sentences.1.3.Link GrammarThe syntactic analysis module used by ExtrAns,Link Grammar(LG),con-sists of a very fast parser and a grammar/dictionary with about60,000word forms(Sleator D.D.&T emperley D.1993).The coverage of the parser was tested using2,781sentences from the manpages and a percentage of76%full parses was found.The remaining sentences are partially parsed by systemat-ically ignoring words,as we will see below.LG returns dependency relations between pairs of words in a sentence by a set of labelled links called linkage. By default,the direction of the dependency is not given explicitly in the linkage. This is a serious shortcoming of LG since information about the direction of the dependency is indispensable for the anaphora resolution algorithm and for the construction of the MLFs.Therefore,the output of LG was extended in ExtrAns by adding the dependency direction to the linkage as the arrows in(1)indicate: (1) copiesfilename1.arg ontofilename2.argSuch directed linkages are called dependency structures in this paper.In the example above,the link connects the subject to the wall. The wall is a dummy word at the beginning of every sentence and has a link-ing requirement like any other word.The link connects the transitive verb with the subject on the left.Thus,the verbal head is at the right hand side of the link.The transitive verb and its direct object that acts as the head of a noun phrase are connected by the link.The link connects the verb to the modifying prepositional phrase.Finally,the link connects the preposition to its object.The LG parser is able to handle unknown words by making guesses from context about syntactic categories.Nevertheless,the result is always better when the words have been categorised in advance.Therefore,we have ad-ded about650domain-specific words to the LG dictionary.Some words that were already classified in the default LG dictionary had to be moved to otherEXTRANS,AN ANSWER EXTRACTION SYSTEM categories because they could be used differently in the Unix domain.An ex-ample is the verb print of the category transitive verbs that had to be moved to the category transitive verbs that may form two-word verbs to exclude ambigu-ity because it is often used as print out in the manpages.In the original LG dictionary,words with multiple entries were distinguish-ed by means of different subscripts.For ExtrAns,a subscript has been added to each single word and the subscript set was refined so that it can be used to tag the syntactic categories of the words and agreement information.T o ease readability,these subscripts do not appear in the linkages,only the subscripts provided by the tokeniser are shown.Substantial changes had to be made in the grammar to deal with some specific syntactic structures like post-nominal modifiers for command names(e.g.an on such a link)or special forms of imperatives with openers(e.g.to quit,type q).LG allows robust parsing by systematically ignoring words until a valid dependency structure is found.These words are represented in a special form, with no links attached to them(Grinberg D.et al.1995).Such“null-linked”words are not lost,they can be used—as we will see later—by the retrieval procedure.1.4.DisambiguatorExtrAns’pruner uses heuristic rules tofilter out all those dependency structures that are obviously wrong in an ambiguous sentence.In a sub-sequent stage,ExtrAns’disambiguator uses a corpus-based approach(Brill E.&Resnik P.1994)to eliminate(some of)those ambiguities that require do-main knowledge.Brill&Resnik’s original disambiguator was designed to solve preposi-tional attachment ambiguity of sentences with a transitive verb and a preposi-tional phrase(PP).The algorithm decides whether the PP should attach to the verb or to the direct object.The algorithm bases its decision on four-tuples (verb,object,preposition,and the object under the preposition)and a set of training rules that are automatically generated from a training corpus.For ex-ample,a sentence like cp copiesfilename1ontofilename2would lead to the template.This means that there exists a PP at-tachment in the sentence,and the main verb is,the head of the direct object is,the preposition is,and the head of the noun phrase in the PP is.There are twofields in front of these four-tuples:Thefirstfield indicates the attachment decision given by the disambiguator.It initially con-tains a default value(attachment to the noun)that may be modified by the disambiguator.The secondfield is defined in the training corpus only,and it reflects the correct attachment during the training and the evaluation.ExtrAns’requirements are different from the original disambiguator since the LG parser multiplies out all the possible attachment variations,giving as a result a list of alternative dependency structures for each sentence.ExtrAns uses Brill’s disambiguator in the following form:for every dependency struc-Diego MOLLÁ,Rolf SCHWITTER,Michael HESS,Rachel FOURNIER ture,the attachment information is translated into four-tuples plus twofields like above where the secondfield is now used to express the actual attach-ment decision in the dependency structure.After the disambiguator is run the first twofields are compared.Equalfields indicate a correct attachment de-cision in the dependency structure.Only the set of dependency structures that has the highest ratio of correct attachments passes thefilter(MolláD.&Hess M.2000).ExtrAns includes—besides transitive verbs—all categories of verbs, multiple PP attachments,gerund and infinitive constructions.First the training was done with the T reebank corpus(Marcus M.et al.1993)(the accuracy is reported to be81.8%)but it turned out that this corpus was not appropriate for the Unix domain(the accuracy was only72,8%).Therefore,the training for ExtrAns was redone with a subset of34manpages(containing a total of 1475attachment decisions).Because there is no automatic way to collect the relevant training data,it was necessary to encode them manually by reading the manpages and writing out the attachment decisions.Not surprisingly,the rules generated from the manpages are more ac-curate in our domain(76.6%correct disambiguations),although the number of training rules(116rules)for the test set is far smaller than those for the T ree-bank corpus(1770rules).For the evaluation we had to split up the subset of 34manpages into a training set of17manpages and a test set of the same size.There are other types of ambiguities that cannot be treated by the disam-biguator.As we will see,in such cases all competing readings of an ambiguous sentence are asserted into the knowledge base where they are fused,graded and presented in context during the retrieval procedure.1.5.Anaphora resolutionAnaphora resolution in the current version of ExtrAns is restricted to pro-nominal cases.The anaphora resolution algorithm used by ExtrAns relies on purely syntactic information(Lappin S.&Leass H.J.1994).Lappin&Leass’algorithm uses the syntactic representations generated by the Slot Grammar parser(McCord M.et al.1992)which contain information about the head-argument and head-adjunct relations and how these relations are realised(e.g. as subject,agent,object,indirect object,or prepositional object).Since Slot Grammar is dependency-based,the relevant information can be emulated in ExtrAns by checking the link types returned by the LG parser.The imple-mented algorithm contains among others the following main components:an intrasentential syntacticfilter,a morphologicalfilter,an anaphora binding al-gorithm,and a salience weighting procedure,very much like the original(Lap-pin S.&Leass H.J.1994).The output of the anaphora resolution algorithm includes a list of equi-valence classes.The equivalence classes group those words that refer to the same object.EXTRANS,AN ANSWER EXTRACTION SYSTEM In contrast to the original algorithm,ExtrAns’implementation restricts in-tersentential anaphora resolution only to the previous sentence.There are two reasons for this decision.First,in the Unix domain a pronoun corefers very rarely with a noun phrase that is not in the same or in the previous sentence. Second,computing the salience measure for all(theoretically)possible can-didates takes a lot of time and space.Lappin&Leass report an accuracy of89%for intrasentential cases.We expect that ExtrAns’anaphora module has similar accuracy,since it is a direct implementation of the original algorithm.1.6.The minimal logical formsAn important requirement of ExtrAns is that it must be fast and robust enough to be able to cope with the manpage sentences.This must apply not only to the processing of the manpages but also to the retrieval procedure to find the answers to the question.The format of the logical forms plays a crucial role in the latter.The logical forms must be simple so that they are easy to construct and to use in the retrieval stage.Still,they must remain expressive enough for the task at hand.For that reason,it is convenient that they allow the addition of more information in a monotonic way(incrementality),so that further refinements of the logical forms can be added without having to modify the notation or destroy any old information.T o fulfill these features,ExtrAns’logical forms consist merely of conjunc-tions of predicates where all the variables are existentially closed with wide scope.This simple notation is easy to build and easy to use.It is also in-cremental,since further extensions to the meaning can be done simply by adding more predicates to the conjunction.T o make the logical forms express-ive enough,we resort to reification and a particular interpretation of existence and of the logical operators.1.6.1.ReificationBy reification we mean that some“abstract”concepts introduced by pre-dicates become“concrete”.The effect of this extension is to provide handles that can be used to refer to these concepts later in the discourse.At the cur-rent stage of ExtrAns we reify predicates derived from open-class words.This is in contrast with(Hobbs J.R.1985),who proposes to reify all the predicates available in a logical form—still,we do not rule out Hobbs’approach in applic-ations that require a more detailed logical form representation.The predicates reified by ExtrAns are classified into three types:Objects.A noun such as cp introduces the predicate,and the meaning is“is the concept that the object is”.The new entity can be used in constructions with adjectives modifying nouns intensionally,or in expressions of identity.Diego MOLLÁ,Rolf SCHWITTER,Michael HESS,Rachel FOURNIER Events.A verb such as copies introduces,and the meaning is“is the concept that copies”.and represent the objects introduced by the arguments of the verb copy.Reification of events is the core of the Davidsonian semantics(Davidson D.1967;Parsons T.1985),and is useful to express the modification of events by means of adverbs,prepositional phrases,etc.Properties.Adjectives and adverbs introduce properties.For example,an ad-jective such as blue introduces the predicate,whose meaning is“is the concept that is”.Reification of properties is useful when we want to modify an adjective,like in the sentence the house is pale blue.Non-reified predicates can be introduced too.For example,the preposition onto would introduce a predicate like.This notation can be used to encode the Minimal Logical Form(MLF) of a sentence,that is,a logical form that expresses the minimal information necessary for the task at hand.Several examples of MLFs are:(2)cp copies longfiles2(3)cp copies possiblefiles(4)cp copies very longfiles(5)cp copiesfiles quicklyHere we can see reification at work.The adjective long in(2)modifies the nounfiles,and accordingly the predicate introduced by the adjective mod-ifies the object.However,the adjective possible in(3)is used intensionally. What is possible is thefileness of,that is,.Accordingly,the predicate introduced by the adjective modifies.Finally,the adverb very in(4)mod-ifies the adjective long(whose reified concept is),whereas quickly in(5) modifies the verb copies(that is,).2We will discuss the predicate in Section1.6.2.1.6.2.Existence and logical operatorsReification can also be used to encode existence of concepts.Reified concepts may or may not exist in the real world.Existential quantification alone only guarantees existence in a Platonic universe of possible entities.T o ex-press that an event actually exists in the world of manpages,ExtrAns uses a specific predicate(Hobbs J.R.1996),thus giving.In those cases where there is not enough information for ExtrAns to conclude that an event exists,nothing is said about it.For example,the copying event in(6)clearly holds in the world of Unix manpages,whereas one cannot say the same in(7) and(8),and it is not clear in(9):(6)cp copiesfiles(7)cp refuses to copy afile onto itself(8)cp does not copy afile onto itself(9)if the user types y,then cp copies thefilesWhat holds in(7)is the refusing event,but we do not say anything about the status of the copying event.One could argue that does not hold because of the lexical meaning of refuse,and therefore we should also add.However,for the time being ExtrAns does not decompose lex-ical meaning and as a consequence the negation cannot be deduced.The information is therefore left underspecified.If needed,and provided that we have enough knowledge to assess negation or assertion,this information can be inferred and added in a later stage.In the same way as with existence of concepts,all logical operators are translated as regular predicates over reified concepts.For example,the neg-ation in(8)is represented as a predicate over the concept of this particular copying event,and the implication in(9)is a predicate over the concepts of a particular typing and a particular copying event.The translation of existence and logical operators into regular predicates is a means to convert embedded structures intoflat structures,allowing the MLFs to be simple conjunctions of predicates.There are some restrictions on the expressivity of the MLFs(for example,the current MLF of cp is not the command that removesfiles would be equivalent to that of cp is not a command。

