Supercritical fluid extraction of Alnus glutinosa (L.) Gaertn.
J. of Supercritical Fluids 61 (2012) 55 61Contents lists available at SciVerse ScienceDirectThe Journal of Supercritical Fluidsjou rn al h om epage: www.elsev ier .comSuperc (LAnik Fe ala Semmelweis Ub Gedeon Richtc Budapest Uni ess Ena r t i c lArticle history:Received 20 JuReceived in reAccepted 5 OcKeywords:Alnus glutinosaPentacyclic triSupercritical GCMSLCMSRP-HPLCon ofin orded byere idrms ole in SFE xane ct, ret yielf trite1. IntroductionImportance of pharmacologically active natural compounds andplant sources has been re-evaluated in the recent years and itbecame onein low concoutstandingrials is supethe 1970s tbroadened istics [1,2].Lupane-compoundswaxes. Amosome of thpromising toxicity andthese chemanti-HIV [6hepatoprotCommondeciduous ttemperate Corresponnosy, 1085 BuE-mail addtreat wounds, ulcers, fever and abdominal pain . Various typesof plant secondary metabolites including anthraquinones, phenolicglycosides, avonol glycoside, terpenoids, xanthones have previ-ously been reported from the barks, buds, leaves and pollens of A.0896-8446/$ doi:10.1016/j. of the most active research elds. They often presententration in the plants and are chemically sensitive. An method to recover these compounds from raw mate-rcritical uid extraction (SFE). It has been applied sinceo isolate natural products and its application eld hasdue to its several well-known advantageous character-type pentacyclic triterpenoids are naturally originated, their main sources are stem barks, leaves and fruitng them, betulin, betulinic acid and lupeol represente most interesting molecules due to their numerouspharmacological effects that are associated with low high selectivity. According to the recent researches,ical agents show anti-tumoral [3,4], antiviral ,,7], antibacterial, anti-inammatory, antioxidant ,ective [9,10] and anxiolitic properties . alder (Alnus glutinosa (L.) Gaertn., Betulaceae) is aree native to a number of countries in northern Africa,Asia and Europe. In the folk medicine it was used toding author at: Semmelweis University, Department of Pharmacog-dapest, lloi t. 26, Hungary. Tel.: +36 1 431 4683.ress: email@example.com (A. Felfldi-Gva).glutinosa .Supercritical uid extraction has previously been used to extracttriterpenoids from various plants. Effect of different supercriticaluid extraction conditions on triterpene content and other com-ponents of chaste berry fruit (Vitex Agnus castus) and dandelionleaves (Taraxacum ofcinale Weber et Wiggers) were studied usinga 32 full factorial design. The pressure and temperature were variedover the ranges of 100450 bar and 3565 C. The extraction yield,the recovery of -sitosterol and -amyrin were compared to thoseobtained by Soxhlet extraction. Similar trends were experiencedin case of both plants. It was revealed that rather pressure thantemperature had signicant effect on recovery. By evaluation theexperiments 450 bar and 6065 C was found to be the best condi-tion within the ranges investigated where the highest yield of thesecompounds was obtained [14,15].The inuence of modiers (methanol and dimethyl sulfoxide)on SFE of triterpenes (ginsenosides) was studied by Wood et al. onNorth American ginseng root (Panax quinquefolius). They examinedtheir effect on the total extraction yield as well as total amountand composition of extracted ginsenosides by combination ofstatic and dynamic extraction with supercritical CO2. Soxhletextraction resulted in 409 mg/g extraction yield and 75.5 mg/g ofginsenosides. Quantities obtained with pure CO2 and with dynamicextraction using modier was negligible compared to that. Several see front matter 2011 Elsevier B.V. All rights reserved.supu.2011.10.003ritical uid extraction of Alnus glutinosalfldi-Gvaa,b,, Szabolcs Szarkaa, Bla Simndic, Bniversity, Department of Pharmacognosy, 1085 Budapest, lloi t 26, Hungaryer Plc, 1103 Budapest, Gymroi t 19-21, Hungaryversity of Technology and Economics, Department of Chemical and Environmental Proc e i n f one 2011vised form 4 October 2011tober 2011 (L.) Gaertn.terpenoidsuid extractiona b s t r a c tSupercritical carbon dioxide extractiT = 40/60 C, EtOH addition = 0/5/10%) composition of extracts were analyzcyclic triterpenes and -sitosterol