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J. of Supercritical Fluids 61 (2012) 55– 61

Contents lists available at SciVerse ScienceDirect

The Journal of Supercritical Fluids

jou rn al h om epage: www.elsev ier .com/ locate /supf lu

upercritical fluid extraction of Alnus glutinosa (L.) Gaertn.

nikó Felföldi-Gávaa,b,∗, Szabolcs Szarkaa, Béla Simándic, Balázs Blazicsa, Blanka Simonc, Ágnes Kérya

Semmelweis University, Department of Pharmacognosy, 1085 Budapest, Ülloi út 26, HungaryGedeon Richter Plc, 1103 Budapest, Gyömroi út 19-21, HungaryBudapest University of Technology and Economics, Department of Chemical and Environmental Process Engineering, 1111 Budapest, Muegyetem rkp. 3, Hungary

r t i c l e i n f o

rticle history:eceived 20 June 2011eceived in revised form 4 October 2011ccepted 5 October 2011

eywords:

a b s t r a c t

Supercritical carbon dioxide extraction of Alnus glutinosa (L.) Gaertn. was performed (P = 300/450 bar,T = 40/60 ◦C, EtOH addition = 0/5/10%) in order to determine preferable process conditions. Phytochemicalcomposition of extracts were analyzed by means of TLC, GC–MS, LC–MS, RP-HPLC. Total of 11 penta-cyclic triterpenes and �-sitosterol were identified. The results indicated that SFE is an advantageousmethod over Soxhlet extraction in terms of yield and recovery of target compounds. Overall extraction

lnus glutinosa (L.) Gaertn.entacyclic triterpenoidsupercritical fluid extractionC–MSC–MSP-HPLC

curves revealed that pressure had little influence on extraction yield, while temperature and amount ofco-solvent increased it. The optimum SFE condition was 300 bar/60 ◦C/10% EtOH, where the amount ofextracts was 3.81% compared to n-hexane (2.56%). Highest amount of betulin, betulinic acid and lupeolwas 3.57, 2.95 and 14.33 g/100 g extract, respectively, depending on the applied SFE condition. EthanolicSoxhlet extraction ensures the highest yield (40.90%), but provides the extracts diluted with undesirablesubstances hence the concentration of triterpenes in the extract was very low.

. Introduction

Importance of pharmacologically active natural compounds andlant sources has been re-evaluated in the recent years and itecame one of the most active research fields. They often present

n low concentration in the plants and are chemically sensitive. Anutstanding method to recover these compounds from raw mate-ials is supercritical fluid extraction (SFE). It has been applied sincehe 1970s to isolate natural products and its application field hasroadened due to its several well-known advantageous character-

stics [1,2].Lupane-type pentacyclic triterpenoids are naturally originated

ompounds, their main sources are stem barks, leaves and fruitaxes. Among them, betulin, betulinic acid and lupeol represent

ome of the most interesting molecules due to their numerousromising pharmacological effects that are associated with lowoxicity and high selectivity. According to the recent researches,hese chemical agents show anti-tumoral [3,4], antiviral [5],nti-HIV [6,7], antibacterial, anti-inflammatory, antioxidant [8],epatoprotective [9,10] and anxiolitic properties [11].

Common alder (Alnus glutinosa (L.) Gaertn., Betulaceae) is aeciduous tree native to a number of countries in northern Africa,emperate Asia and Europe. In the folk medicine it was used to

∗ Corresponding author at: Semmelweis University, Department of Pharmacog-osy, 1085 Budapest, Ülloi út. 26, Hungary. Tel.: +36 1 431 4683.

E-mail address: ganiko@mailbox.hu (A. Felföldi-Gáva).

896-8446/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.supflu.2011.10.003

© 2011 Elsevier B.V. All rights reserved.

treat wounds, ulcers, fever and abdominal pain [12]. Various typesof plant secondary metabolites including anthraquinones, phenolicglycosides, flavonol glycoside, terpenoids, xanthones have previ-ously been reported from the barks, buds, leaves and pollens of A.glutinosa [13].

Supercritical fluid extraction has previously been used to extracttriterpenoids from various plants. Effect of different supercriticalfluid extraction conditions on triterpene content and other com-ponents of chaste berry fruit (Vitex Agnus castus) and dandelionleaves (Taraxacum officinale Weber et Wiggers) were studied usinga 32 full factorial design. The pressure and temperature were variedover the ranges of 100–450 bar and 35–65 ◦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 significant effect on recovery. By evaluation theexperiments 450 bar and 60–65 ◦C was found to be the best condi-tion within the ranges investigated where the highest yield of thesecompounds was obtained [14,15].