Current to June 12, 2014Last amended on June 20, 2011À jour au 12 juin 2014Dernière modification le 20 juin 2011Published by the Minister of Justice at the following address:http://laws-lois.justice.gc.ca Publié par le ministre de la Justice à l’adresse suivante :http://lois-laws.justice.gc.caCANADACONSOLIDATION Glazed Ceramics and Glassware RegulationsCODIFICATIONRèglement sur les produits céramiques émaillés et les produits deverre émaillésSOR/98-176DORS/98-176OFFICIAL STATUS OF CONSOLIDATIONS CARACTÈRE OFFICIEL DES CODIFICATIONSSubsections 31(1) and (3) of the Legislation Revision and Consolidation Act, in force on June 1, 2009, provide as follows:Les paragraphes 31(1) et (3) de la Loi sur la révision et la codification des textes législatifs, en vigueur le 1er juin 2009, prévoient ce qui suit :Published consolidation is evidence31. (1) Every copy of a consolidated statute orconsolidated regulation published by the Ministerunder this Act in either print or electronic form is ev-idence of that statute or regulation and of its contentsand every copy purporting to be published by theMinister is deemed to be so published, unless thecontrary is shown.31. (1) Tout exemplaire d'une loi codifiée ou d'unrèglement codifié, publié par le ministre en vertu dela présente loi sur support papier ou sur support élec-tronique, fait foi de cette loi ou de ce règlement et deson contenu. Tout exemplaire donné comme publiépar le ministre est réputé avoir été ainsi publié, saufpreuve contraire.Codificationscomme élémentde preuve ...[...]Inconsistencies in regulations(3) In the event of an inconsistency between aconsolidated regulation published by the Ministerunder this Act and the original regulation or a subse-quent amendment as registered by the Clerk of thePrivy Council under the Statutory Instruments Act,the original regulation or amendment prevails to theextent of the inconsistency.(3) Les dispositions du règlement d'origine avecses modifications subséquentes enregistrées par legreffier du Conseil privé en vertu de la Loi sur lestextes réglementaires l'emportent sur les dispositionsincompatibles du règlement codifié publié par le mi-nistre en vertu de la présente loi.Incompatibilité— règlementsNOTE NOTEThis consolidation is current to June 12, 2014. Thelast amendments came into force on June 20, 2011.Any amendments that were not in force asof June 12, 2014 are set out at the end of this docu-ment under the heading “Amendments Not inForce”.Cette codification est à jour au 12 juin 2014. Lesdernières modifications sont entrées en vigueurle 20 juin 2011. Toutes modifications qui n'étaientpas en vigueur au 12 juin 2014 sont énoncées à la finde ce document sous le titre « Modifications non envigueur ».TABLE OF PROVISIONS TABLE ANALYTIQUESection Page Article PageGlazed Ceramics and Glassware Regulations Règlement sur les produitscéramiques émaillés et les produits de verre émaillés1INTERPRETATION11DÉFINITIONS1 2AUTHORIZATION22AUTORISATION24LEACHABILITY LIMITS FORLEAD AND CADMIUM24TAUX MAXIMAL DELIBÉRATION DE PLOMB ET DECADMIUM26IDENTIFICATION ANDWARNING36ÉLÉMENT DISTINCTIF ET MISEEN GARDE37REPEAL37ABROGATION3 8COMING INTO FORCE38ENTRÉE EN VIGUEUR3SCHEDULETEST METHODS4ANNEXEMÉTHODES D’ESSAI4Registration EnregistrementSOR/98-176 March 19, 1998DORS/98-176 Le 19 mars 1998CANADA CONSUMER PRODUCT SAFETY ACT LOI CANADIENNE SUR LA SÉCURITÉ DES PRODUITSDE CONSOMMATIONGlazed Ceramics and Glassware Regulations Règlement sur les produits céramiques émaillés et lesproduits de verre émaillésP.C. 1998-400 March 19, 1998 C.P. 1998-400 Le 19 mars 1998His Excellency the Governor General in Council, on the recommendation of the Minister of Health, pursuant to section 5a of the Hazardous Products Act, hereby makes the annexed Hazardous Products (Glazed Ceram-ics and Glassware) Regulations.Sur recommandation du ministre de la Santé et en ver-tu de l’article 5a de la Loi sur les produits dangereux, Son Excellence le Gouverneur général en conseil prend le Règlement sur les produits dangereux (produits céra-miques émaillés et produits de verre), ci-après.a R.S., c. 24 (3rd Supp.), s. 1a L.R., ch. 24 (3e suppl.), art. 1GLAZED CERAMICS AND GLASSWARE REGULATIONS RÈGLEMENT SUR LES PRODUITS CÉRAMIQUES ÉMAILLÉS ET LES PRODUITS DE VERRE ÉMAILLÉSINTERPRETATION DÉFINITIONS1. The definitions in this section apply in these Regu-lations.“cups and mugs” means small hollow-ware used for the consumption of liquids. (tasses et chopes)“drinking vessel” means any hollow-ware from which one can drink liquids. (récipient à boire)“flatware” means a product having an internal depth not exceeding 25 mm, measured vertically from the lowest interior point to a horizontal plane passing through the point of overflow. (vaisselle plate)“good laboratory practices” means practices similar to those set out in the OECD Principles of Good Laborato-ry Practice, Number 1 of the OECD Series on Principles of Good Laboratory Practice and Compliance Monitor-ing, ENV/MC/CHEM (98) 17, the English version of which is dated January 21, 1998 and the French version of which is dated March 6, 1998. (bonnes pratiques de laboratoire)“hollow-ware” means a product having an internal depth greater than 25 mm, measured vertically from the lowest interior point to a horizontal plane passing through the point of overflow. (vaisselle creuse)“large hollow-ware” means hollow-ware with a capacity of 1.1 L or more. (grande vaisselle creuse)“permanent warning” means a warning that remains legi-ble for the lifetime of the product under normal condi-tions of use and cleaning. (mise en garde permanente)“pitcher” means a large hollow-ware vessel that is com-monly used for storing and dispensing liquids but does not include a creamer, a coffeepot or a teapot. (pichet)“product” means a product that has all of the following characteristics:(a) it is completely or partly made of ceramic or glass;1. Les définitions qui suivent s’appliquent au présent règlement.« bonnes pratiques de laboratoire » S’entend des pra-tiques analogues à celles énoncées dans les Principes de l’OCDE de bonnes pratiques de laboratoire, Série sur les principes de bonnes pratiques de laboratoire et vérifica-tion du respect de ces principes, numéro 1, ENV/MC/ CHEM(98)17, daté du 6 mars 1998 dans sa version fran-çaise et du 21 janvier 1998 dans sa version anglaise. (good laboratory practices)« grande vaisselle creuse » Vaisselle creuse d’une capaci-té de 1,1 L ou plus. (large hollow-ware)« mise en garde permanente » Mise en garde qui demeure lisible durant toute la vie du produit dans des conditions normales d’utilisation et de nettoyage. (permanent warn-ing)« petite vaisselle creuse » Vaisselle creuse d’une capacitéde moins de 1,1 L. (small hollow-ware)« pichet » Article de grande vaisselle creuse utilisé habi-tuellement pour conserver et pour servir des liquides. Sont exclues de la présente définition les crémières, les cafetières et les théières. (pitcher)« produit » Produit qui satisfait aux exigences suivantes : a) il est fait entièrement ou partiellement en céra-mique ou verre;b) il est recouvert en totalité ou en partie d’un revête-ment, d’un émail ou d’une décoration contenant du plomb ou du cadmium;c) il est utilisé pour conserver, préparer ou servir un aliment au sens de l’article 2 de la Loi sur les aliments et drogues. (product)« récipient à boire » Article de vaisselle creuse dans le-quel on peut boire des liquides. (drinking vessel)« tasses et chopes » Articles de petite vaisselle creuse uti-lisés pour consommer des liquides. (cups and mugs)(b) it is completely or partly covered with a coating, glaze or decoration that contains lead or cadmium; and(c) it is used in storing, preparing or serving food, as defined in section 2 of the Food and Drugs Act. (pro-duit)“small hollow-ware” means hollow-ware with a capacity of less than 1.1 L. (petite vaisselle creuse)SOR/2007-30, s. 2; SOR/2009-179, s. 6.« vaisselle creuse » Ensemble de produits dont la profon-deur, mesurée verticalement du point intérieur le plus bas jusqu’à un plan horizontal traversant le point de dé-bordement, est supérieure à 25 mm. (hollow-ware)« vaisselle plate » Ensemble de produits dont la profon-deur, mesurée verticalement du point intérieur le plus bas jusqu’à un plan horizontal traversant le point de dé-bordement, n’excède pas 25 mm. (flatware)DORS/2007-30, art. 2; DORS/2009-179, art. 6.AUTHORIZATION AUTORISATION2. A product may be advertised, sold or imported if it meets the requirements of these Regulations.SOR/2009-179, s. 7.2. La vente, l’importation et la publicité du produit sont autorisées si celui-ci satisfait aux exigences du pré-sent règlement.DORS/2009-179, art. 7.3. [Repealed, SOR/2009-179, s. 7] 3. [Abrogé, DORS/2009-179, art. 7]LEACHABILITY LIMITS FOR LEAD ANDCADMIUM TAUX MAXIMAL DE LIBÉRATION DE PLOMB ETDE CADMIUM4. No product of a product category set out in col-umn 1 of an item of the table to this section, when tested in accordance with the method set out in item 1 of the schedule, shall release lead in excess of the leachability limit set out in column 2 of that item or cadmium in ex-cess of the leachability limit set out in column 3 of that item, unless the product is identified or displays a per-manent warning in accordance with section 6.4. Un produit d’une catégorie de produits mentionnée à la colonne 1 du tableau du présent article qui est mis àl’essai conformément à la méthode établie à l’article 1 de l’annexe ne doit libérer ni plomb ni cadmium à un taux supérieur au taux maximal indiqué à la colonne 2 ou à la colonne 3, à moins de comporter un élément distinctif ou une mise en garde permanente conformément à l’ar-ticle 6.TABLEItem Column 1Column 2Column 3Product CategoryLeachability Limitfor Lead (milligramsper litre)Leachability Limit forCadmium (milligramsper litre)1.Flatware 3.00.502.Small hollow-ware,other than cups ormugs 2.00.50 rge hollow-ware,other than pitchers 1.00.254.Cups and mugs0.50.505.Pitchers0.50.25TABLEAUArticleColonne 1Colonne 2Colonne 3Catégorie deproduitsTaux maximal delibération de plomb(milligrammes parlitre)Taux maximal delibération de cadmium(milligrammes parlitre)1.Vaisselle plate3,00,502.Petite vaissellecreuse, sauf lestasses et les chopes2,00,503.Grande vaissellecreuse, sauf lespichets1,00,254.Tasses et chopes0,50,505.Pichets0,50,255. No drinking vessel with a distinctive exterior deco-rative pattern within 20 mm of the rim, when tested in accordance with the method set out in item 2 of the schedule, shall release lead in excess of 4 mg/L or cad-mium in excess of 0.4 mg/L.SOR/2007-30, s. 3.5. Un récipient à boire orné, à l’extérieur, d’un motif décoratif caractéristique situé à 20 mm ou moins du bord qui est mis à l’essai conformément à la méthode établie àl’article 2 de l’annexe ne doit pas libérer plus de 4 mg/L de plomb ou 0,4 mg/L de cadmium.DORS/2007-30, art. 3.IDENTIFICATION AND WARNINGÉLÉMENT DISTINCTIF ET MISE EN GARDE6. A product that is not for food use and that releases lead or cadmium in excess of the leachability limits set out in the table to section 4 shall:(a) be identified by a design feature, such as a hole or a mounting hook, that renders the product unsuitable for the storing, preparing or serving of food; or(b) display, in both official languages and in capital letters of not less than 3 mm in height, a permanent warning consisting of one of the following texts or a similar text, as may be appropriate:6. Un produit qui n’est pas destiné à être utilisé pour les aliments et qui libère du plomb ou du cadmium à un taux supérieur au taux maximal indiqué au tableau de l’article 4 doit comporter :a) soit un élément distinctif, tel un trou ou un crochet, qui le rend inutilisable pour conserver, préparer ou servir des aliments;b) soit, dans les deux langues officielles et en majus-cules d’au moins 3 mm de hauteur, une mise en garde permanente portant l’une des mentions suivantes ou une mention semblable, selon le cas :DANGER!CONTAINS LEAD — CONTIENT DU PLOMB DO NOT USE FOR FOOD — NE PAS UTILISER POURLES ALIMENTSDANGER!CONTIENT DU PLOMB — CONTAINS LEADNE PAS UTILISER POUR LES ALIMENTS — DO NOTUSE FOR FOODDANGER!CONTAINS CADMIUM — CONTIENT DU CADMIUM DO NOT USE FOR FOOD — NE PAS UTILISER POURLES ALIMENTSDANGER!CONTIENT DU CADMIUM — CONTAINS CADMIUM NE PAS UTILISER POUR LES ALIMENTS — DO NOTUSE FOR FOODDANGER!CONTAINS LEAD AND CADMIUM — CONTIENT DUPLOMB ET DU CADMIUMDO NOT USE FOR FOOD — NE PAS UTILISER POURLES ALIMENTSDANGER!CONTIENT DU PLOMB ET DU CADMIUM — CON-TAINS LEAD AND CADMIUMNE PAS UTILISER POUR LES ALIMENTS — DO NOTUSE FOR FOODREPEAL ABROGATION 7. [Repeal]7. [Abrogation]COMING INTO FORCE ENTRÉE EN VIGUEUR8. These Regulations come into force on March 19, 1998.8. Le présent règlement entre en vigueur le 19 mars 1998.SCHEDULE (Sections 4 and 5)ANNEXE (articles 4 et 5)TEST METHODS MÉTHODES D’ESSAI1. To determine whether a product releases lead or cadmium, the following method, which is based on the International Organization for Standardization standard ISO 6486-1:1999, Ceramic ware, glass-ceramic ware and glass dinnerware in contact with food — Release of lead and cadmium — Part 1: Test Method, second edition, 1999-12-15, shall be used:(a) handwash the product using a non-acidic detergent solution, rinse with distilled water and air dry;(b) fill the product to within 5 mm of the level of overflowing with an extraction solution of 4% (volume/volume) of acetic acid in wa-ter;(c) cover the product with an inert opaque cover and allow to stand for 24 hours at 22°C ± 2°C;(d) stir the extraction solution to ensure homogeneity taking care not to abrade the surface of the product;(e) take an aliquot of the extraction solution and, within 8 hours, analyze the solution using an analytical technique that is in accor-dance with good laboratory practices; and(f) express the results as the quantity of the lead or cadmium in milligrams per litre of the extraction solution.1. Pour établir si un produit libère du plomb ou du cadmium, utili-ser la méthode ci-après, basée sur la norme ISO 6486-1:1999 de l’Or-ganisation internationale de normalisation, intitulée Vaisselle en céra-mique, vaisselle