wmethod over Soxhlet extraction in tecurves revealed that pressure had littco-solvent increased it. The optimumextracts was 3.81% compared to n-hewas 3.57, 2.95 and 14.33 g/100 g extraSoxhlet extraction ensures the highessubstances hence the concentration o/ locate /supf lu.) Gaertn.zs Blazicsa, Blanka Simonc, gnes Kryagineering, 1111 Budapest, Muegyetem rkp. 3, Hungary Alnus glutinosa (L.) Gaertn. was performed (P = 300/450 bar,er to determine preferable process conditions. Phytochemical means of TLC, GCMS, LCMS, RP-HPLC. Total of 11 penta-entied. The results indicated that SFE is an advantageousf yield and recovery of target compounds. Overall extractionuence on extraction yield, while temperature and amount ofcondition was 300 bar/60 C/10% EtOH, where the amount of(2.56%). Highest amount of betulin, betulinic acid and lupeolspectively, depending on the applied SFE condition. Ethanolicd (40.90%), but provides the extracts diluted with undesirablerpenes in the extract was very low. 2011 Elsevier B.V. All rights reserved.56 A. Felfldi-Gva et al. / J. of Supercritical Fluids 61 (2012) 55 61experimental conditions were studied using CO2 + MeOH(20.748.3 MPa, 50110 C, 530% MeOH mole percentage) incombined extraction. Modier usage was found to have the mostsignicant effect on the quantity of ginsenosides extracted. Onlya small amamount (1at 30% Memately 90%ginsenosideSome au(Calendula extraction The extracdiol esters (T = 29931oresin by Can increasinincrease in complex dusure that rephysical meevaluated tmodels. Thelogistic modSFE alsoous oilseedroselle seedide extractiof 200400ical uid in roselle scholesterolvariable withe extractephytosterol40 C, a highrate of 20 mBetulin by Zhang etlike modiand extractmum betul1.5 mL g1 bthe extractiIn our pan alternatiin addition plant in varus a good poof differenttion of targSolvent extable SFE conthe plant, fo2. Materia2.1. MateriThe aldenia. Plant sdeposited inversity, Buddry and dar(Hungary). from Carlo Erba (Italy) and acetic acid of LC grade was fromFluka (Switzerland). Methanol of LC super gradient grade wasfrom SigmaAldrich (Germany). All other chemicals of analytical-reagent grade were obtained from Reanal (Hungary). Water usedC stusysteurchethodPrepa drie partto thre dacopainede divalu00 e0 = 1Prepandarddivi of 0tulinrt RC in resitosncenSoxh extrient illede cotracless. tion Supe rawre pred b00 g (essund 6 10%n wraturout and on used t phyty anolarread-solvts wiSapordermentountount of ginsenosides was extracted at low modier2 mg/g at 1 g modier/g ginseng). On the contrary,OH mole percentage (4.1 g mod/g ginseng) approxi-, while with DMSO (3.6 g mod/g ginseng) 64% of thes obtained in Soxhlet extraction were extracted .thors investigated SFE on triterpenoids of marigoldofcinalis). Hamburger et al. applied supercritical uidfor purication of faradiol esters under 500 bar/50 C.tion yield was 5% and approximately 85% of fara-were extracted. Also, SFE under different conditions3 K, P = 1220 MPa) was investigated on marigold ole-ampos et al. According to their results, pressure hadg effect on the yield at constant temperature due tosolvent density. However, the effect of temperature wase to the combined effect of density and vapour pres-sulted crossover of the yield isotherms at 15 MPa. Thechanisms involved in the mass transfer process werehrough the application of ve different mathematical best t to the experimental data was obtained for theel [17,18]. has been used to extract -sitosterol from vari-s and other products. Recovery of phytosterol froms (Hibiscus sabdariffa L.) via supercritical carbon diox-on modied with ethanol was investigated at pressures bar, temperatures from 40 to 80 C and at supercrit-ow rates from 10 to 20 mL min1. The major sterolseed oil were -sitosterol, campesterol, stigmasterol, and 5-avenasterol. Pressure was determined to be theth the highest inuence on phytosterol composition ind seed oil. The highest extraction yield and the highest composition were obtained at the low temperature of pressure of 400 bar and a high supercritical uid owL min1 .was extracted by SFE from bark of Betula platyphylla al. The authors investigated and analyzed parameterser dosage (12 mL), extraction pressure (1535 MPa)ion temperature (3575 C). It was found that the