The influence of modifiers (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 of

static 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 modifier was negligible compared to that. Several

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6 A. Felföldi-Gáva et al. / J. of Su

xperimental conditions were studied using CO2 + MeOH20.7–48.3 MPa, 50–110 ◦C, 5–30% MeOH mole percentage) inombined extraction. Modifier usage was found to have the mostignificant effect on the quantity of ginsenosides extracted. Only

small amount of ginsenosides was extracted at low modifiermount (1–2 mg/g at 1 g modifier/g ginseng). On the contrary,t 30% MeOH mole percentage (4.1 g mod/g ginseng) approxi-ately 90%, while with DMSO (3.6 g mod/g ginseng) 64% of the

insenosides obtained in Soxhlet extraction were extracted [16].Some authors investigated SFE on triterpenoids of marigold

Calendula officinalis). Hamburger et al. applied supercritical fluidxtraction for purification of faradiol esters under 500 bar/50 ◦C.he extraction yield was 5% and approximately 85% of fara-iol esters were extracted. Also, SFE under different conditionsT = 299–313 K, P = 12–20 MPa) was investigated on marigold ole-resin by Campos et al. According to their results, pressure hadn increasing effect on the yield at constant temperature due toncrease in solvent density. However, the effect of temperature wasomplex due to the combined effect of density and vapour pres-ure that resulted crossover of the yield isotherms at 15 MPa. Thehysical mechanisms involved in the mass transfer process werevaluated through the application of five different mathematicalodels. The best fit to the experimental data was obtained for the

ogistic model [17,18].SFE also has been used to extract �-sitosterol from vari-

us oilseeds and other products. Recovery of phytosterol fromoselle seeds (Hibiscus sabdariffa L.) via supercritical carbon diox-de extraction modified with ethanol was investigated at pressuresf 200–400 bar, temperatures from 40 to 80 ◦C and at supercrit-cal fluid flow rates from 10 to 20 mL min−1. The major sterolsn roselle seed oil were �-sitosterol, campesterol, stigmasterol,holesterol and �5-avenasterol. Pressure was determined to be theariable with the highest influence on phytosterol composition inhe extracted seed oil. The highest extraction yield and the highesthytosterol composition were obtained at the low temperature of0 ◦C, a high pressure of 400 bar and a high supercritical fluid flowate of 20 mL min−1 [19].

Betulin was extracted by SFE from bark of Betula platyphyllay Zhang et al. The authors investigated and analyzed parameters

ike modifier dosage (1–2 mL), extraction pressure (15–35 MPa)nd extraction temperature (35–75 ◦C). It was found that the opti-um betulin recovery is achieved when the modifier dosage was

.5 mL g−1 bark powder, the extractive pressure was at 20 MPa, andhe extractive temperature was at 55 ◦C [20].

In our previous work, we described A. glutinosa (L.) Gaertn. asn alternative source of lupane-type triterpenes and phytosterolsn addition to Betula species [21]. As these compounds occur in thelant in various chemical forms like alcohols, carbonic acids, it gaves a good possibility to use as a model plant investigating the effectsf different SFE conditions on the extraction yield and concentra-ion of target compounds compared to those obtained by classicalolvent extraction. Objective of this work was to determine prefer-ble SFE condition and to identify valuable bioactive compounds inhe plant, focusing on pentacyclic triterpenoids.

. Materials and methods

.1. Materials

The alder bark was collected and dried in Mures County, Roma-ia. Plant sample was authenticated and voucher specimen is

eposited in the Department of Pharmacognosy, Semmelweis Uni-ersity, Budapest. The collected plant material was stored in ary and dark place. Pure supercritical CO2 was supplied by LindeHungary). Acetonitrile and methanol of LC grade were purchased

tical Fluids 61 (2012) 55– 61

from Carlo Erba (Italy) and acetic acid of LC grade was fromFluka (Switzerland). Methanol of LC super gradient grade wasfrom Sigma–Aldrich (Germany). All other chemicals of analytical-reagent grade were obtained from Reanal (Hungary). Water usedin HPLC studies was deionised by Millipore Direct Q5 water purifi-cation system (USA). Betulin, betulinic acid and lupeol standardswere purchased from Biomarker Kft. (Hungary).