en vitrocéramique et vaisselle de table en verre en contact avec les aliments — Émission de plomb et de cadmium —Partie 1 : Méthode d’essai, deuxième édition, dans sa version du15 décembre 1999 :a) laver le produit à la main avec une solution détersive non acide, le rincer à l’eau distillée et le faire sécher à l’air;b) remplir le produit d’une solution d’extraction constituée de 4 % (volume/volume) d’acide acétique dans de l’eau, à 5 mm ou moins du niveau de débordement;c) couvrir le produit d’un couvercle opaque fait de matériel inerte et laisser reposer 24 heures à une température de 22 °C ± 2 °C; d) remuer la solution d’extraction pour l’homogénéiser, en prenant soin de ne pas abraser la surface du produit;e) prélever une aliquote de la solution d’extraction et, dans les 8 heures qui suivent, la soumettre à l’analyse en appliquant une mé-thode conforme aux bonnes pratiques de laboratoire;f) exprimer les résultats en milligrammes de plomb ou de cad-mium par litre de solution d’extraction.2. To determine whether a drinking vessel bearing a distinctive ex-terior decorative pattern within 20 mm of the rim releases lead or cadmium, the following method, which is based on a standard of the American Society for Testing and Materials (ASTM C927-80), reap-proved 2004, Standard Test Method for Lead and Cadmium Extract-ed from the Lip and Rim Area of Glass Tumblers Externally Decorat-ed with Ceramic Glass Enamels, shall be used:(a) handwash the drinking vessel using a non-acidic detergent so-lution, rinse with distilled water and air dry;(b) measure the internal volume of the drinking vessel in millil-itres by filling with distilled water to within 5 mm of the level of overflowing and record the internal volume (V2);(c) discard the water and invert the drinking vessel in an appropri-ate laboratory glassware container whose diameter is a minimum of 1.25 times and a maximum of 2 times the external diameter of the test specimen as measured at the rim;(d) add to the laboratory glassware container the volume of an ex-traction solution of 4% (volume/volume) of acetic acid in water that is sufficient to permit the drinking vessel to be submerged in 20 mm of the solution and record the volume of extraction solution used (V1);(e) cover the laboratory glassware container with an inert opaque cover and allow to stand for 24 hours at 22°C ± 2°C;(f) remove the drinking vessel and stir the extraction solution to ensure homogeneity;2. Pour établir si un récipient à boire orné, à l’extérieur, d’un motif décoratif caractéristique situé à 20 mm ou moins du bord libère du plomb ou du cadmium, utiliser la méthode ci-après, basée sur la norme ASTM C927-80 de l’American Society for Testing and Mate-rials, réapprouvée en 2004 et intitulée Standard Test Method for Lead and Cadmium Extracted from the Lip and Rim Area of Glass Tum-blers Externally Decorated with Ceramic Glass Enamels:a) laver le récipient à boire à la main avec une solution détersive non acide, le rincer à l’eau distillée et le faire sécher à l’air;b) mesurer le volume interne du récipient à boire, en millilitres, en le remplissant d’eau distillée à 5 mm ou moins du niveau de débor-dement et noter le volume interne (V2);c) jeter l’eau et déposer le récipient à boire, inversé, dans un réci-pient de laboratoire en verre approprié, dont le diamètre est d’au moins 1,25 fois et d’au plus 2 fois le diamètre externe du produit mesuré sur le bord;d) ajouter dans le récipient de laboratoire en verre un volume suf-fisant d’une solution d’extraction constituée de 4 % (volume/vo-lume) d’acide acétique dans de l’eau de telle sorte que le récipient à boire soit immergé dans 20 mm de cette solution et noter le vo-lume de solution d’extraction utilisé (V1);e) couvrir le récipient de laboratoire en verre avec un couvercle opaque fait de matériel inerte et laisser reposer pendant 24 heures àune température de 22 °C ± 2 °C;f) enlever le récipient à boire et remuer la solution d’extraction pour l’homogénéiser;(g) take an aliquot of the extraction solution and, within 8 hours, analyze the solution using an analytical technique that is in accor-dance with good laboratory practices; and(h) express the results as the quantity of the lead or cadmium in milligrams per litre of the extraction solution relative to the inter-nal volume of the drinking vessel as follows:(C × V1) / V2whereC is the concentration of lead or cadmium in milligrams per litrein the extraction solution,V1is the volume in millilitres of the extraction solution used, andV2is the internal volume in millilitres of the drinking vessel. SOR/2007-30, ss. 4, 5.g) prélever une aliquote de la solution d’extraction et, dans les 8 heures qui suivent, la soumettre à l’analyse en appliquant une mé-thode conforme aux bonnes pratiques de laboratoire;h) exprimer les résultats, en milligrammes de plomb ou de cad-mium par litre de solution d’extraction, en tenant compte du vo-lume interne du récipient à boire, selon la formule suivante :(C × V1) / V2où :C représente la concentration de plomb ou de cadmium expriméeen milligrammes par litre de la solution d’extraction utilisée,V1le volume en millilitres de la solution d’extraction utilisée,V2le volume interne en millilitres du récipient à boire.DORS/2007-30, art. 4 et 5.。

Extraction of Fucoxanthin from Undaria Pinnatifida using enzymatic pre-treatment followed by DME & EtoH co-solvent extraction Jagan M Billakanti*1, Owen Catchpole1, Tina Fenton1 and Kevin Mitchell11Industrial Research Limited, Integrative Bioactive Technologies, 69 Gracefield Road, PO Box 31310, Lower Hutt5040, New Zealand.Corresponding author: j.billakanti@; Phone: (+64) 4 931 3285; Fax: (+64) 4 566 6004 ABSTRACTThe brown seaweed (Undaria pinnatifida) has been recognized as a potential source of biologically active lipid compounds and in particular, fucoxanthin. Fucoxanthin has been reported to have anti-cancer, anti-obesity and anti-inflammatory effects. Like other carotenoids, fucoxanthin is a fat soluble compound and usually requires organic solvents for its extraction. The extraction of fucoxanthin poses a number of challenges since it is unstable with respect to high and low pH, and also to light; and the extraction of fucoxanthin from dry materials gives poor yields. This study was carried out to improve the yields of total lipids and fucoxanthin from Undaria pinnatifida using an enzyme-assisted extraction process followed by dimethyl ether (DME) extraction. Enzymatic pre-treatment was employed to degrade cell wall polysaccharides to oligosaccharides prior to the extraction of fucoxanthin from the residual biomass using near critical dimethyl ether with and without ethanol as a co-solvent. The residual biomass was separated from hydrolyzed polysaccharides by filtration or centrifugation. Optimal enzyme pre-treatment conditions were achieved using 0.05% (g/g dry weight) alginase lyase, a pH of 6.2, temperature of 37°C, reaction time of 2 hrs and 5% biomass solid content (dry basis). Lipids extracted by DME & ethanol co-solvent after the enzymatic pre-treatment of Undaria pinnatifida were recovered at >95% of the yield obtained using classical solvent extraction techniques, a 15-20% improvement over non-enzyme treated wet biomass. Similarly, the amount of fucoxanthin in the enzyme pre-treated (0.64% g/g of extract) biomass showed >50% improvement over non-treated biomass (0.30%, g/g of extract). Thus, an enzyme-assisted DME extraction process appears to be a good method for recovering biologically active fucoxanthin from Undaria pinnatifida with improved yields.Keywords: Fucoxanthin, brown seaweed, dimethyl ether extraction, Alginase lyase, Undaria pinnatifida INTRODUCTIONUndaria pinnatifida is a brown seaweed and also known as Wakame. This is the most commonly consumed dietary seaweed in Japan due in part to its potential health promoting benefits [1-5]. Undaria kelp is present in almost all marine regions of the world, and is an invasive pest species in New Zealand marine aquaculture operations [6-7]. Undaria seaweed is a good source of natural bioactive compounds such as complex and neutral lipids rich in essential ω-3 fatty acids, carotenoids (fucoxanthin), dietary fiber, proteins, vitamins, polyphenolic compounds, sulphated polysaccharides and fucoidans [8-9]. Fucoxanthin isolated from brown seaweed has been reported to have several health benefits including anti-obesity, anti-diabetic and anti cancer properties [4-5]. Therefore, brown seaweed is considered as very interesting source of natural compounds with numerous biological activities that could potentially be used as functional ingredients in many industrial applications. Fucoxanthin is a fat soluble carotenoid compound that usually requires organic solvents for its extraction. In addition, fucoxanthin is sensitive to light, temperature, pH and strong acidic & alkaline conditions. Therefore, extraction procedures for fucoxanthin must be carried out under a controlled environment to minimize the degradation and hydrolysis of carotenoids [10-11]. The most common method of extraction is by using liquid organic solvents such as toluene, hexane, dimethyl sulfoxide (DMSO), acetone, methanol and combinations thereof [11-15]. However, many of these solvents are not suitable for the production of food ingredients or nutraceuticals and removal of solvents at elevated temperatures can also damage functional properties of bioactive compounds [16-18]. In addition, recovery yields of these methods varied widely and not suitable for industrial applications due to the presence of large quantities of cell wall polysaccharides, which greatly reduce the extraction efficiencies. Supercritical fluid extraction (SFE) using CO2provides an alternative technology with potentially better efficiencies and improved recoveries. The extract obtained from SFE contains fewer polar impurities than by conventional organic solvent extraction. Because SFE has a favorable criticaltemperature and pressure that enables heat liable bioactive compounds recovery [18]. However, one of the main drawbacks of conventional SFE using CO2 is the need to process dry feed materials. Bioactive compounds present in brown seaweed are greatly reduced by conventional (air) drying methods and freeze-drying is very expensive. In order to avoid the loss of bioactive compounds in brown seaweed the materials should be processed in its native state. Recent developments in SFE using near critical dimethyl ether (DME) facilitates the processing of wet feed materials [17]. In addition, recent research into the recovery of bioactive compounds from seaweed using microwave-assisted or enzyme assisted extraction techniques appeared to give improved yields over classical methods [19-21]. Interestingly, enzymatic digestion of algal cell wall polysaccharides prior to the extraction gains more attention due to its potency in proving the recovery yields of bioactive compounds.In this report, we have investigated the use of enzyme-assisted DME and DME-EtOH co-solvent extraction of bioactive lipids and carotenoids from Undaria pinnatifida seaweed, and compared this with SFE-CO2and DME extraction of dry seaweed. The enzyme and DME extraction process could be a potential alternative method to achieve improved yields of industrially useful bioactive compounds from brown seaweed.MATERIALS AND METHODSUndaria pinnatifida seaweed material used in this study was obtained by Tai Tipu, Malborough, New Zealand. The seaweed was water washed and ground and then frozen. Frozen seaweed (wet) was stored in the freezer (-20°C) until it used in the experiments. Freeze and air dried seaweed was also supplied by Tai Tipu. Until unless stated, all chemicals and solvents used in this study were analytical grade. Fucoxanthin and a mixture of carotenoid standards were purchased from DHI Lab, Denmark. Alginase lyase enzyme was purchased from Sigma Aldrich, Australia. LIPID CONTENT ESTIMATIONTotal lipid (TL) content of Undaria pinnatifida seaweed was estimated using the Bligh and Dyer [22] method with modifications necessary for dealing with seaweed. Typically, 10 grams of brown seaweed (wet) was transferred into a 50 mL Falcon tube and mixed thoroughly for an hour with dH2O, MeOH and CHCl3 (1:1:1). The phases were the separated and lipids were extracted in the chloroform phase by phase separation using centrifugation at 4000 rpm for 5 min, and the chloroform layer containing the lipids was then recovered. The residual biomass was re-extracted a further 2-3 times using the same procedure until the maximum lipid extraction achieved. All solvent + extract solutions were pooled together and the solvent was removed by rotary evaporation at 45°C until complete dryness. Total lipid content was estimated by subtracting the empty flask weight and lipid content was represented in wt% (g/g of wet seaweed).CAROTENOID COMPOUNDS ANALYSISSample solutions were prepared by accurately weighing samples and dissolving in ethanol to a known volume, typically, 100 mg of sample made up to 20.0 ml in a volumetric flask. Sample solutions were then analysed directly by HPLC method following membrane filtration (0.20 µm). A flow rate of 0.15 mL/min was employed and an injection volume of 5 µL was used. Solvent A was 20mM ammonium acetate (prepared in DI water) and solvent B methanol. Initial solvent conditions were 75% solvent B. These conditions were held for 0.5 min followed by a linear increase to 100% solvent B at 39 min following injection. Solvent composition was held at this level for 10 min before being changed back to the initial conditions over 1 min and the column was then allowed to equilibrate for 10 min before a subsequent injection was made. The analysis was carried out using a Waters Acuity UPLC equipped with diode array detector and column oven. Chromatography was carried out using a Waters Acquity UPLC BEH C18 1.7 µm 2x150mm column, Waters Acquity UPLC BEH C18 1.7 µm VanGuard Pre-Column and oven temperature of 60ºC.Samples were maintained at 15ºC prior to analysis in the auto sampler compartment. Peak areas in chromatograms recorded at 449 nm were used for quantification