opti-in recovery is achieved when the modier dosage wasark powder, the extractive pressure was at 20 MPa, andve temperature was at 55 C .revious work, we described A. glutinosa (L.) Gaertn. asve source of lupane-type triterpenes and phytosterolsto Betula species . As these compounds occur in theious chemical forms like alcohols, carbonic acids, it gavessibility to use as a model plant investigating the effects SFE conditions on the extraction yield and concentra-et compounds compared to those obtained by classicalraction. Objective of this work was to determine prefer-dition and to identify valuable bioactive compounds incusing on pentacyclic triterpenoids.ls and methodsalsr bark was collected and dried in Mures County, Roma-ample was authenticated and voucher specimen is the Department of Pharmacognosy, Semmelweis Uni-apest. The collected plant material was stored in ak place. Pure supercritical CO2 was supplied by LindeAcetonitrile and methanol of LC grade were purchasedin HPLcation were p2.2. M2.2.1. ThebiggerPrior als wePharmthe grcle sizwere eR(x) = 1were d2.2.2. Staof an intrationand beMinisastoredThe -at a co2.2.3. Theat amband muntil thwas excolourdistilla2.2.4. Thepressu(delivewith 8tion pr40 C a5% andadditiotemperate ablected carrieddecreaTheviscosiwith apeasy spas a coextrac2.2.5. In oexperiAn amdies was deionised by Millipore Direct Q5 water puri-m (USA). Betulin, betulinic acid and lupeol standardsased from Biomarker Kft. (Hungary).sration of raw material for extractiond bark was milled and contained some smaller andicles. The colour of the samples was cocoa brown.e extraction, the dry residue of the raw materi-etermined by method 2.8.16 described in Europeanoeia (5th ed., 2005). The dry matter content of alder barks were 91.33 0.07 (w/w %). The parti-stribution was analyzed by sieving. Analysis resultsated by the RosinRammlerBennet (RRB) distribution:xp((d/d0)n). The average size distribution parameters.415 0.057 mm and n = 1.326 0.079 mm.ration of standard solutions stock solutions were made by dissolving 0.403.30 mgdual compound in 0.403.30 mL methanol at a concen-.50 mg mL1 for betulinic acid, 1.0 mg mL1 for lupeol and ltered through a single use syringe lter (0.20 m, 15, Vivascience AG, Germany). The solutions werefrigerator and brought to room temperature before use.terol standard used in TLC was dissolved in chloroformtration of 0.54 mg mL 1.let extractionaction was carried out in a laboratory scale apparatuspressure at the boiling point of the solvent used. Dried barks were extracted with n-hexane on a water bathlour of the extract seemed to be fading. Then the drugted again, with 96% ethanol until this solvent becameThe extracts were evaporated to dry mass by vacuumand weighed.rcritical uid extraction material was extracted using carbon-dioxide in a highilot plant equipped with 5 L volume extractor vessely NATEX Austria). The extraction vessel was suppliedexactly weighed) of raw material. The designed extrac-res were 300 and 450 bar. The temperature was set to0 C. The ethanol concentration was varied between 0%,. The extraction at 300 bar and 60 C, with 5% EtOHas performed in triplicate. After adjusting the desirede and pressure, the CO2 feed was started with a ow7 kg h1. The accumulated product samples were col-weighed at certain time intervals. The extraction wasntil the amount of the product sample collected for 1 ho under 0.1% of the raw material.sical characteristics of both extracts including colour,d odour depended on the solvent used. Those extracted solvents had a yellowish-green colour, gentle odour and consistency. Application of ethanol either as solvent orent resulted in dark green-black coloured highly viscousth strong smell.nication of the extracts to eliminate fatty acids that may interfere with thes, non-saponiable fractions of samples were prepared. of 0.20 g of the extracts was reuxed in alcoholicA. Felfldi-Gva et al. / J. of Supercritical Fluids 61 (2012) 55 61 57Table 1Extraction yield of Alnus glutinosa (L.) Gaertn. obtained by Soxhlet and supercritical uid extraction and unsaponiable residue of extracted plant material.Soxhlet extraction SFEExperiment Yield (%) Non-saponiable part (%) Experiment P [bar]/T [C] EtOH (%) Yield (%) Non-saponiable part (%)n-Hexane 2.30 