J. of Supercritical Fluids 61 (2012) 55 61
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The Journal of Supercritical Fluids
jou rn al h om epage: www.elsev ier .com
Anik Fe ala Semmelweis Ub Gedeon Richtc Budapest Uni ess En
a r t i c l
Article history:Received 20 JuReceived in reAccepted 5 Oc
Keywords:Alnus glutinosaPentacyclic triSupercritical GCMSLCMSRP-HPLC
on ofin orded byere idrms ole in
SFE xane ct, ret yielf trite
Importance 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 [6hepatoprot
Corresponnosy, 1085 Bu
treat 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 represent
e 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 to
ding 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 extract
triterpenoids 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 glutinosa
lfldi-Gvaa,b,, Szabolcs Szarkaa, Bla Simndic, Bniversity, Department of Pharmacognosy, 1085 Budapest, lloi t 26, Hungary
er Plc, 1103 Budapest, Gymroi t 19-21, Hungaryversity of Technology and Economics, Department of Chemical and Environmental Proc
e i n f o
ne 2011vised form 4 October 2011tober 2011
(L.) Gaertn.terpenoidsuid extraction
a b s t r a c t
Supercritical 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
zs Blazicsa, Blanka Simonc, gnes Krya
gineering, 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 61
experimental 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%ginsenoside
Some au(Calendula extraction The extracdiol esters (T = 29931oresin by Can increasinincrease in complex dusure that rephysical meevaluated tmodels. Thelogistic mod
SFE alsoous oilseedroselle seedide extractiof 200400ical uid in roselle scholesterolvariable withe extractephytosterol40 C, a highrate of 20 m
Betulin by Zhang etlike modiand extractmum betul1.5 mL g1 bthe extracti
In our pan alternatiin addition plant in varus a good poof differenttion of targSolvent extable SFE conthe plant, fo
The 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 used
partto thre dacopainede divalu00 e0 = 1
of 0tulinrt RC
Soxh extrient illede cotracless. tion
Supe rawre pred b00 g (essund 6
10%n wraturout and
on used t
mentountount 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 marigold
ofcinalis). 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 to
solvent 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 methods
r 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 purchased
in HPLcation were p
biggerPrior als wePharmthe grcle sizwere eR(x) = 1were d
of an intrationand beMinisastoredThe -at a co
at amband muntil thwas excolourdistilla
pressu(delivewith 8tion pr40 C a5% andadditiotemperate ablected carrieddecrea
Theviscosiwith apeasy spas a coextrac
2.2.5. In o
experiAn amdies was deionised by Millipore Direct Q5 water puri-m (USA). Betulin, betulinic acid and lupeol standardsased from Biomarker Kft. (Hungary).
ration 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 mg
dual 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 were
frigerator 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% EtOH
as 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 alcoholic
A. Felfldi-Gva et al. / J. of Supercritical Fluids 61 (2012) 55 61 57
Table 1Extraction yield of Alnus glutinosa (L.) Gaertn. obtained by Soxhlet and supercritical uid extraction and unsaponiable residue of extracted plant material.
Soxhlet extraction SFE
Experiment 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 50.70
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 38.458 300/60 10 3.81 33.00
a 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 (L
Identicchromatogr(GCMS) anThe TLC wative comparthe same as
126.96.36.199. GCsaponied einto septumbis-(trimethwas addedThereafter diluted witcomponent(Santa Claraperformed column (SuSigmaAldrfor 1.0 min,mal); then f5 min. Analhelium at aThe injectio
splitless 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-selective
of f sta
. LColvinnol aatogrer, GA coed (as acze: 1
at 40ing g
, 12 matoged bess spquadlogi
tempre: 3Full over
of c valural ire w
Fig. 1. Ovponied residues were weighed and stock solutionsred of them by dissolving the samples in HPLC grade
ication 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...