following subtraction of a blank run chromatogram generated by running the above gradient with an ethanol blank solution being injected. The system was calibrated using standard solutions of carotenoid compounds of known concentrations purchased from DHI Lab, Denmark. Individual carotenoids were identified by comparison of retention times and on line UV-Vis. absorption spectra with those of the authentic standards. Fucoxanthin content in the brown seaweed extracts was estimated using the above HPLC method and represented in wt% (g/g lipid extract basis).ETHANOL EXTRACTION METHODEthanol was used as a solvent for recovering lipid soluble compounds from brown seaweed because EtOH was used because of it is a food grade (FG) solvent. Generally, 10 grams of brown seaweed (wet) was mixed with 90 mL of EtOH (FG) for an hour at room temperature and centrifuged at 4000 rpm for 20 minutes. The ethanol phase was separated and transferred into a weighed round bottom flask and residual biomass was re-extracted twice more with another 90 mL of EtOH each time. All EtOH extracts were pooled together and lipid-rich extract was concentrated by rotary evaporation (50ºC) until dryness. Some EtOH extracts were re-extracted using CHCl3: MeOH: dH2O solvent system to remove any non-lipid materials present and thus to determine the lipid content.PACKED BED EXTRACTION USING DME AND CO2Extraction of lipids and carotenoids from wet brown seaweed was carried out using near critical dimethylether (DME); and DME+ ethanol so-solvent system for achieving better recovery yields. The seaweed (wet or dry) is packed in a basket which has porous plates at the top and bottom. The basket is then placed in the 2L extraction vessel and lipid extraction was carried out with DME or DME+EtOH (FG) co-solvent (10% mass ratio) at a pressure of 40 bar and temperature of 60ºC (Fig. 1), or CO2 and CO2+EtOH co-solvent (10% mass ratio) at 300 bar and 60ºC. The DME or CO2(and co-solvent) flowed upwards through the bed, before passing through a pressure reduction valve and into the separator. The extract and co-solvent is recovered from the separator (held at ~ 6 bars for DME or ~ 55 bar for CO2 and at the same temperature) at regular time intervals. The DME or CO2 is then recycled via a condensing heat exchanger and compressor (Haskel AG72). The co-solvent flow is stopped after a given extraction time period, and the extraction continued until the amount of extract recovered per time interval drops to almost zero. The plant is then depressurized, the extracted solids recovered and then the separator and control valve rinsed with solvent to recover any additional extract. The total recovered extract and ethanol is then rotary evaporated to recover the lipid-rich extract and to remove any solvent. Lipid extracts were analysed for fucoxanthin quantities in each extract by HPLC method and ω-3 fatty acids by GC. Lipid profiles were determined by TLC..ENZYME PRE-TREATMENT PRIOR TO EXTRACTION OF FUCOXANTHINAlginase lyase enzyme selected for this work has the ability to hydrolyze specific polymer bonds present in the intact cell wall of brown seaweed, which assists in obtaining the maximum recovery of fucoxanthin. There are several factors that directly influence the enzyme performance during the digestion process. These factors were adjusted to find the optimal reaction conditions in small scale experiments. The incubation time-temperature combination of Alginase lyase treatment was one of the most important factors to be optimized. During the brown seaweed cell wall polysaccharide digestion, Alginase lyase hydrolysis was carried out at mild reaction conditions to minimize the loss of fucoxanthin, which can be greatly influenced by pH, temperature and light. Typical polysaccharide hydrolysis was performed at 5% (w/v) solids, pH 6.2, 37°C, 2 hrs and 0.05% (w/w) enzyme at continuous mixing conditions. Hydrolyzed polysaccharide (water soluble compounds) materials was separated from the seaweed biomass by centrifugation at 4000 rpm for 20 minutes and stored at 4°C until further processing.EXTRACTION USING ENZYME DIGESTION AND CONTINUOUS DME EXTRACTIONA combined extraction method was derived using firstly enzyme digestion as above (without the final centrifugation step) followed by direct DME processing of the enzyme digest solution. Typically, brown seaweed was defrosted and mixed with water to a final solids content of 5%. The resultant seaweed slurry was adjusted to pH 6.2, temperature 37°C (water bath) and then enzymatic hydrolysis was initiated by the addition of 0.05% (w/w) Alginase lyase enzyme. Lipid soluble compounds from the enzyme treated brown seaweed biomass were then extracted using a continuous DME+EtOH co-solvent extraction method directly from the enzyme digest slurry. An empty, pre-weighed extraction basket was placed into the extraction vessel, the vessel was then closed, and then the system was pressurized with DME up to the desired operating pressure of 40 bar and temperature of 60°C. The slurry was pumped into the top of the extraction vessel through a nozzle, while simultaneously DME or DME with FG ethanol at a 10 % addition rate was passed upwards thyrough the extraction vessel and basket. The DME or DME + cos-solvent simultaneously extracted the lipids and water from the enzyme digest solution, giving a dry residual biomass (and enzyme) that collected in the bottom of the basket. The DME, ethanol, water and lipid extract then passes through the pressure reduction valve as per usual, and the water, ethanol and lipid are collected at regular time intervals from the separator (held at ~ 6 bar and 60°). The DME is then recycled via a condensing heat exchanger and compressor (Haskel AG72). The slurry feed pump is stopped at a desired throughput, and then the co-solventflow is stopped after a further 30 minutes. The extraction carries on until the amount of extract recovered per time interval drops to almost zero. The plant is then depressurized, the extracted solids recovered and then the separator and control valve rinsed with solvent to recover any additional extract. The total recovered extract, water and ethanol is then rotary evaporated to recover the lipid-rich extract and to remove any solvent. Lipid extracts were analysed for fucoxanthin by HPLC and ω-3 fatty acids by GC. Lipid profiles were determined by TLCAlgae(wet)Aqueousextractsupercritical extraction technology.RESULTS AND DISCUSSIONLIPID EXTRACTION OF WET UNDARIA PINNATIFIDA SEAWEED USING ETHANOLTotal lipid extraction of wet Undaria pinnatifida (wet) was performed by repeated extraction of brown seaweed biomass using CHCl3: MeOH: H2O (1:1:1) solvent system and lipid content was determined to be 0.64% (w/w of wet seaweed or 6.4% on dry weight basis). Initial extracts (3 cycles using CHCl3: MeOH: H2O) appeared to contain a higher lipid content than expected and re-extraction of initial extracts using CHCl3-MeOH removed approximately10-20% of non-lipid materials. This indicates that repeated extraction caused the extraction of some non-lipid materials into the chloroform-rich phase. Hence re-extraction of initial extract using the same solvent system was routinely performed to remove non-lipid extracts. Residual biomass of CHCl3-MeOH extracted Undaria seaweed appeared slightly green and this indicates that complete lipid extraction was not achieved even after repeated extraction and could be because of intact cell wall polysaccharides present in brown seaweed.Lipid soluble compounds can be extracted from brown seaweed using food grade EtOH. Lipid extract (after re-extraction with CHCl3-MeOH) obtained from EtOH extraction (3 cycles) was 0.3-0.35%, which accounts for 40-50%of feed material lipid content (Table 1). The residual biomass after exhaustive EtOH extraction appeared to retain a significant green colour (due to chlorophyll), and this is indicative of incomplete lipid extraction. Increasing the number of EtOH extraction cycles did not improve recovery. During the EtOH extraction of Undaria seaweed, large amounts (30-50%) of non-lipid materials were also extracted into the EtOH phase. On the other hand, pre-treatment of Undaria using Alginase lyase digested the cell wall polysaccharides into oligosaccharides and hydrolyzed oligosaccharides were easily separated from the seaweed biomass by centrifugation. Enzymatic hydrolysis of Undaria seaweed helped to remove approximately 30-40% of non-lipid solids from feed materials. Lipid extraction followed by EtOH extraction (3 times) and CHCl3-MeOH partitioning showed improved recovery yields (40-50% improvement over untreated, lipid yield 0.57 %). Enzyme pre-treated extracts also showed significantly reduced levels of non-lipid materials in the EtOH extract. However, processing large volumes of Undaria seaweed using this combined method for industrial application will require huge amounts of EtOH and significant costs for ethanol recovery and reuse. Both extracts, with and without enzyme pre-treatment contained similar fucoxanthin concentrations (Table 1), but fucoxanthin recovery increased more than 40% using the enzyme pre-treatment.LIPID EXTRACTION OF WET UNDARIA PINNATIFIDA SEAWEED USING DMEIn order to improve the recovery of bioactive compounds from brown seaweed and overcome some of the limitations of ethanol as a solvent, DME extraction with and without EtOH co-solvent was employed and achieved good lipid yields (0.55%). The residual biomass after DME-EtOH co-solvent extraction was almost completely dry, a light green/brown colour, and contained a small amount of lipids (lipid extract from the residue was a light green/yellow colour, and contained more polar than neutral lipids). The DME-EtOH co-solvent extraction method gave better lipid yields (40-50%) compared to EtOH (Table 1 & 2). However, around 40 % of the total extract was non-lipids (Table 2). DME-EtOH co-solvent SFE extraction was further improved using enzyme pre-treatment. Undaria seaweed (wet biomass) was pre-treated with Alginate lyase enzyme, which digests complex polysaccharides into oligosaccharides, prior to the extraction of bioactive compounds using DME-EtOH co-solvent extraction. Enzymatic hydrolysis of Undaria seaweed helped to remove approximately 30-40% solids from the feed materials (mainly polysaccharides) by means of centrifugation, which accounts for 50% improvement over untreated material. Lipid extraction of enzyme treated seaweed biomass using DME-EtOH co-solvent extraction gave a total lipid yield of 0.68%; and the crude extract contained only 20% of non-lipid materials. In addition, this method achieved almost complete fat soluble bioactive compounds recovery (6.8% g/g dry seaweed) from Undaria pinnatifida seaweed (residual biomass contains only 0.175%, g/g dry seaweed of lipids and mostly polar lipids). On the other hand, fucoxanthin concentration in the enzyme pre-treated lipid extract contain >50% than that of untreated DME-EtOH extract (Table 2). Removal of cell wall polysaccharides from Undaria seaweed by enzyme pre-treatment not only improved the recovery of lipids and fucoxanthin, it also reduced the amount of biomass (≈40-50%) to be processed in DME-EtOH co-solvent extraction, which results in less solvent usage and this potentially provides economic benefits in industrial applications.Table 1: Summary of lipid extracts obtained from Undaria pinnatifida seaweed (wet) by different extraction methods. Lipid extracts given in this table were re-extracted using CHCl3: MeOH: dH2O after initial extraction with respected solvent system. Until unless stated, all extracts were repeated and average values are given. SW-seaweed.Extraction method Seaweed(g, wet)Lipid extract(g)Lipid extract(g/g wet SW)Fucoxanthin(g/g lipid extract)Fucoxanthin(mg/g wet SW)CHCl3: MeOH: dH2O 20 0.128 0.0064 0.0130 0.083 EtOH only 200 0.624 0.00312 0.0129 0.040 EtOH & Enzyme 200 1.148 0.00574 0.0131 0.075 Table 2: Summary of lipid extracts obtained from DME extraction with EtOH co-solvent SFE technologyExtraction method Seaweed(g, wet)EtOHextract (g)Lipidextract (g)Lipid extract(g/g wet SW)Fucoxanthin(g/g EtOH extract)Fucoxanthin(mg/g wet SW)DME+EtOH 620 5.73 3.44 0.0055 0.0030 0.028Enzyme/DME+EtOH800 6.86 5.48 0.0068 0.0064 0.055Note*: EtOH extract is the concentrate of DME-EtOH extract and lipid extract is then obtained from the re-extraction of the DME-EtOH extract using CHCl3: MeOH: dH2O solvent. Fucoxanthin content analysis was carried out on EtOH extract. SW-seaweed.CONCLUSIONIn this paper, the extraction of lipid soluble bioactive compounds from Undaria pinnatifida seaweed using enzyme-assisted DME-EtOH co-solvent extraction has been successfully demonstrated at a laboratory scale. Results indicate that enzyme pre-treatment improved the yield of fucoxanthin by >50% and total lipid by >10% over untreated feed materials; and that a continuous process for extraction of the enzyme digest solution is possible using DME + EtOH co-solvent system. Enzymatic digestion of intact cell wall polysaccharides assisted in release of lipid soluble compounds from brown seaweed. In addition, enzyme pre-treatment also helped to remove 30-40% polysaccharides from feed materials and this could greatly reduce the amount of solvents used per gram of seaweed processed. The proposed enzyme assisted continuous DME-EtOH extraction method is a potential alternative to ethanol for the extraction of fucoxanthin from Undaria pinnatifida seaweed that also gives good yields of complex lipids rich in ω-3. ACKNOWLEDGEMENTSThe authors thank MSI for providing funding for this work through grant CO8X0305.REFERENCES1.SE-KWON, K., RATIH, P., Advances in Food and Nutrition Research, Academic Press, Vol 64, 2011, p. 1112.HAYATO, M., MASASHI, H., TOKUTAKE, S., KATSURA, F., KAZUO, M., Biochem &mun, Vol 332, 2005, p 392.3.PRABHASANKAR, P., GANESAN, P., BHASKAR, N., HIROSE, A., NIMISHMOL, S., LALITHA, R G.,HOSOKAWA, M., MIYASHITA, K., Food Chem. Vol 115, 2009, p.5014.RYZA OIL & FAT CHEMICAL CO., LTD. ver.2.0 SJ5.MAEDA, H., TSUKUI, T., SASHIMA, T., HOSOKAWA, M., MIYASHITA, K., Asia Pac J Clin Nutr. Vol 17,2008, p. 1966.PARSONS, M. J., Landcare research contract report: LC 9495/61, 19947.KEIJI, I., KANJI, H., Food Rev. Int. Vol. 5, 1989, p.1018.SERGE, M., JOËL, F., Tren in Food Sci. & Tech., Vol 4, 1993, p. 1039.YOHEI, I., MASAYUKI, K., MAI, W., CHIE, M., HIROYUKI, K., SHIGERU, I., YASUHIRO, K.,KAZUHIKO, S., Bioch Biophys. Acta (BBA) - Bioenergetics, Vol 1777, 2008, p. 35110.J.T.O. KIRK., Plant Sci.Let., Vol 9, 1977, p. 37311.ARKOYO AND ELIFI A.S., Makara, Tech. Vol 15, 2011, p.512.SIEW-LING, H., POOI-YI, C., KWAN-KIT, W., CHING-LEE, W., Aus. J. Bas & App. Sci., Vol 4, 2010, p.458013.NOVIENDRI, D., JASWIR, I., SALLEH, H.M., TAHER, M., MIYASHITA, K., RAMLI, N., J. Med. Plan. Res.,Vol 5, 2011, p. 2405.14.MISE, T., UEDA, M., YASUMOTO, T. Adv. J. Food Sci. and Tech., Vol 3, 2011, p.7315.JARLE, A. H., SYNNØVE, L.J., Phytochem. Vol 28, 1989, p. 279716.YA, F. S., SANG, M. K., WON, J. L., BYUNG-HUN, U., J. Biosci. Bioeng. Vol 111, 2011, p. 23717.OWEN, C., JASON, R., YIN, Z., KRISTINA, F., JOHN, G., MIKHAIL, V., ANDREW, M., EDUARD, N.,KEVIN, M., J. Sup. Flu., Vol 53, 2010, p.3418.MYONG-KYUN, R., SALIM, U., BYUNG-SOO, C. Biotech. Bioproc. Eng. Vol 13, 2008, p.72419.W.A.J.P. WIJESINGHE., YOU-JIN, J., Fitoterapia, Vol 83, 2012, p. 620.PAULA, M., ROBERTO, E. A., Eur. J. Lipid Sci. Technol. Vol 113, 2011, p.53921.GACESA, P., Int.J. Bioche, Vol 24, 1992, p.54522.BLIGH, E. G., DYER, W. J., Can.J. Biochem. Physiol. Vol37, 1959, p.911。