44.20 1 300/40 0 1.70 62.15EtOH 40.90 1.70 2 300/60 0 1.96 50.703 450/40 0 1.51 61.454 450/60 0 2.56 44.905 300/40 5 2.70 13.406a 300/60 5 2.87 0.08 35.907 300/40 10 3.52 38.458 300/60 10 3.81 33.00a Performed in triplicate.KOH (0.50 g KOH + 20.0 mL 96% EtOH) on water bath for 1 h. Thensolutes were cool down by adding deionised water and extractedthree times with petroleum ether. The unied organic phaseswere washed with water to neutral. Petroleum ether was des-iccated with Na2SO4 and evaporated in vacuum after ltration.The non-sawere prepamethanol.2.2.6. Identglutinosa (LIdenticchromatogr(GCMS) anThe TLC wative comparthe same as184.108.40.206. GCsaponied einto septumbis-(trimethwas addedThereafter diluted witcomponent(Santa Claraperformed column (SuSigmaAldrfor 1.0 min,mal); then f5 min. Analhelium at aThe injectiosplitless mode. The injector was programmed from 140 to 300 Cat 720 C min1 using a 40 psi pressure pulse for 1.0 min.The mass selective detector equipped with quadrupole massanalyser was operated in electron ionisation mode at 70 eV in fullscan mode (41500 amu). The temperature of the mass-selectiveor wa of f sta5 libr. LColvinnol aatogrer, GA coed (as acze: 1 at 40ing g min, 12 matoged bess spquadlogitempre: 3Full over of c valural ire wFig. 1. Ovponied residues were weighed and stock solutionsred of them by dissolving the samples in HPLC gradeication of triterpenes and phytosterols in bark of A..) Gaertn.ation of compounds was performed by thin-layeraphy (TLC), gas chromatographymass spectrometryd liquid chromatographymass spectrometry (LCMS).s used for preliminary qualitative and rough quantita-ison of the samples. The experimental conditions were previously described .MS conditions. For derivatisation, 100 L aliquot ofxtracts dissolved in 5 mL chloroform were transferred-capped vials and evaporated to dryness. A 100 mL ofylsilyl)-triuoroacetamide (BSTFA) silylation reagent then left standing at room temperature overnight.the content of the vial was evaporated to dry andh chloroform to an exact volume. Analysis of sterols was carried out with Agilent 6890N/5973N GC-MSD, CA, USA) system. Gas chromatographic separation wason a 30 m 250 m 0.25 m lm thickness capillarypelco 28471-U, SLB-5ms 5% phenyl-methyl siloxane,ich). The oven temperature was maintained at 120 C then increased to 270 C at 20 C min1 (20 min isother-urther increased to 300 C at 10 C min 1 and held forysis time was 36.5 min. The carrier gas was high-purity constant ow rate of 1.0 mL min1 throughout the run.n volume was 1.0 L. The inlet was operated in pulseddetectparisonthose oNIST 02.2.6.2by dissmethachromdegassG1316was ustion wpore sitainedfollow0.2 mL(v/v) BChromrecordMatriple Technolows: pressu120 V. mode spectraenergystructusoftwaerall extraction curves of Alnus glutinosa bark supercritical extracts under different conds 300 C. The main components were identied by com-their retention time and recorded mass spectra withndards. Mass spectra of known literature data and theary were also consulted.MS conditions. Sample stock solutions were preparedg the saponied extracts in LC super gradient gradet different concentrations (1.65.9 mg mL1). For theaphic separation an Agilent 1100 LC system (G1379A1312A binary gradient pump, G1329A autosampler,lumn thermostat and G1315C diode array detector)Agilent Technologies, Waldbronn, Germany). Separa-hieved on a Kinetex C18 (100 mm 2.1 mm, 2.6 m,00 A) column (Phenomenex, Torrance, CA, USA), main-C. Eluent A was water, eluent B was acetonitrile. Theradient elution program was applied at a ow rate of1; 0 min: 65% (v/v) B, 3 min: 80% (v/v) B, 10 min: 100%in: 100% (v/v) B, 13 min: 65% (v/v) B, 16 min: 65% (v/v) B.rams were acquired at 207 and 340 nm, UV spectra weretween 200 and 400 nm. Injection volume was 2 L.ectral analyses were performed with an Agilent 6410rupole system equipped with ESI ionsource (Agilentes, Waldbronn, Germany). ESI conditions were as fol-erature: 350 C, drying gas ow: 8 L min1, nebulizer0 psi, capillary voltage: 3500 V, fragmentor voltage:mass scan spectra were recorded in positive ionisationa range of m/z 50700 (scan time: 800 ms). CID massompounds were recorded applying different collisiones between 10 and 30 eV in order to obtain as muchnformation as it was possible. The