2.2. Methods

2.2.1. Preparation of raw material for extractionThe dried bark was milled and contained some smaller and

bigger particles. The colour of the samples was cocoa brown.Prior to the extraction, the dry residue of the raw materi-als were determined by method 2.8.16 described in EuropeanPharmacopoeia (5th ed., 2005). The dry matter content ofthe grained alder barks were 91.33 ± 0.07 (w/w %). The parti-cle size distribution was analyzed by sieving. Analysis resultswere evaluated by the Rosin–Rammler–Bennet (RRB) distribution:R(x) = 100 exp(−(d/d0)n). The average size distribution parameterswere d0 = 1.415 ± 0.057 mm and n = 1.326 ± 0.079 mm.

2.2.2. Preparation of standard solutionsStandard stock solutions were made by dissolving 0.40–3.30 mg

of an individual compound in 0.40–3.30 mL methanol at a concen-tration of 0.50 mg mL−1 for betulinic acid, 1.0 mg mL−1 for lupeoland betulin and filtered through a single use syringe filter (0.20 �m,Minisart RC 15, Vivascience AG, Germany). The solutions werestored in refrigerator and brought to room temperature before use.The �-sitosterol standard used in TLC was dissolved in chloroformat a concentration of 0.54 mg mL − 1.

2.2.3. Soxhlet extractionThe extraction was carried out in a laboratory scale apparatus

at ambient pressure at the boiling point of the solvent used. Driedand milled barks were extracted with n-hexane on a water bathuntil the colour of the extract seemed to be fading. Then the drugwas extracted again, with 96% ethanol until this solvent becamecolourless. The extracts were evaporated to dry mass by vacuumdistillation and weighed.

2.2.4. Supercritical fluid extractionThe raw material was extracted using carbon-dioxide in a high

pressure pilot plant equipped with 5 L volume extractor vessel(delivered by NATEX Austria). The extraction vessel was suppliedwith 800 g (exactly weighed) of raw material. The designed extrac-tion pressures were 300 and 450 bar. The temperature was set to40 ◦C and 60 ◦C. The ethanol concentration was varied between 0%,5% and 10%. The extraction at 300 bar and 60 ◦C, with 5% EtOHaddition was performed in triplicate. After adjusting the desiredtemperature and pressure, the CO2 feed was started with a flowrate about 7 kg h−1. The accumulated product samples were col-lected and weighed at certain time intervals. The extraction wascarried on until the amount of the product sample collected for 1 hdecreased to under 0.1% of the raw material.

The physical characteristics of both extracts including colour,viscosity and odour depended on the solvent used. Those extractedwith apolar solvents had a yellowish-green colour, gentle odour andeasy spread consistency. Application of ethanol either as solvent oras a co-solvent resulted in dark green-black coloured highly viscousextracts with strong smell.

2.2.5. Saponification of the extractsIn order to eliminate fatty acids that may interfere with the

experiments, non-saponifiable fractions of samples were prepared.An amount of 0.20 g of the extracts was refluxed in alcoholic

A. Felföldi-Gáva et al. / J. of Supercritical Fluids 61 (2012) 55– 61 57

Table 1Extraction yield of Alnus glutinosa (L.) Gaertn. obtained by Soxhlet and supercritical fluid extraction and unsaponifiable residue of extracted plant material.

Soxhlet extraction SFE

Experiment Yield (%) Non-saponifiable part (%) Experiment P [bar]/T [◦C] EtOH (%) Yield (%) Non-saponifiable 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 50.70

3 450/40 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 38.45

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OH (0.50 g KOH + 20.0 mL 96% EtOH) on water bath for 1 h. Thenolutes were cool down by adding deionised water and extractedhree times with petroleum ether. The unified organic phasesere washed with water to neutral. Petroleum ether was des-

ccated with Na2SO4 and evaporated in vacuum after filtration.he non-saponified residues were weighed and stock solutionsere prepared of them by dissolving the samples in HPLC gradeethanol.

.2.6. Identification of triterpenes and phytosterols in bark of A.lutinosa (L.) Gaertn.