Separation and Purification Technology56 (2007) 291–295Extraction and purification of solanesol from tobacco(I).Extraction and silica gel column chromatographyseparation of solanesolDe-Song Tang a,Lin Zhang a,∗,Huan-Lin Chen a,Yue-Rong Liang b,Jian-Liang Lu b,Hui-Ling Liang c,Xin-Qiang Zheng ba Institute of Bioengineering,College of Materials Science and Chemical Engineering,Zhejiang University,Hangzhou310027,Chinab Tea Research Institute,Zhejiang University,Hangzhou310029,Chinac College of Horticulture,South China Agriculture University,Guangzhou510642,ChinaReceived13June2006;received in revised form2January2007;accepted17January2007AbstractSolanesol,a main polyprenol occurred in tobacco,has gained the attention of biochemists because of its value as a source of isoprene units for the synthesis of metabolically active quinones and vitamin K analogs.This article deals with extraction and purifyication of solanesol.Ground tobacco leaf was extracted with petroleum ether in50◦C water bath under reflux for2h,and thenfiltrated,the powder leaf was re-extracted successively with petroleum ether and the extracts were pooled and concentrated to get pasty residue.The residue was saponified and subsequently loaded onto silica gel column chromatography.The chemical was eluted using a binary solvent mixture of petroleum ether–acetone(90:10, v/v)and the eluent was collected by fraction size of5ml.Tentative identification was carried out using TLC.The fractions mainly contained solanesol were pooled and dried by rotary evaporation.The yield of solenosol was0.38%of the dry weight of tobacco leaf,and the purity was83.04%.© 2007 Elsevier B.V. All rights reserved.Keywords:Solanesol;Extraction;Separation;Silica gel column chromatography1.IntroductionSolanesol,a polyisoprenoid alcohol(Fig.1),wasfirst isolated fromflue-cured tobacco in1956by Rowland et al.[1].Solanesol occurs in both free and esterified forms[2],numerous authors [3–9]have investigated both the free and esterified solanesol content of tobacco and found concentrations ranging up to4% of the dry leaf weight for the free alcohol,making solanesol the most abundant tobacco terpenoid.Furthermore,solanesol has been shown to be present in the lamina of tobacco leaf while absent from the stem and stalk[4].The solanesol content of tobacco depends upon a number of factors including the type and variety of tobacco,stalk position, duration of growth and method of curing.The increase in free solanesol closely corresponds to the decrease in solanesyl esters ∗Corresponding author.Tel.:+8657187952121;fax:+8657187952121.E-mail address:linzhang@(L.Zhang).during curing[6].Burton et al.[10]made a similar study of Ken-tucky14burley tobacco and found that free solanesol increases monotonically during growth and continues to increase during air curing.Polyisoprenoid alcohols are found in diverse life forms including higher plants,mammalian tissues and microorgan-isms.Many other polyprenols consist of both cis and trans units but solanesol is recognized to be composed of all trans isoprene units.Solanesol is primarily found in plants from the Solanaceae family,particularly tobacco.A total synthesis of solanesol has been reported[11,12],in which cis and trans forms of the alcohol were obtained.The nat-ural occured isomer was trans form[12].Solanesol has gained much attention nowadays because of its value as a source of iso-prene units for the synthesis of metabolically active quinones, such as coenzyme Q10[13,14].The objective of this investiga-tion is to develop a method of extraction and separation solanesol from tobacco.1383-5866/$–see front matter© 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2007.01.040292 D.-S.Tang et al./Separation and Purification Technology56 (2007) 291–295Fig.1.Molecular structure of solanesol.2.Materials and methods2.1.Chemicals and materialsThe analytical standard of solanesol was obtained from J&K Chemical Ltd.,with purity of>90%;solvents for HPLC mobile phases were of HPLC grade,obtained from Tianjin Shield Co.,China;silica gel was from Yandangshan Chemical Co., Zhejiang,China;other reagents were of analytical grade.The flue-cured tobacco leaves were obtained from Laifeng County, China.2.2.Extraction of solanesolFlue-cured tobacco leaves were dried at80◦C for4h,ground and passed through a40-mesh sieve.About500g ground leaf powder were extracted using3l petroleum ether in a water bath at50◦C under reflux for2h,andfiltered.The residue was re-extracted successively with petroleum ether.The petroleum ether extracts were combined and concentrated using rotary vacuum evaporator at40◦C to get pasty residue.The residue was saponified with10%ethanolic potassium hydroxide(50ml) overnight at room temperature and then extracted with petroleum ether,washed free of alkali,concentrated and again dried by rotary evaporation.The dried extract was stored at−20◦C in an air tight container.2.3.Silica gel low-pressure chromatographyThe crude solanesol was dissolved in petroleum ether at a ratio of10:1(v/w)of petroleum ether-to-crude solanesol.The solution was applied to a silica gel column(30cm×2.0cm i.d.),which was preconditioned with petroleum ether.The col-umn was eluted employing petroleum ether–acetone(90:10, v/v).The eluent was collected by fraction size of5ml and tentative identification was carried out using TLC.The frac-tions containing solanesol were pooled and dried by rotary evaporation.2.4.TLC detectionSilica gel plates were home made and activated at120◦C for 1h before used.Ten microliters of each fraction collected from column chromatography along with solanesol standard solution was loaded to the marked points about10mm from the bottom of silica plate.The plates were developed in petroleum ether–ethyl acetate(80:20,v/v)at room temperature and the separated spots were visualized by iodine fume[15].Ingredients of each fraction were compared with standard for identification.Recordation was made by digital camera(NIKON-COOLPIX2100).2.5.HPLC analysisThe Shimadzu HPLC system(Shimadzu SCL-10A,Shi-madzu Cooperation,Tokyo,Japan)with auto sampler was employed in the present study.A Diamonsil TM C18column (150mm×4.6mm,i.d.)was used at column temperature of 30◦C and the injection volume was10␮l.A binary solvent mixture of acetonitrile–isopropanol(60:40,v/v)was used as mobile phase and measurements were performed at aflow rate of 1.0ml/min,Detection was done by a Shimadzu SPD ultraviolet detector set at210nm.2.6.ESI-MS experimentESI-MS experiments were performed on a Bruker Esquire 3000plus(Bremen,Germany)in positive ionization mode ana-lyzing ions from50m/z up to2200m/z.2.7.Data statisticsAll tests in the present paper were carried out in duplicate and the mean values were presented.Statistics was carried out on Statistical Analysis System(Version8.01,SAS Institute Inc.).3.Results and discussion3.1.Selection of extractantNumerous classical methods have been reported on the of solanesol extraction from tobacco leaves[16–21]and many sol-vents were employed such as petroleum ether[16],acetone[17] and n-hexane[21].In order to evaluate the solvents’extract ability,1g of ground leaf powder were extracted using80ml solvent in a water bath at50◦C under reflux for2h,and filtered.The residue was washed.The extract and washings were combined and dried by rotary evaporation.The dried extract was carefully weighed and the solanesol content was determined by HPLC analysis.The percentage extraction of solanesol(w/w)was counted as:extract rate(%)=(weight of extracted solanesol/weight of tobacco leaf samples)×100%. The solanesol content in extract was counted as:solanesol con-tent in extract(%)=(weight of extracted solanesol/total weight of tobacco extract)×100%.Results indicated that petroleum ether and hexane have good extract ability for solanesol and the content of solanesol in the crude tobacco extract was higher than those obtained using other solvents(Table1).It suggested that solanesol could be selec-tively extracted by petroleum ether and hexane with much less impurities than extracted using other solvents,which could make the subsequent purification easier.Petroleum ether is preferred because it much cheaper than hexane.3.2.TLC detection of solanesolTLC is routinely used for qualitative examination of solanesol [22,23].TLC used here was also helpful in tentative identifica-tion of solanesol purity in each fraction(Fig.2),and was efficientD.-S.Tang et al./Separation and Purification Technology 56 (2007) 291–295293Table 1Extract ability comparison of different solvents on extraction of solanesol ExtractantPetroleum ether Hexane Acetone 80%acetone in water Extract rate (%)0.75A a 0.72B 0.71B 0.69B Solanesol content in extract (%)5.76A5.75A2.58B0.98CaValues with the same letters (A–C)are not significantly different (compared using Tukey’s Test in Statistical AnalysisSystem).Fig.2.Thin-layer co-chromatogram of standard (0),original sample (0 )and eluted fractions from silica gel column chromatography (from 1to 25).to detect many samples in one time.Real-time monitoring is use-ful in column chromatography separation process,but TLC is a cheap alterative method to analyze the eluent fractions in the absence of such a monitor.Visualization could be carried out by spraying with sulfuric acid–anisaldehyde–glacial acetic acid (5:5:90,v/v/v)at 110◦C [23],Using iodine fume to visualize is time saving with heating unnecessary,but recordation should be made soon since iodine is prone to evaporation.3.3.Free and total solanesol content in tobacco leaves Solanesol occurs in both free and esterified forms [2].Scholtzhauer et al.[24]isolated solanesyl esters fractions.Saponification of the solanesyl esters fractions in 10%KOH/ethanol yielded a series of fatty acids and solanesol.Rowland and Latimer [25]found that solanesol combined with palmitic,linoleic,linolenic,myristic and oleic acids and two unidentified acids.It was also reported that solanesol occurs as the acetate,octanoate,decanoate and in combination with fatty acids such as stigmasterol and ␤-sitosterol [2].The extract of tobacco employed in this experiment was ana-lyzed before and after saponification to give the free and total solanesol content.The content of free and total solanesol were 0.66%(Fig.3A)and 0.79%(Fig.3B),respectively,in the mate-rial of the experiment.3.4.Selection of eluentAfter the crude solanesol solution was loaded onto the col-umn,petroleum ether was added when the sample solution layer went down almost to the surface of the column bed.The objec-tive is to keep the solanesol in as small a volume as possible to diminish band broadening and to prevent the separation from initiating before the entire sample solution has reached the adsor-bent top.The eluent could be selected according to the method mentioned by Rodriguez-Amaya [26].After the petroleum etherreached the surface of the column bed,develop the silica gel col-umn successively with petroleum ether and then 2,5,8,10,15and 20%acetone in petroleum ether.Solanesol could not be eluted by petroleum ether,and with lower concentration of acetone in petroleum,it could be eluted but large amount of eluent was needed.In present investigation,10%acetone in petroleum ether was good at separation of solanesol on the silica gel column,whiletheFig.3.HPLC chromatograms of:(A)before and (B)after saponification of petroleum ether extract of tobacco and (C)solanesol isolated using silica gel column chromatography.294 D.-S.Tang et al./Separation and Purification Technology 56 (2007) 291–295 acetone concentration in petroleum ether higher than10%,solanesol moved downed too quickly and could not be separatedfrom other components.3.5.Purity and yield of solanesol by silica columnchromatographySilica gel chromatography had been investigated for sepa-rating the hexane extract offlue-cured tobacco[27].Pigmentsand some impurities in crude solanesol were eluted beforesolanesol using solvent of petroleum ether–acetone(90:10,v/v).The fractions which mainly contained solanesol were combined,concentrated and1.92g solanesol was obtained with purity of83.04%(Fig.3C),the yield was0.38%calculated from theformula as:Y(%)=W ST×100,(1)where W S and W T are the weight of extracted solanesol and ground tobacco leaves,respectively.The total content of solanesol in tobacco leaves employed in this study was0.79%(Fig.3B)and recovery rate of solanesol was42.31%calculated from the formula as:R(%)=W S×p SW T×p T×100,(2)where p S and p T are the purity of solanesol in the extracted sample and in the ground tobacco leaves,respectively.3.6.Molecular validation for the isolateThe ESI-MS of the isolate indicated a molecular ion at m/z 653.6[M–H–Na]+.It was in agreement with that reported by Du et al.