Masshunter B.01.03as used for data acquisition and qualitative analysis.itions: (A) with supercritical CO2 and (B) with ethanol addition.58 A. Felfldi-Gva et al. / J. of Supercritical Fluids 61 (2012) 55 61Fig. 2. (A and B) Scatterplot of yields against EtOH concentration under different pressure and temperature.Table 2Mean percentage distribution of components in Alnus glutinosa bark extracts identied and/or characterised by GCMS.Rt (min) Identied component Mean percentage distribution (%)n-Hexane extract Ethanolic extracts SFE extracts SFE + EtOH extracts25.32 Taraxerone 3.00 3.80 3.78 3.8026.25 -Sitosterol 9.00 7.50 6.66 8.2328.11 Lupenon 20.90 23.10 26.00 20.0329.03 Lupenylacetate 12.60 16.30 14.50 13.2029.26 Lupeol 6.60 6.60 9.20 16.2229.42 Simiarenol 14.50 14.50 14.10 16.2232.75 Betulin 6.40 4.40 3.05 8.0533.69 Betulinic acid 2.40 2.00 1.85 2.43Fig. 3. Total ion chromatograms (TIC) of silylated investigated compounds present in extracts of Alnus glutinosa L. (Gaertn.): (A) n-hexane and (B) SFE (450 bar/60 C).A. Felfldi-Gva et al. / J. of Supercritical Fluids 61 (2012) 55 61 59Table 3List of components in Alnus bark extracts identied and/or characterised by LCMS method.Rt (min) Identied component Basic ion (m/z) SFE experiment Soxhlet extraction1 4 6 7 EtOH n-Hexane4.68 Uvaol (?) 425.4 + + + + +7.61 Betulinic aldehid (?) 441.4 + + +8.40 Betulinic acid 439.4 ++ ++ +8.78 Ursolic acid 439.4 + + + 9.33 Betulin 425.4 ++ ++ ++ ++ ++ ++11.97 -Amyrin 423.4 ++ ++ + + +++: detected in a lower ratio; ++: detected in a high ratio; : minor or absent compound; ?: identied theoretically based on literature data For numbering of the experiments,see Table 1.Fig. 4. LC-DAD ). Num3 = betulin, 4 =Identicatiohensive intspectroscopwith those 2.2.7. Quanglutinosa (LReverse HPLC) methtriterpenes prepared byferent concsingle use sGermany). to room temvious RP-Hmodicatiowe manageelution wasacetic acid The volume205 nm. Antained at 2acid and lupretention tition as wellinto the chr, betal staults tractcess vity Table 4Calculated lineAnalyte Betulinic aciBetulinLupeol chromatogram of Alnus glutinosa L. (Gaertn.) supercritical extract (experiment 7 -amyrin.n of the components was carried out by the compre-erpretation of their respective chromatographic andic (MS, MS/MS and UV) data and by the comparisonof literature data and authentic standards.tication of lupane-type triterpenes in bark of A..) Gaertn.phased high-performance liquid chromatography (RP-betulinextern3. Res3.1. ExProselectiod was applied for quantitative analysis of lupane-typein Alnus bark extracts. Sample stock solutions were dissolving the saponied extracts in methanol at dif-entrations (3.417.96 mg mL1) and ltered through ayringe lter (0.20 m, Minisart RC 15, Vivascience AGThe solutions were stored in refrigerator and broughtperature and diluted before use. Compared to our pre-PLC conditions referred above , we made a slightn in composition and ratio of the mobile phase. Thusd to detect betulinic acid and lupeol as well. Isocratic performed with mobile phase acetonitrile/0.2% (v/v)in water (87/13, v/v%) with a 1.0 mL min1 ow rate. of injection was 20 L, detection wavelength was set atalysis took 30 min; the column temperature was main-5 C. The chromatographic peaks of betulin, betuliniceol in the samples were conrmed by comparing theirme with the respective standards and by standard addi-. Each standard and the sample solutions were injectedomatograph and peak areas were recorded. Amount ofextraction obtained w(300 and 45and ethanoof the SFE wthe similar (sc-CO2) anextract wahexane extthe whole and n-hexaponents onOverall uid extracfunction of The shape oof the extraIn SFE, tperature anar regression parameters for calibration curves.A B r2d 198,402.26 114,8887.34 0.998203,136.22 836,041.40 0.998285,572.35 1,444,742 0.998bering of peaks are the following: 1 = betulinic acid, 2 = ursolic acid,ulinic acid and lupeol in the extracts were calculated byndard calibration.and discussionion yieldscharacteristics, such as extraction yield, solubility andare the function of pressure and temperature. Theyields (mass of the extract/mass of the dry matter)ith supercritical CO2 at different operational conditions0 bar, 40 and 60 C, ethanol addition) and with n-hexanel with