Identification of compounds was performed by thin-layerhromatography (TLC), gas chromatography–mass spectrometryGC–MS) and liquid chromatography–mass spectrometry (LC–MS).he TLC was used for preliminary qualitative and rough quantita-ive comparison of the samples. The experimental conditions werehe same as previously described [21].

.2.6.1. GC–MS conditions. For derivatisation, 100 �L aliquot ofaponified extracts dissolved in 5 mL chloroform were transferrednto septum-capped vials and evaporated to dryness. A 100 mL ofis-(trimethylsilyl)-trifluoroacetamide (BSTFA) silylation reagentas added then left standing at room temperature overnight.

hereafter the content of the vial was evaporated to dry andiluted with chloroform to an exact volume. Analysis of sterolomponents was carried out with Agilent 6890N/5973N GC-MSDSanta Clara, CA, USA) system. Gas chromatographic separation waserformed on a 30 m × 250 �m × 0.25 �m film thickness capillaryolumn (Supelco 28471-U, SLB-5ms 5% phenyl-methyl siloxane,igma–Aldrich). The oven temperature was maintained at 120 ◦Cor 1.0 min, then increased to 270 ◦C at 20 ◦C min−1 (20 min isother-

al); then further increased to 300 ◦C at 10 ◦C min − 1 and held for min. Analysis time was 36.5 min. The carrier gas was high-purityelium at a constant flow rate of 1.0 mL min−1 throughout the run.he injection volume was 1.0 �L. The inlet was operated in pulsed

Fig. 1. Overall extraction curves of Alnus glutinosa bark supercritical extracts under

300/60 10 3.81 33.00

splitless mode. The injector was programmed from 140 to 300 ◦Cat 720 ◦C min−1 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 (41–500 amu). The temperature of the mass-selectivedetector was 300 ◦C. The main components were identified by com-parison of their retention time and recorded mass spectra withthose of standards. Mass spectra of known literature data and theNIST 05 library were also consulted.

2.2.6.2. LC–MS conditions. Sample stock solutions were preparedby dissolving the saponified extracts in LC super gradient grademethanol at different concentrations (1.6–5.9 mg mL−1). For thechromatographic separation an Agilent 1100 LC system (G1379Adegasser, G1312A binary gradient pump, G1329A autosampler,G1316A column thermostat and G1315C diode array detector)was used (Agilent Technologies, Waldbronn, Germany). Separa-tion was achieved on a Kinetex C18 (100 mm × 2.1 mm, 2.6 �m,pore size: 100 A) column (Phenomenex, Torrance, CA, USA), main-tained at 40 ◦C. Eluent A was water, eluent B was acetonitrile. Thefollowing gradient elution program was applied at a flow rate of0.2 mL min−1; 0 min: 65% (v/v) B, 3 min: 80% (v/v) B, 10 min: 100%(v/v) B, 12 min: 100% (v/v) B, 13 min: 65% (v/v) B, 16 min: 65% (v/v) B.Chromatograms were acquired at 207 and 340 nm, UV spectra wererecorded between 200 and 400 nm. Injection volume was 2 �L.

Mass spectral analyses were performed with an Agilent 6410triple quadrupole system equipped with ESI ionsource (AgilentTechnologies, Waldbronn, Germany). ESI conditions were as fol-lows: temperature: 350 ◦C, drying gas flow: 8 L min−1, nebulizerpressure: 30 psi, capillary voltage: 3500 V, fragmentor voltage:120 V. Full mass scan spectra were recorded in positive ionisationmode over a range of m/z 50–700 (scan time: 800 ms). CID mass

spectra of compounds were recorded applying different collisionenergy values between 10 and 30 eV in order to obtain as muchstructural information as it was possible. The Masshunter B.01.03software was used for data acquisition and qualitative analysis.

different conditions: (A) with supercritical CO2 and (B) with ethanol addition.

58 A. Felföldi-Gáva et al. / J. of Supercritical Fluids 61 (2012) 55– 61

Fig. 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 identified and/or characterised by GC–MS.

Rt (min) Identified component Mean percentage distribution (%)

n-Hexane extract Ethanolic extracts SFE extracts SFE + EtOH extracts

25.32 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 2.43

Fig. 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. Felföldi-Gáva et al. / J. of Supercritical Fluids 61 (2012) 55– 61 59

Table 3List of components in Alnus bark extracts identified and/or characterised by LC–MS method.