[28].4.ConclusionOne method of extraction and separation of solanesol from tobacco was established.Flue-cured tobacco extracts was sub-jected to silica gel column chromatography and was eluted using a binary solvent mixture of petroleum ether–acetone(90:10, v/v).The eluent was collected by fraction size of5ml and ten-tative identification was carried out using TLC.The fractions containing solanesol were pooled and dried by rotary evapora-tion and obtained solanesol with purity of83.04%,the yield was 0.38%.But further purification is necessary before it was used for the synthesis of ubiquinones and vitamin K analogs. AcknowledgementThe authors gratefully acknowledge the donation offlue-cured tobacco from Mr.Zihua Xiang,Laifeng County. References[1]R.L.Rowland,timer,J.A.Giles,Flue-cured,I.Tobacco,Isolationof solanesol,an unsaturated alcohol,J.Am.Chem.Soc.78(18)(1956) 4680–4683.[2]R.L.Stedman,Chemical composition of tobacco and tobacco smoke,Chem.Rev.68(2)(1968)153–207.[3]B.H.Woolen,D.H.Jones,Analytical methods for tobacco lipids.1.Arapid method for the estimation of solanesol by thin-layer densitometry,J.Chromatogr.61(1971)180–182.[4]R.F.Severson,J.J.Ellington,P.F.Schlotzhauer,R.F.Arrendale,A.I.Schep-artz,Gas chromatographic method for the determination of free and total solanesol in tobacco,J.Chromatogr.139(1977)269–282.[5]J.J.Ellington,P.F.Schlotzhauer,A.I.Schepartz,Quantitation of hexane-extractable lipids in serial samples offlue-cured tobaccos,J.Agric.Food Chem.26(1)(1978)270–273.[6]S.J.Sheen,D.L.Davis,D.W.DeJong,J.F.Chaplin,Gas–liquid chromato-graphic quantification of solanesol in chlorophyll mutants of tobacco,J.Agric.Food Chem.26(1)(1978)259–262.[7]R.F.Severson,J.J.Ellington,R.F.Arrendale,M.E.Snook,Quantitativegas chromatographic method for the analysis of aliphatic hydrocarbons, terpenes,fatty alcohols,fatty acids and sterols in tobacco,J.Chromatogr.160(1978)155–168.[8]R.F.Severson,R.F.Arrendale,J.F.Chaplin,E.Ralph,Williamson,Use ofpale-yellow tobacco to reduce smoke polynuclear aromatic hydrocarbons, J.Agric.Food Chem.27(4)(1979)896–900.[9]J.B.Wooten,Direct detection of solanesol in tobacco by proton and carbon-13magic angle spinning NMR,J.Agric.Food Chem.33(3)(1985) 419–425.[10]A.R.Burton,G.Childs,J.L.Sims,L.Walton,38th Tobacco Chemists’Research Conference,Atlanta,GA,1984.[11]R.R¨u egg,U.Gloor,R.N.Goel,G.Ryser,O.Wiss,O.Isler,Synthesevon Ubichinon(45)und Ubichinon(50),Helv.Chim.Acta42(7)(1959) 2616–2621.[12]R.R¨u egg,U.Gloor,ngemann,M.Kofler,C.von Planta,G.Ryser,O.Isler,Die totalsynthese von Solanesol,Helv.Chim.Acta43(6)(1960) 1745–1751.[13]B.H.Lipshutz,P.Mollard,S.S.Pfeiffer,W.Chrisman,A.Short,Highlyefficient synthesis of coenzyme Q10,J.Am.Chem.Soc.124(2002) 14282–14283.[14]B.H.Lipshutz,A.Lower,V.Berl,K.Schein,F.Wetterich,An improvedsynthesis of the“miracle nutrient”coenzyme Q10,Org.Lett.7(19)(2005) 4095–4097.[15]W.Xia,D.H.Yu,X.Z.Chen,Isolation of solanesol from discarded tobaccoextracts,Chin.J.Pharmaceut.34(7)(2003)328–329.[16]B.Cen,W.Duan,S.Zhao,Study on new technology of extracting solanesolfrom abandoned tobacco leaves,J.Guangxi Univ.(Nat.Sci.Ed.)27(3) (2002)240–242.[17]X.Q.Sun,J.Zhao,C.J.Wang,B.S.Chen,Study on extraction of solanesolfrom discard tobacco leaves,J.Henan Univ.(Nat.Sci.)25(2)(1995) 37–40.[18]H.Wei,H.M.Mi,Z.L.Liu,Extraction and separation of solanesol fromNicotiana tobacum by supercriticalfluid extraction and silica gel col-umn chromatography,Chin.Tradit.Herbal Drugs36(5)(2005)690–692.[19]M.Keca,S.Gross,I.Malnar,Z.Kalodera,R.Malojcic,Isolation ofsolanesol from tobacco(Nicotiana tabacum L.)by classic extraction and ultrasound extraction,Farmaceutski Glasnik53(6)(1997)173–182.[20]W.Yao,R.Wu,H.Luan,Extraction of solanesol with SOT from abandonedtobacco dross,Sub-Inst.Fine Chem.Eng.4(4)(1995)93–96.[21]O.S.Park,Isolation of solanesol from Korean native tobacco,SaengyakHakhoechi12(4)(1981)211–214.[22]L.Li,Z.Y.Ma,Q.X.Qian,X.D.Cong,Determining the content of solanesolin tobacco leaves using TLC scanning,Chin.Tradit.Herbal Drugs33(5) (2002)420–421.[23]B.H.Woollen,D.H.Jones,Analytical methods for tobacco lipids.Rapidmethod for the estimation of solanesol by thin-layer densitometry,J.Chro-matogr.61(1)(1971)180–182.[24]W.S.Scholtzhauer,R.F.Severson,O.T.Chortyk,R.F.Arrendale,Pyrolyticformation of polynuclear aromatic hydrocarbons from petroleum ether extractable constituents offlue-cured tobacco leaf,J.Agric.Food Chem.24(5)(1976)992–997.D.-S.Tang et al./Separation and Purification Technology 56 (2007) 291–295295[25]R.L.Rowland,timer,Flue-cured tobacco.IV.Isolation of solanesylesters,Tobacco Int.3(1959)1–3.[26]D.B.Rodriguez-Amaya,A Guide to Carotenoid Analysis in Foods,ILSIPress,2001.[27]C.E.Cook,E.T.Margaret,C.R.Tallent,H.Ingeborg,G.Heunisch,J.B.Lewis,M.E.Wall,An examination of the hexane extract offlue-curedtobacco involving gel permeation chromatograph,Phytochemistry8(6) (1969)1025–1033.[28]Q.Du,D.Wang,I.Yoichiro,Preparation of solanesol from a tobaccoleaf extract using high speed countercurrent chromatography,J.Liquid Chromatogr.Relat.Technol.29(2006)2587–2592.。

绪论Strengthening basic researches on Chinese Medicinal Plants and its relations to realizing the modernization of CMM加强中国药用植物基础研究及其与中药现代化的关系Traditional Chinese Medicine(TCM)中药health care卫生健康Chinese traditional medicinal herbs中草药inciting side-effects明显的副作用go back to the nature回归自然the modernization of Chinese Materia Medica(CMM)中药现代化Surveys调查special projects专项调查scientific identification科学鉴定chemical constituents化学成分pharmacological experiments药理实验clinical applications临床适应性monographs各论、专论manuals手册Pharmacopoeia药典therapeutical efficacy疗效黄花蒿(Artemisia annua)，青蒿(Artemisia apiacea)chloroquine resistant malaria/抗氯喹宁疟疾Pernicious（有害的）malaria/恶性疟疾cerebral（大脑的,脑的）malaria/脑疟疾derivatives/衍生物quinine/喹宁harringtonine三尖杉酯碱homoharringtonine高三尖杉酯碱extracted from提取leukemia白血病malignant lymphoma恶性淋巴瘤ginkgetin银杏黄酮criteria标准revise修正，校订systematic studies系统研究revising修订common-used Chinese materia medica常用中药材品种整理和质量研究impact n.冲击，碰撞era时代public health care公共健康事业traditional Chinese patent medicines and preparations中成药和中药制剂basic researches基础研究production生产marketing流通research研究①Identification of species/品种鉴定identifying鉴别clarifying澄清，阐明confused varieties易混淆的变种false matters伪品益胃生津，滋阴清热benefit the stomach,promote the production of body fluid and remove the excessive heat②Quality control and evaluation/质量的控制和评价intensive加强的，透彻的contents=components,constituentswith great care:小心翼翼地habitat：产地qualitative and quantitative analysis定性定量分析③The methods of research研究方法biotechnology生物技术molecular biology分子生物学remedy:n.药物，治疗法，赔偿，v.补救，矫正modern scientific methodologies现代科学方法①Strengthen the study of medicinal plant resources加强药用植物基源研究exploring new resources开发新的资源re-producting resources可持续性利用资源conservation of germplasm of the rare and endangered medicinal plants珍稀濒危药用植物的种质资源保护②Carry out the researches on specific biology of medicinal plantsspecific adj.种的，明确的，特殊的，具有特效的n.特效药，详情，特性③Map out GAP(Good Agriculturing Practice)in medicinal cultivationmap out规划seed quality standards种子的质量标准processing rules and regulations加工方法及规范④Established standards of quality control and renew methodologycriteria标准protocol草案foreign matter杂质⑤Apply modern comprehensive multidisciplinary studies on Chinese medicinal plants comprehensive：综合的，广泛的multidisciplinary：多学科⑥Establish information systems in modern research of Chinese medicinal herbsEthnobotany人类植物学Ethnopharmacology民族药理学Ethno-民族，种族semi-colonial and semi-feudal nation/半殖民半封建的国家constraint and devastation/压迫与毁坏the inheritance and development of Chinese troditional medicine……/继承和发扬many difficult and complicated diseases疑难杂症TERMINOLOGY部分要求的词汇中药Traditional Chinese Medicines中药材Chinese Materia Medica(CMM)Traditional Chinese Medicinal MaterialsChinese Crude Drugs中草药Traditional Chinese Medicinal HerbsChinese Herbal Medicines药用植物学Medicinal Plants民间药物Folklore Medicaments Folk Herbs Indigenous Drugs中成药Traditional Chinese Patent Medicines中药制剂Chinese Medicinal Preparations药用植物学Pharmaceutical Botany生药学Pharmacognosy药用植物分类学Pharmaceutical Plant Taxonomy植物化学Phytochemistry植物化学分类学Plant Chemotaxonomy药用植物志Flora of Medicinal Plants中药药剂学Traditional Chinese Pharmaceutics中药炮制学Science of Processing Chinese Crude Drugs显微生药学Microscopical Pharmacognosy本草学herbals药典pharmacopoeia细胞cell细胞壁cell wall初生壁primary wall次生壁secondary wall细胞核nucleus质体plastid叶绿体chloroplast染色体chromoplast纹孔pit结晶体crystal淀粉粒starch granule（grain）脐点hilum(pi.hila)层纹striation单粒single starch granule复粒compound starch granule半复粒semi-compound starch granule 蛋白质protein脂类化合物lipid挥发油volatile oil苷glycoside 生物碱alkaloid皂苷saponin萜terpene三萜皂苷triterpenoid黄铜flavonoid蒽醌anthraquinone酚phenol香豆素coumarin鞣质rannin氨基酸amino acid菊糖inulin草酸钙结晶calcium cxalate crystal 簇晶clusrer crystal(cluster,druse)针晶acicular crystal(needle) Parenchyma薄壁组织Parenchymatous cell包庇细胞Epidermal tissue表皮组织Epidermis表皮Cuticle角质层stoma(pl.stomata)气孔guard cell保卫细胞subsidiary cell副卫细胞非腺毛non-glandular hair腺毛glandular hair腺鳞glandular scale周皮periderm木栓层cork木栓形成层cork cambium(phellogen)栓内层phelloderm皮层cortex皮孔lenticel形成层cambium次生木质部secondary xylem次生韧皮部secondary phloem分泌组织secretory tissue树脂道resin canal厚角组织collenchyma纤维fibre(fiber)韧皮纤维phloem fibre木纤维xylem fibre晶鞘纤维crystal fibre层纹striation木化lignified增厚thickening增厚细胞壁thickened wall 导管vessel网纹导管reticulated vessel具缘纹孔导管bordered pitted vessel 筛管sieve tube维管束vascular bundle外韧维管束collateral bundle异性维管束abnormal vascular bundle 形成层成环cambium ring主根main(tap)root定根normal root须根fibrous root不定根adventitious root细根rootler初生构造primary structure次生构造secondary structure皮层cortex中柱鞘pericycle初生木质部primary xylem髓部pith射线ray木栓层cork植物phyta科aceae丹参Pubescent软毛Stem4-angled茎4棱Angled棱Leaves opposite叶对生Verticillate轮生pseudo-假的Racemes总状花序stamens雄蕊Gynobasic着生于基部的Nutlets坚果Ellipsoid椭圆形Vermillion朱红色serrate锯齿状的Retuse浅凹形Falcate钩状的Filament花丝Style花柱Anther花粉囊fragile:脆的astringent:收敛性的ferric chloride:三氯化铁TS：test solutiondull:暗的cortex:栓皮，皮层phelloderm:栓内层a stoppered test tube:具塞试管stand for：静置residue:残渣reference drug:对照药材CRS：Chemical Reference Standard养心安神：clear heart-fire and remove restlessnessRhizomes short and stout,sometimes with remains of a stem at the apex.根茎短而粗壮，Externally brownish-red or dark brownish-red,rough,longitudinally wrinkled.dissolved on mounting in chloral hydrate solution.Boil5g of the powder in50ml of water for15～20minutes,cool and filter.Concentrate the filtrate on a water bath,dissolve the extract in3～5ml of ethanol,filter.Apply several drops of the filtrate to a piece of filter paper,allow it to dry and examine under ultra-violet light(365nm), a bright bluish-grey fluorescence is produced.Expose the filter paper to ammonia vapour for20 minutes,remove the filter paper and examine again under ultra-violet light(365nm),a pale bluish-green fluorescence is produced.置5g粉末于50ml水中煎煮15~20分钟，冷却后过滤.在水浴中浓缩滤液，用3~5ml乙醇溶解浓缩产物，过滤。

Glider Flying Handbook2013U.S. Department of TransportationFEDERAL AVIATION ADMINISTRATIONFlight Standards Servicei iPrefaceThe Glider Flying Handbook is designed as a technical manual for applicants who are preparing for glider category rating and for currently certificated glider pilots who wish to improve their knowledge. Certificated flight instructors will find this handbook a valuable training aid, since detailed coverage of aeronautical decision-making, components and systems, aerodynamics, flight instruments, performance limitations, ground operations, flight maneuvers, traffic patterns, emergencies, soaring weather, soaring techniques, and cross-country flight is included. Topics such as radio navigation and communication, use of flight information publications, and regulations are available in other Federal Aviation Administration (FAA) publications.The discussion and explanations reflect the most commonly used practices and principles. Occasionally, the word “must” or similar language is used where the desired action is deemed critical. The use of such language is not intended to add to, interpret, or relieve a duty imposed by Title 14 of the Code of Federal Regulations (14 CFR). Persons working towards a glider rating are advised to review the references from the applicable practical test standards (FAA-G-8082-4, Sport Pilot and Flight Instructor with a Sport Pilot Rating Knowledge Test Guide, FAA-G-8082-5, Commercial Pilot Knowledge Test Guide, and FAA-G-8082-17, Recreational Pilot and Private Pilot Knowledge Test Guide). Resources for study include FAA-H-8083-25, Pilot’s Handbook of Aeronautical Knowledge, FAA-H-8083-2, Risk Management Handbook, and Advisory Circular (AC) 00-6, Aviation Weather For Pilots and Flight Operations Personnel, AC 00-45, Aviation Weather Services, as these documents contain basic material not duplicated herein. All beginning applicants should refer to FAA-H-8083-25, Pilot’s Handbook of Aeronautical Knowledge, for study and basic library reference.It is essential for persons using this handbook to become familiar with and apply the pertinent parts of 14 CFR and the Aeronautical Information Manual (AIM). The AIM is available online at . The current Flight Standards Service airman training and testing material and learning statements for all airman certificates and ratings can be obtained from .This handbook supersedes FAA-H-8083-13, Glider Flying Handbook, dated 2003. Always select the latest edition of any publication and check the website for errata pages and listing of changes to FAA educational publications developed by the FAA’s Airman Testing Standards Branch, AFS-630.This handbook is available for download, in PDF format, from .This handbook is published by the United States Department of Transportation, Federal Aviation Administration, Airman Testing Standards Branch, AFS-630, P.O. Box 25082, Oklahoma City, OK 73125.Comments regarding this publication should be sent, in email form, to the following address:********************************************John M. AllenDirector, Flight Standards Serviceiiii vAcknowledgmentsThe Glider Flying Handbook was produced by the Federal Aviation Administration (FAA) with the assistance of Safety Research Corporation of America (SRCA). The FAA wishes to acknowledge the following contributors: Sue Telford of Telford Fishing & Hunting Services for images used in Chapter 1JerryZieba () for