Soxhlet extraction are compared in Table 1. Yieldas comparable to the n-hexane extraction, because ofdissolving capacity of the supercritical carbon dioxided n-hexane. At the same time, the yield of alcoholics approximately 20 times higher than the SFE or n-racts. It can be explained by that the ethanol dissolvedunwanted soluble polar compounds, while the sc-CO2ne, as non-polar solvents, dissolved the non-polar com-ly; thus resulting more selective extraction.extraction curves (OEC) representing the supercriticaltion yields of Alnus bark under different conditions as aCO2 usage (kg CO2/kg dry material) are shown in Fig. 1.f the extraction curves indicates that at different stagesction, different mechanisms control the mass transfer.he process yield and selectivity is determined by tem-d pressure. The solvent density is inuenced by theirRange (mg mL1) Rt (min)7 0.0730.170 5.100 0.0730.113 6.019 0.1020.170 25.9360 A. Felfldi-Gva et al. / J. of Supercritical Fluids 61 (2012) 55 61alteration which affects the solubility of solute in solvent phase andconsequently the extraction yield and selectivity. From Fig. 1A it canbe seen that pressure alone had little inuence on the yield howeverthe interaction of pressure and temperature is not negligible.While at lower temperature similar yields were obtained atboth pressures, almost 0.6% enhancement in extraction yield wasachieved with increase in pressure at 60 C. This pressure effect canbe explainethe solubiliThe temcombined esolute vapobility, whileand solubiltemperaturwas more sthe same atthe dominasure. We alsaddition offacilitates thsolvent polshows the tions at 300concentratithe aspect oare supportFrom thethanol conabove resulbe optimal respect to e3.2. Ratio oSamplesmaterials. Itriterpenessaponied pThe highby supercripressure waslightly less(experimention of ethato supercritcan observethe ratio ofwas, the ratvents increextract thatnents to be matter was3.3. IdentiIdenticformed witpossibility ftitative comlupeol and son of theirRF values wTable 5Betulin, betulinic acid and lupeol content of Alnus glutinosa bark extracts.Experiment Triterpene content (g/100 g extract)Soxhlet extrn-HexaneEtOH detecIdentmicaMS.ramsnents ide, betr lupl as erceuentig. 3t in tIdento Soxis wenoidic ac wouat walysiulin -Ametuls whdentiDADtulinHPLCrmints. To determine linearity, the calibration curve of betulin,ic acid and lupeol standard were drawn and evaluated bysion analysis. From the stock standard solutions a mixturerepared which contained all three standards in the sametration (0.5 mg mL1). Seven concentrations of a freshly pre-working solution of this mixture were injected in the range60.170 mg mL1. Three replicate injections at each con-tion level were performed. The calculated linear regressioneters for calibration curves are given in Table 4, Y = Ax + B. betulin, betulinic acid and lupeol content of A. glutinosats is presented in Table 5.ong the three examined components, lupeol occured in mostant amount in the extracts. The highest lupeol content wasred at 450 bar/60 C (experiment 4). Decreasing the pressured by the increase in the solvent density which enhancessation.perature effect in the process yield is complex due to theffect of solvent density and solute vapour pressure. Theur pressure increases with temperature raising the solu- temperature has opposite effect on the solvent densityity. In Fig. 1A it is seen that the yield increased withe at both studied pressure however this enhancementignicant at 450 bar (from 1.51% to 2.56%) compared to 300 bar. This pattern suggests that at higher pressurent effect which inuences the yield is the vapour pres-o investigated the effect of co-solvent and observed that ethanol further increased the yield. Ethanol additione dissolvation of more polar compounds by enhancingarity which explains the increase in the yield. Fig. 1Bextraction curves under different temperature condi- bar after applying ethanol as co-solvent. The higher itson was the higher yield was achieved. Difference fromf temperature can also be observed. These experiencesed by Fig. 2.e scatter plots it can be seen that the temperature andtent had determinant effect on the yield. Based on thets, experiment 8 (300 bar/60 C/10% ethanol) proved tofor Alnus bark among the studied