Rt (min) Identified component Basic ion (m/z) SFE experiment Soxhlet extraction

1 4 6 7 EtOH n-Hexane

4.68 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; ?: identified theoretically based on literature data For numbering of the experiments,see Table 1.

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dentification of the components was carried out by the compre-ensive interpretation of their respective chromatographic andpectroscopic (MS, MS/MS and UV) data and by the comparisonith those of literature data and authentic standards.

.2.7. Quantification of lupane-type triterpenes in bark of A.lutinosa (L.) Gaertn.

Reverse phased high-performance liquid chromatography (RP-PLC) method was applied for quantitative analysis of lupane-type

riterpenes in Alnus bark extracts. Sample stock solutions wererepared by dissolving the saponified extracts in methanol at dif-erent concentrations (3.4–17.96 mg mL−1) and filtered through aingle use syringe filter (0.20 �m, Minisart RC 15, Vivascience AGermany). The solutions were stored in refrigerator and brought

o room temperature and diluted before use. Compared to our pre-ious RP-HPLC conditions referred above [21], we made a slightodification in composition and ratio of the mobile phase. Thuse managed to detect betulinic acid and lupeol as well. Isocratic

lution was performed with mobile phase acetonitrile/0.2% (v/v)cetic acid in water (87/13, v/v%) with a 1.0 mL min−1 flow rate.he volume of injection was 20 �L, detection wavelength was set at05 nm. Analysis took 30 min; the column temperature was main-ained at 25 ◦C. The chromatographic peaks of betulin, betulinic

cid and lupeol in the samples were confirmed by comparing theiretention time with the respective standards and by standard addi-ion as well. Each standard and the sample solutions were injectednto the chromatograph and peak areas were recorded. Amount of

able 4alculated linear regression parameters for calibration curves.

Analyte A B

Betulinic acid 198,402.26 114,8887.34

Betulin 203,136.22 836,041.40

Lupeol 285,572.35 1,444,742

iment 7). Numbering of peaks are the following: 1 = betulinic acid, 2 = ursolic acid,

betulin, betulinic acid and lupeol in the extracts were calculated byexternal standard calibration.

3. Results and discussion

3.1. Extraction yields

Process characteristics, such as extraction yield, solubility andselectivity are the function of pressure and temperature. Theextraction yields (mass of the extract/mass of the dry matter)obtained with supercritical CO2 at different operational conditions(300 and 450 bar, 40 and 60 ◦C, ethanol addition) and with n-hexaneand ethanol with Soxhlet extraction are compared in Table 1. Yieldof the SFE was comparable to the n-hexane extraction, because ofthe similar dissolving capacity of the supercritical carbon dioxide(sc-CO2) and n-hexane. At the same time, the yield of alcoholicextract was approximately 20 times higher than the SFE or n-hexane extracts. It can be explained by that the ethanol dissolvedthe whole unwanted soluble polar compounds, while the sc-CO2and n-hexane, as non-polar solvents, dissolved the non-polar com-ponents only; thus resulting more selective extraction.

Overall extraction curves (OEC) representing the supercriticalfluid extraction yields of Alnus bark under different conditions as afunction of CO2 usage (kg CO2/kg dry material) are shown in Fig. 1.

The shape of the extraction curves indicates that at different stagesof the extraction, different mechanisms control the mass transfer.

In SFE, the process yield and selectivity is determined by tem-perature and pressure. The solvent density is influenced by their

r2 Range (mg mL−1) Rt (min)

0.9987 0.073–0.170 5.100.9980 0.073–0.113 6.010.9989 0.102–0.170 25.93

6 percritical Fluids 61 (2012) 55– 61

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Table 5Betulin, betulinic acid and lupeol content of Alnus glutinosa bark extracts.

Experiment Triterpene content (g/100 g extract)

Betulinic acid Betulin Lupeol

Soxhlet extractionn-Hexane n.d. 1.2 6.22EtOH n.d. 0.062 n.d.

SFE1 n.d. 2.69 13.502 n.d. 2.90 8.603 n.d. 1.92 14.334 n.d. 2.03 7.015 n.q. 1.34 0.716 1.55 3.57 0.627 2.95 3.17 2.985

0 A. Felföldi-Gáva et al. / J. of Su

lteration which affects the solubility of solute in solvent phase andonsequently the extraction yield and selectivity. From Fig. 1A it cane seen that pressure alone had little influence on the yield howeverhe interaction of pressure and temperature is not negligible.