images used in Chapter 2Tim Mara () for images used in Chapters 2 and 12Uli Kremer of Alexander Schleicher GmbH & Co for images used in Chapter 2Richard Lancaster () for images and content used in Chapter 3Dave Nadler of Nadler & Associates for images used in Chapter 6Dave McConeghey for images used in Chapter 6John Brandon (www.raa.asn.au) for images and content used in Chapter 7Patrick Panzera () for images used in Chapter 8Jeff Haby (www.theweatherprediction) for images used in Chapter 8National Soaring Museum () for content used in Chapter 9Bill Elliot () for images used in Chapter 12.Tiffany Fidler for images used in Chapter 12.Additional appreciation is extended to the Soaring Society of America, Inc. (), the Soaring Safety Foundation, and Mr. Brad Temeyer and Mr. Bill Martin from the National Oceanic and Atmospheric Administration (NOAA) for their technical support and input.vv iPreface (iii)Acknowledgments (v)Table of Contents (vii)Chapter 1Gliders and Sailplanes ........................................1-1 Introduction....................................................................1-1 Gliders—The Early Years ..............................................1-2 Glider or Sailplane? .......................................................1-3 Glider Pilot Schools ......................................................1-4 14 CFR Part 141 Pilot Schools ...................................1-5 14 CFR Part 61 Instruction ........................................1-5 Glider Certificate Eligibility Requirements ...................1-5 Common Glider Concepts ..............................................1-6 Terminology...............................................................1-6 Converting Metric Distance to Feet ...........................1-6 Chapter 2Components and Systems .................................2-1 Introduction....................................................................2-1 Glider Design .................................................................2-2 The Fuselage ..................................................................2-4 Wings and Components .............................................2-4 Lift/Drag Devices ...........................................................2-5 Empennage .....................................................................2-6 Towhook Devices .......................................................2-7 Powerplant .....................................................................2-7 Self-Launching Gliders .............................................2-7 Sustainer Engines .......................................................2-8 Landing Gear .................................................................2-8 Wheel Brakes .............................................................2-8 Chapter 3Aerodynamics of Flight .......................................3-1 Introduction....................................................................3-1 Forces of Flight..............................................................3-2 Newton’s Third Law of Motion .................................3-2 Lift ..............................................................................3-2The Effects of Drag on a Glider .....................................3-3 Parasite Drag ..............................................................3-3 Form Drag ...............................................................3-3 Skin Friction Drag ..................................................3-3 Interference Drag ....................................................3-5 Total Drag...................................................................3-6 Wing Planform ...........................................................3-6 Elliptical Wing ........................................................3-6 Rectangular Wing ...................................................3-7 Tapered Wing .........................................................3-7 Swept-Forward Wing ..............................................3-7 Washout ..................................................................3-7 Glide Ratio .................................................................3-8 Aspect Ratio ............................................................3-9 Weight ........................................................................3-9 Thrust .........................................................................3-9 Three Axes of Rotation ..................................................3-9 Stability ........................................................................3-10 Flutter .......................................................................3-11 Lateral Stability ........................................................3-12 Turning Flight ..............................................................3-13 Load Factors .................................................................3-13 Radius of Turn ..........................................................3-14 Turn Coordination ....................................................3-15 Slips ..........................................................................3-15 Forward Slip .........................................................3-16 Sideslip .................................................................3-17 Spins .........................................................................3-17 Ground Effect ...............................................................3-19 Chapter 4Flight Instruments ...............................................4-1 Introduction....................................................................4-1 Pitot-Static Instruments ..................................................4-2 Impact and Static Pressure Lines................................4-2 Airspeed Indicator ......................................................4-2 The Effects of Altitude on the AirspeedIndicator..................................................................4-3 Types of Airspeed ...................................................4-3Table of ContentsviiAirspeed Indicator Markings ......................................4-5 Other Airspeed Limitations ........................................4-6 Altimeter .....................................................................4-6 Principles of Operation ...........................................4-6 Effect of Nonstandard Pressure andTemperature............................................................4-7 Setting the Altimeter (Kollsman Window) .............4-9 Types of Altitude ......................................................4-10 Variometer................................................................4-11 Total Energy System .............................................4-14 Netto .....................................................................4-14 Electronic Flight Computers ....................................4-15 Magnetic Compass .......................................................4-16 Yaw String ................................................................4-16 Inclinometer..............................................................4-16 Gyroscopic Instruments ...............................................4-17 G-Meter ........................................................................4-17 FLARM Collision Avoidance System .........................4-18 Chapter 5Glider Performance .............................................5-1 Introduction....................................................................5-1 Factors Affecting Performance ......................................5-2 High and Low Density Altitude Conditions ...........5-2 Atmospheric Pressure .............................................5-2 Altitude ...................................................................5-3 Temperature............................................................5-3 Wind ...........................................................................5-3 Weight ........................................................................5-5 Rate of Climb .................................................................5-7 Flight Manuals and Placards ..........................................5-8 Placards ......................................................................5-8 Performance Information ...........................................5-8 Glider Polars ...............................................................5-8 Weight and Balance Information .............................5-10 Limitations ...............................................................5-10 Weight and Balance .....................................................5-12 Center of Gravity ......................................................5-12 Problems Associated With CG Forward ofForward Limit .......................................................5-12 Problems Associated With CG Aft of Aft Limit ..5-13 Sample Weight and Balance Problems ....................5-13 Ballast ..........................................................................5-14 Chapter 6Preflight and Ground Operations .......................6-1 Introduction....................................................................6-1 Assembly and Storage Techniques ................................6-2 Trailering....................................................................6-3 Tiedown and Securing ................................................6-4Water Ballast ..............................................................6-4 Ground Handling........................................................6-4 Launch Equipment Inspection ....................................6-5 Glider Preflight Inspection .........................................6-6 Prelaunch Checklist ....................................................6-7 Glider Care .....................................................................6-7 Preventive Maintenance .............................................6-8 Chapter 7Launch and Recovery Procedures and Flight Maneuvers ............................................................7-1 Introduction....................................................................7-1 Aerotow Takeoff Procedures .........................................7-2 Signals ........................................................................7-2 Prelaunch Signals ....................................................7-2 Inflight Signals ........................................................7-3 Takeoff Procedures and Techniques ..........................7-3 Normal Assisted Takeoff............................................7-4 Unassisted Takeoff.....................................................7-5 Crosswind Takeoff .....................................................7-5 Assisted ...................................................................7-5 Unassisted...............................................................7-6 Aerotow Climb-Out ....................................................7-6 Aerotow Release.........................................................7-8 Slack Line ...................................................................7-9 Boxing the Wake ......................................................7-10 Ground Launch Takeoff Procedures ............................7-11 CG Hooks .................................................................7-11 Signals ......................................................................7-11 Prelaunch Signals (Winch/Automobile) ...............7-11 Inflight Signals ......................................................7-12 Tow Speeds ..............................................................7-12 Automobile Launch ..................................................7-14 Crosswind Takeoff and Climb .................................7-14 Normal Into-the-Wind Launch .................................7-15 Climb-Out and Release Procedures ..........................7-16 Self-Launch Takeoff Procedures ..............................7-17 Preparation and Engine Start ....................................7-17 Taxiing .....................................................................7-18 Pretakeoff Check ......................................................7-18 Normal Takeoff ........................................................7-19 Crosswind Takeoff ...................................................7-19 Climb-Out and Shutdown Procedures ......................7-19 Landing .....................................................................7-21 Gliderport/Airport Traffic Patterns and Operations .....7-22 Normal Approach and Landing ................................7-22 Crosswind Landing ..................................................7-25 Slips ..........................................................................7-25 Downwind Landing ..................................................7-27 After Landing and Securing .....................................7-27viiiPerformance Maneuvers ..............................................7-27 Straight Glides ..........................................................7-27 Turns.........................................................................7-28 Roll-In ...................................................................7-29 Roll-Out ................................................................7-30 Steep Turns ...........................................................7-31 Maneuvering at Minimum Controllable Airspeed ...7-31 Stall Recognition and Recovery ...............................7-32 Secondary Stalls ....................................................7-34 Accelerated Stalls .................................................7-34 Crossed-Control Stalls ..........................................7-35 Operating Airspeeds .....................................................7-36 Minimum Sink Airspeed ..........................................7-36 Best Glide Airspeed..................................................7-37 Speed to Fly ..............................................................7-37 Chapter 8Abnormal and Emergency Procedures .............8-1 Introduction....................................................................8-1 