SFE conditions withxtraction yield and rate.f non-saponiable part were saponied to be cleaned from interfering oilyn these unsaponied residues the phytosterols and were present in free form. The ratio of the non-art in the extracts is shown in Table 1.est amount of non-saponiable residue was obtainedtical uid extraction when temperature was 40 C ands set to 300 bar and 450 bar, respectively. This ratio was in case of the same pressures but higher temperaturets 2 and 4) and further decrease was observed by addi-nol as co-solvent. The n-hexane extract was comparableical CO2 extracts due to the similar solvent powers. We that rather temperature than pressure had inuence on non-saponiable matters. The higher the temperatureio of these matters decreased. Also, addition of polar sol-ased the amount of polar or bonded compounds in the explain the decrease in the ratio of free apolar compo-present in the residue. As a result, the less unsaponied extracted from the ethanolic extract.cation of components in Alnus barkation of components in the Alnus bark extracts was per-h TLC, LCMS and GCMS methods. TLC gave us theor a rapid evaluation of the qualitative and rough quan-position prole of plant extracts. Betulin, betulinic acid,-sitosterol were identied in the samples by compari- retention times with those of the respective standards.ere as described previously .SFE12 3 4 5678 n.d.: not3.3.1. Cheby GCmatogcompopoundbetulinfurtheas welmean pconstitIn Fpresen3.3.2. Twanalystriterpbetulinlupeolfor whthis anBetples. while bsamplewere iLC-Fig. 4.3.4. BeAn to deteextracbetulinregreswas pconcenpared of 0.04centraparamTheextracAmsignicmeasuBetulinic acid Betulin Lupeolaction n.d. 1.2 6.22n.d. 0.062 n.d.n.d. 2.69 13.50n.d. 2.90 8.60n.d. 1.92 14.33n.d. 2.03 7.01n.q. 1.34 0.711.55 3.57 0.622.95 3.17 2.9851.74 3.30 1.42ed, n.q.: identied but not quantied.ication with GCMSl composition prole of the extracts was investigated The percentage data of the total ion current chro- (TIC) were calculated. The percentile values of thes represent their distribution in the plant bark. Com-ntied by TLC were justied by GCMS results. Besideulinic acid, lupeol and -sitosterol, we identied twoeol derivatives, namely lupenone and lupenylacetate;other triterpenes like taraxerone and simiarenol. Thentage distribution rate and retention time of the singles are presented in Table 2., TIC chromatograms of TMS derivatives of compoundswo extracts (n-hexane, SFE) are shown.ication with LCMShlet and four SFE extracts were subjected to LCMShich resulted in the identication of six pentacyclics as shown in Table 3. For identication of betulin andid, authentic standards were used. As identication ofld have been required different instrumental conditionse had no access, this compound was not subjected intos.as a major compound occurred in all examined sam-yrin was identied mainly in the apolar SFE extracts,inic acid and ursolic acid were detected rather in thoseere modier was applied. Uvaol and betulinic aldehydeed as minor components based on literature data. prole of A. glutinosa (L.) bark SFE extract is shown in, betulinic acid and lupeol content of the extracts-UV method developed by Zhao et al.  was adaptede betulin, betulinic acid and lupeol content in alder barkA. Felfldi-Gva et al. / J. of Supercritical Fluids 61 (2012) 55 61 61Fig. 5. HPLC-U(experiment 53 = lupeol.and temperits recoveryits recoveryCO2 only. Inenhanced ibetulin wascharacteristwhen ethan300 bar/40 7). Increasinresulted lesWe needdue to theirtime was aptheir peak earlier elutiand in SFE earea of the oextract. TheHPLC-UV ch4. ConclusSupercrithe effect oftion yield anof experimtemperaturyield. An aming the temlower pressmodier amwas also inon the abovdened as achieved con-hexane (acid and lutively, depeof ethanoliccentration othat SFE is a capable method for obtaining valuable triterpenoidsfrom the bark of A. glutinosa (L.) Gaertn.Composition of extracts was investigated with chromatographicmethods. We identied A. glutinosa as a valuable raw materialfor pharmacologically active pentacyclic triterpenoids. Total of 11compounds were identied. In addition to previously described, betde, luenol .) Gawleds worncesHerre: rec525ang, Cdies ger, Htributint ext. Laszools inong, K, S.L. Splex