While at lower temperature similar yields were obtained atoth pressures, almost 0.6% enhancement in extraction yield waschieved with increase in pressure at 60 ◦C. This pressure effect cane explained by the increase in the solvent density which enhanceshe solubilisation.

The temperature effect in the process yield is complex due to theombined effect of solvent density and solute vapour pressure. Theolute vapour pressure increases with temperature raising the solu-ility, while temperature has opposite effect on the solvent densitynd solubility. In Fig. 1A it is seen that the yield increased withemperature at both studied pressure however this enhancementas more significant at 450 bar (from 1.51% to 2.56%) compared to

he same at 300 bar. This pattern suggests that at higher pressurehe dominant effect which influences the yield is the vapour pres-ure. We also investigated the effect of co-solvent and observed thatddition of ethanol further increased the yield. Ethanol additionacilitates the dissolvation of more polar compounds by enhancingolvent polarity which explains the increase in the yield. Fig. 1Bhows the extraction curves under different temperature condi-ions at 300 bar after applying ethanol as co-solvent. The higher itsoncentration was the higher yield was achieved. Difference fromhe aspect of temperature can also be observed. These experiencesre supported by Fig. 2.

From the scatter plots it can be seen that the temperature andthanol content had determinant effect on the yield. Based on thebove results, experiment 8 (300 bar/60 ◦C/10% ethanol) proved toe optimal for Alnus bark among the studied SFE conditions withespect to extraction yield and rate.

.2. Ratio of non-saponifiable part

Samples were saponified to be cleaned from interfering oilyaterials. In these unsaponified residues the phytosterols and

riterpenes were present in free form. The ratio of the non-aponified part in the extracts is shown in Table 1.

The highest amount of non-saponifiable residue was obtainedy supercritical fluid extraction when temperature was 40 ◦C andressure was set to 300 bar and 450 bar, respectively. This ratio waslightly less in case of the same pressures but higher temperatureexperiments 2 and 4) and further decrease was observed by addi-ion of ethanol as co-solvent. The n-hexane extract was comparableo supercritical CO2 extracts due to the similar solvent powers. Wean observe that rather temperature than pressure had influence onhe ratio of non-saponifiable matters. The higher the temperatureas, the ratio of these matters decreased. Also, addition of polar sol-

ents increased the amount of polar or bonded compounds in thextract that explain the decrease in the ratio of free apolar compo-ents to be present in the residue. As a result, the less unsaponifiedatter was extracted from the ethanolic extract.

.3. Identification of components in Alnus bark

Identification of components in the Alnus bark extracts was per-ormed with TLC, LC–MS and GC–MS methods. TLC gave us theossibility for a rapid evaluation of the qualitative and rough quan-itative composition profile of plant extracts. Betulin, betulinic acid,upeol and �-sitosterol were identified in the samples by compari-on of their retention times with those of the respective standards.F values were as described previously [21].

8 1.74 3.30 1.42

n.d.: not deteced, n.q.: identified but not quantified.

3.3.1. Identification with GC–MSChemical composition profile of the extracts was investigated

by GC–MS. The percentage data of the total ion current chro-matograms (TIC) were calculated. The percentile values of thecomponents represent their distribution in the plant bark. Com-pounds identified by TLC were justified by GC–MS results. Besidebetulin, betulinic acid, lupeol and �-sitosterol, we identified twofurther lupeol derivatives, namely lupenone and lupenylacetate;as well as other triterpenes like taraxerone and simiarenol. Themean percentage distribution rate and retention time of the singleconstituents are presented in Table 2.

In Fig. 3, TIC chromatograms of TMS derivatives of compoundspresent in two extracts (n-hexane, SFE) are shown.

3.3.2. Identification with LC–MSTwo Soxhlet and four SFE extracts were subjected to LC–MS

analysis which resulted in the identification of six pentacyclictriterpenoids as shown in Table 3. For identification of betulin andbetulinic acid, authentic standards were used. As identification oflupeol would have been required different instrumental conditionsfor what we had no access, this compound was not subjected intothis analysis.

Betulin as a major compound occurred in all examined sam-ples. �-Amyrin was identified mainly in the apolar SFE extracts,while betulinic acid and ursolic acid were detected rather in thosesamples where modifier was applied. Uvaol and betulinic aldehydewere identified as minor components based on literature data.