Porpoising ......................................................................8-2 Pilot-Induced Oscillations (PIOs) ..............................8-2 PIOs During Launch ...................................................8-2 Factors Influencing PIOs ........................................8-2 Improper Elevator Trim Setting ..............................8-3 Improper Wing Flaps Setting ..................................8-3 Pilot-Induced Roll Oscillations During Launch .........8-3 Pilot-Induced Yaw Oscillations During Launch ........8-4 Gust-Induced Oscillations ..............................................8-5 Vertical Gusts During High-Speed Cruise .................8-5 Pilot-Induced Pitch Oscillations During Landing ......8-6 Glider-Induced Oscillations ...........................................8-6 Pitch Influence of the Glider Towhook Position ........8-6 Self-Launching Glider Oscillations During Powered Flight ...........................................................8-7 Nosewheel Glider Oscillations During Launchesand Landings ..............................................................8-7 Tailwheel/Tailskid Equipped Glider Oscillations During Launches and Landings ..................................8-8 Aerotow Abnormal and Emergency Procedures ............8-8 Abnormal Procedures .................................................8-8 Towing Failures........................................................8-10 Tow Failure With Runway To Land and Stop ......8-11 Tow Failure Without Runway To Land BelowReturning Altitude ................................................8-11 Tow Failure Above Return to Runway Altitude ...8-11 Tow Failure Above 800' AGL ..............................8-12 Tow Failure Above Traffic Pattern Altitude .........8-13 Slack Line .................................................................8-13 Ground Launch Abnormal and Emergency Procedures ....................................................................8-14 Abnormal Procedures ...............................................8-14 Emergency Procedures .............................................8-14 Self-Launch Takeoff Emergency Procedures ..............8-15 Emergency Procedures .............................................8-15 Spiral Dives ..................................................................8-15 Spins .............................................................................8-15 Entry Phase ...............................................................8-17 Incipient Phase .........................................................8-17 Developed Phase ......................................................8-17 Recovery Phase ........................................................8-17 Off-Field Landing Procedures .....................................8-18 Afterlanding Off Field .............................................8-20 Off-Field Landing Without Injury ........................8-20 Off-Field Landing With Injury .............................8-20 System and Equipment Malfunctions ..........................8-20 Flight Instrument Malfunctions ................................8-20 Airspeed Indicator Malfunctions ..........................8-21 Altimeter Malfunctions .........................................8-21 Variometer Malfunctions ......................................8-21 Compass Malfunctions .........................................8-21 Glider Canopy Malfunctions ....................................8-21 Broken Glider Canopy ..........................................8-22 Frosted Glider Canopy ..........................................8-22 Water Ballast Malfunctions ......................................8-22 Retractable Landing Gear Malfunctions ..................8-22 Primary Flight Control Systems ...............................8-22 Elevator Malfunctions ..........................................8-22 Aileron Malfunctions ............................................8-23 Rudder Malfunctions ............................................8-24 Secondary Flight Controls Systems .........................8-24 Elevator Trim Malfunctions .................................8-24 Spoiler/Dive Brake Malfunctions .........................8-24 Miscellaneous Flight System Malfunctions .................8-25 Towhook Malfunctions ............................................8-25 Oxygen System Malfunctions ..................................8-25 Drogue Chute Malfunctions .....................................8-25 Self-Launching Gliders ................................................8-26 Self-Launching/Sustainer Glider Engine Failure During Takeoff or Climb ..........................................8-26 Inability to Restart a Self-Launching/SustainerGlider Engine While Airborne .................................8-27 Self-Launching Glider Propeller Malfunctions ........8-27 Self-Launching Glider Electrical System Malfunctions .............................................................8-27 In-flight Fire .............................................................8-28 Emergency Equipment and Survival Gear ...................8-28 Survival Gear Checklists ..........................................8-28 Food and Water ........................................................8-28ixClothing ....................................................................8-28 Communication ........................................................8-29 Navigation Equipment ..............................................8-29 Medical Equipment ..................................................8-29 Stowage ....................................................................8-30 Parachute ..................................................................8-30 Oxygen System Malfunctions ..................................8-30 Accident Prevention .....................................................8-30 Chapter 9Soaring Weather ..................................................9-1 Introduction....................................................................9-1 The Atmosphere .............................................................9-2 Composition ...............................................................9-2 Properties ....................................................................9-2 Temperature............................................................9-2 Density ....................................................................9-2 Pressure ...................................................................9-2 Standard Atmosphere .................................................9-3 Layers of the Atmosphere ..........................................9-4 Scale of Weather Events ................................................9-4 Thermal Soaring Weather ..............................................9-6 Thermal Shape and Structure .....................................9-6 Atmospheric Stability .................................................9-7 Air Masses Conducive to Thermal Soaring ...................9-9 Cloud Streets ..............................................................9-9 Thermal Waves...........................................................9-9 Thunderstorms..........................................................9-10 Lifted Index ..........................................................9-12 K-Index .................................................................9-12 Weather for Slope Soaring .......................................9-14 Mechanism for Wave Formation ..............................9-16 Lift Due to Convergence ..........................................9-19 Obtaining Weather Information ...................................9-21 Preflight Weather Briefing........................................9-21 Weather-ReIated Information ..................................9-21 Interpreting Weather Charts, Reports, andForecasts ......................................................................9-23 Graphic Weather Charts ...........................................9-23 Winds and Temperatures Aloft Forecast ..............9-23 Composite Moisture Stability Chart .....................9-24 Chapter 10Soaring Techniques ..........................................10-1 Introduction..................................................................10-1 Thermal Soaring ...........................................................10-2 Locating Thermals ....................................................10-2 Cumulus Clouds ...................................................10-2 Other Indicators of Thermals ................................10-3 Wind .....................................................................10-4 The Big Picture .....................................................10-5Entering a Thermal ..............................................10-5 Inside a Thermal.......................................................10-6 Bank Angle ...........................................................10-6 Speed .....................................................................10-6 Centering ...............................................................10-7 Collision Avoidance ................................................10-9 Exiting a Thermal .....................................................10-9 Atypical Thermals ..................................................10-10 Ridge/Slope Soaring ..................................................10-10 Traps ......................................................................10-10 Procedures for Safe Flying .....................................10-12 Bowls and Spurs .....................................................10-13 Slope Lift ................................................................10-13 Obstructions ...........................................................10-14 Tips and Techniques ...............................................10-15 Wave Soaring .............................................................10-16 Preflight Preparation ...............................................10-17 Getting Into the Wave ............................................10-18 Flying in the Wave .................................................10-20 Soaring Convergence Zones ...................................10-23 Combined Sources of Updrafts ..............................10-24 Chapter 11Cross-Country Soaring .....................................11-1 Introduction..................................................................11-1 Flight Preparation and Planning ...................................11-2 Personal and Special Equipment ..................................11-3 Navigation ....................................................................11-5 Using the Plotter .......................................................11-5 A Sample Cross-Country Flight ...............................11-5 Navigation Using GPS .............................................11-8 Cross-Country Techniques ...........................................11-9 Soaring Faster and Farther .........................................11-11 Height Bands ..........................................................11-11 Tips and Techniques ...............................................11-12 Special Situations .......................................................11-14 Course Deviations ..................................................11-14 Lost Procedures ......................................................11-14 Cross-Country Flight in a Self-Launching Glider .....11-15 High-Performance Glider Operations and Considerations ............................................................11-16 Glider Complexity ..................................................11-16 Water Ballast ..........................................................11-17 Cross-Country Flight Using Other Lift Sources ........11-17 Chapter 12Towing ................................................................12-1 Introduction..................................................................12-1 Equipment Inspections and Operational Checks .........12-2 Tow Hook ................................................................12-2 Schweizer Tow Hook ...........................................12-2x。

文章篇号:1007-2764(2006)02-0138-044仙人掌多糖的提取、分离纯化及GPC法测定其分子量金鑫，赖凤英(华南理工大学轻工与食品学院,广东广州510640)摘要：研究了仙人掌多糖水提法的最佳工艺条件,以及仙人掌多糖的分离纯化。

试验结果表明,提取液用Savag法脱蛋白后，采用Sephacryl S-400柱层析纯化，得到仙人掌多糖OP，经过高效凝胶渗透色谱和常压凝胶柱色谱分析表明，OP为均一多糖，高效液相凝胶色谱法测定OP分子量(Mw)为172591Da。

关键词：仙人掌多糖；提取；分离纯化；分子量测定Extraction、Isolation and Purification of Polysaccharides in Opuntia and Determination of its molecular weight by GPC methodJin Xin, Lai Feng-ying(College of light industry and Food, South China University of Technology, Guangzhou 510640, China) Abstract The extraction technology、separation and purification of the polysaccharide from opuntia was studied in this paper. By using Savag method, almost all protein was removed from the extracted juice. Then the polysaccharide was purified by Sephacryl S-400 gelcolumn chromatography, and OP was gained. Its homogeneity is verified by HPGPC and Sephadex G-200 column chromatography. And the molecular weight of OP is 172591Da by the determination of HPGPC.Keywords: Opuntia; Polysaccharide; Extraction; Isolation; Purification; Determination of molecular weights仙人掌，别名仙巴掌、观音掌、霸王树、龙舌等，为仙人掌科仙人掌属植物，原产于美洲、墨西哥一带，广泛分布于非洲、亚洲、美洲等热带和亚热带地区[1]。

冯琳，罗世博，古丽格娜·皮达买买提，等. 响应面法优化准噶尔山楂总三萜提取工艺及其纯化工艺研究[J]. 食品工业科技，2024，45（9）：196−204. doi: 10.13386/j.issn1002-0306.2023070139FENG Lin, LUO Shibo, PIDAMAIMAITI Guligena, et al. Optimization of Extraction Process of Total Triterpenoids from Crataegus songarica by Response Surface Methodology and Its Purification Process[J]. Science and Technology of Food Industry, 2024, 45(9):196−204. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023070139· 工艺技术 ·响应面法优化准噶尔山楂总三萜提取工艺及其纯化工艺研究冯　琳1,2，罗世博1,2，古丽格娜·皮达买买提1，刘媛梦1,3，高红艳1,2,*（1.伊犁师范大学化学化工学院，新疆伊宁 835000；2.新疆普通高等学校天然产物化学与应用重点实验室，新疆伊宁 835000；3.污染物化学与环境治理重点实验室，新疆伊宁 835000）摘　要：为了提高准噶尔山楂的高值化利用，以准噶尔山楂为原料，通过单因素实验、正交试验和响应面设计优化复合酶辅助提取准噶尔山楂总三萜的工艺，并对纯化工艺进行优化。

结果表明，准噶尔山楂总三萜的最优提取工艺为纤维素酶、果胶酶和木瓜蛋白酶的添加量分别为4%、4%和2%，料液比为1:24 g/mL ，温度51 ℃，pH 为4.5，酶解76 min ，此条件下准噶尔山楂总三萜的提取量为（36.570±0.332）mg/g ，通过超声再次提取，总三萜的提取量达到（53.782±0.673）mg/g 。