v. Cicheentialctioniken,leculalakures of nces unitheol linatoto. Kim, antioatotoucenrkovaiconvu08) 71. Neve notesortugRourker, HtulaceossutV chromatogram of Alnus glutinosa L. (Gaertn.) supercritical extract). Numbering of peaks are the following: 1 = betulinic acid, 2 = betulin,ature, as well as modier usage negatively inuenced. Regarding the content of betulin, we observed that was decreased at higher pressure when using purecrease in temperature and addition of ethanol howeverts recovery. The most optimal extraction condition of 300 bar/60 C + 5%EtOH (experiment 5). Due to its acidicic, betulinic acid was detected in measurable amountol was used. The most optimal extraction condition wasC by addition 10% of ethanol as co-solvent (experimentg the temperature or decreasing the amount of ethanols extraction yield. to mention two unidentied but detected components signicant pattern in the samples. Their mean retentionproximately 7.0 and 20.0 min, respectively. Comparingarea in each sample, we observed that amount of theng compound extensively increased in ethanolic extractxperiment performed at 450 bar/40 C, while the peakther compound was signicantly high in the same SFEir identication requires further studies. An example ofromatogram of alder bark extract is presented in Fig. 5.ionstical uid extraction of alder bark was performed andbetulinaldehysimiarnosa (LAcknoThiRefere M. tion249 Q. Lstu S. Jdispla M.Nas t Y. Gsonsim R.Hpotinfe C. AMo S. AertiScie S. Sluphep S.Tandhep R. MZhaant(20 J.Micalof P C. OSar(Be D. C pressure, temperature and modier addition on extrac-d composition of the extracts were studied. Evaluationental results showed that interaction of pressure ande as well as ethanol addition improved the extractionount of 1.04% enhancement was achieved by increas-perature at 450 bar, while this ratio was 0.2% only at theure. Similar trends were experienced by increasing theount. The recovery of selected lupane-type triterpenesuenced by the extraction conditions applied. Basede observations, the optimum extraction condition was300 bar/60 C + 10% EtOH where the highest yield wasmpared to that obtained by Soxhlet extraction with3.81% vs 2.56%). Highest amount of betulin, betulinicpeol was 3.57; 2.95 and 14.33 g/100 g extract, respec-nding on the applied SFE condition. Although the yield Soxhlet extraction was about 10 times higher, the con-f the active constituents was very low. We can concludeT. Keve, SFluids 47 B. Simnduid extr J.A. Woodsenosidesdioxide, J M. Hambcation of(Calendul L.M.A.S. Cdata andofcinalis K.L. Nyamroselle seProcessin Z. Yu-Hophylla by202204 A. Gva, Btanaceaecomposit671681ulinic acid, lupeol and -sitosterol, presence of betulinicpenone, lupenyl-acetate, -amyrin, uvaol, ursolic acid,and taraxerone was described rst in the bark of A. gluti-ertn.gementk was supported by GVOP 3.1.1.-2004-05-0397/3.0.ro, J.A. Mendiola, A. Cifuentes, E. Ibnez, Supercritical uid extrac-ent advances and applications, J. Chromatography A 1217 (2010)11..M. Wai, Supercritical uid extraction in herbal and natural product a practical review, Talanta 53 (2001) 771782.. Trojan, T. Kopp, M.N. Laszczyk, A. Schefer, Pentacyclic triterpeneon in various plants rich sources for a new group of multi-potentracts, Molecules 14 (2009) 20162031.czyk, Pentacyclic triterpenes of the lupane, oleanane and ursane group cancer therapy, Planta Medica 75 (2009) 15491560..M. Raj, C.A. Luscombe, I. Gadawski, T. Tam, J. Chu, D. Gibson, R. 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Gaertn.1 Introduction2 Materials and methods2.1 Materials2.2 Methods2.2.1 Preparation of raw material for extraction2.2.2 Preparation of standard solutions2.2.3 Soxhlet extraction2.2.4 Supercritical fluid extraction2.2.5 Saponification of the extracts2.2.6 Identification of triterpenes and phytosterols in bark of A. glutinosa (L.) Gaertn.220.127.116.11 GCMS conditions18.104.22.168 LCMS conditions2.2.7 Quantification of lupane-type triterpenes in bark of A. glutinosa (L.) Gaertn.3 Results and discussion3.1 Extraction yields3.2 Ratio of non-saponifiable part3.3 Identification of components in Alnus bark3.3.1 Identification with GCMS3.3.2 Identification with LCMS3.4 Betulin, betulinic acid and lupeol content of the extracts4 ConclusionsAcknowledgementReferences