LC-DAD profile of A. glutinosa (L.) bark SFE extract is shown inFig. 4.

3.4. Betulin, betulinic acid and lupeol content of the extracts

An HPLC-UV method developed by Zhao et al. [20] was adaptedto determine betulin, betulinic acid and lupeol content in alder barkextracts. To determine linearity, the calibration curve of betulin,betulinic acid and lupeol standard were drawn and evaluated byregression analysis. From the stock standard solutions a mixturewas prepared which contained all three standards in the sameconcentration (0.5 mg mL−1). Seven concentrations of a freshly pre-pared working solution of this mixture were injected in the rangeof 0.046–0.170 mg mL−1. Three replicate injections at each con-centration level were performed. The calculated linear regressionparameters for calibration curves are given in Table 4, Y = Ax + B.

The betulin, betulinic acid and lupeol content of A. glutinosa

extracts is presented in Table 5.

Among the three examined components, lupeol occured in mostsignificant amount in the extracts. The highest lupeol content wasmeasured at 450 bar/60 ◦C (experiment 4). Decreasing the pressure

A. Felföldi-Gáva et al. / J. of Supercri

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ig. 5. HPLC-UV chromatogram of Alnus glutinosa L. (Gaertn.) supercritical extractexperiment 5). Numbering of peaks are the following: 1 = betulinic acid, 2 = betulin,

= lupeol.

nd temperature, as well as modifier usage negatively influencedts recovery. Regarding the content of betulin, we observed thatts recovery was decreased at higher pressure when using pureO2 only. Increase in temperature and addition of ethanol howevernhanced its recovery. The most optimal extraction condition ofetulin was 300 bar/60 ◦C + 5%EtOH (experiment 5). Due to its acidicharacteristic, betulinic acid was detected in measurable amounthen ethanol was used. The most optimal extraction condition was

00 bar/40 ◦C by addition 10% of ethanol as co-solvent (experiment). Increasing the temperature or decreasing the amount of ethanolesulted less extraction yield.

We need to mention two unidentified but detected componentsue to their significant pattern in the samples. Their mean retentionime was approximately 7.0 and 20.0 min, respectively. Comparingheir peak area in each sample, we observed that amount of thearlier eluting compound extensively increased in ethanolic extractnd in SFE experiment performed at 450 bar/40 ◦C, while the peakrea of the other compound was significantly high in the same SFExtract. Their identification requires further studies. An example ofPLC-UV chromatogram of alder bark extract is presented in Fig. 5.

. Conclusions

Supercritical fluid extraction of alder bark was performed andhe effect of pressure, temperature and modifier addition on extrac-ion yield and composition of the extracts were studied. Evaluationf experimental results showed that interaction of pressure andemperature as well as ethanol addition improved the extractionield. An amount of 1.04% enhancement was achieved by increas-ng the temperature at 450 bar, while this ratio was 0.2% only at theower pressure. Similar trends were experienced by increasing the

odifier amount. The recovery of selected lupane-type triterpenesas also influenced by the extraction conditions applied. Based

n the above observations, the optimum extraction condition wasefined as 300 bar/60 ◦C + 10% EtOH where the highest yield waschieved compared to that obtained by Soxhlet extraction with-hexane (3.81% vs 2.56%). Highest amount of betulin, betulinic

cid and lupeol was 3.57; 2.95 and 14.33 g/100 g extract, respec-ively, depending on the applied SFE condition. Although the yieldf ethanolic Soxhlet extraction was about 10 times higher, the con-entration of the active constituents was very low. We can conclude

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tical Fluids 61 (2012) 55– 61 61

that SFE is a capable method for obtaining valuable triterpenoidsfrom the bark of A. glutinosa (L.) Gaertn.

Composition of extracts was investigated with chromatographicmethods. We identified A. glutinosa as a valuable raw materialfor pharmacologically active pentacyclic triterpenoids. Total of 11compounds were identified. In addition to previously describedbetulin, betulinic acid, lupeol and �-sitosterol, presence of betulinicaldehyde, lupenone, lupenyl-acetate, �-amyrin, uvaol, ursolic acid,simiarenol and taraxerone was described first in the bark of A. gluti-nosa (L.) Gaertn.

Acknowledgement

This work was supported by GVOP 3.1.1.-2004-05-0397/3.0.

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