萃取技术比较

Journal of Chromatography A,1105(2006)
115–118
Comparison of soxhlet,ultrasound-assisted and pressurized liquid
extraction of terpenes,fatty acids and Vitamin E from
Piper gaudichaudianum Kunth
Val´e ria Flores P´e res a ,b ,Jenifer Saffia ,Maria Inˆe s S.Melecchi c ,Fernanda C.Abad c ,
Rosˆa ngela de Assis Jacques c ,Migdalia M.Martinez b ,Eniz Conceic ¸˜a
o Oliveira d ,Elina B.Caram˜a o c ,∗
a
Laborat´o rio de Gen´e tica Toxicol´o gica,ULBRA,Canoas,RS-Brazil b Universidad de La Habana,Cuba
c
Instituto de Qu´ımica,UFRGS,Porto Alegre,RS,Av.Bento Gon¸c alves,9500,90160-970,
Porto Alegre,RS,Brazil
d Center III,Univates,Lageado,RS,Brazil
Available online 16August 2005
Abstract
This paper describes a comparative study of extraction methods of terpenes (terpenic alcohols and phytosterols),fatty acids and Vitamin E from leaves of Piper gaudichaudianum Kunth.The analysis of extracts was done by gas chromatography with mass spectrometric detection.The identification and quantification was made by co-injections of the extract with certified standards.The use of pressurized liquid extraction (PLE;Dionex trade name:ASE,for accelerated solvent extraction)decrease significantly the total time of extraction,the amount of solvent and the manipulation of sample and solvents in comparison with soxhlet (SE)and ultrasound-assisted (USE).In addition,PLE was more effective for the extractions of terpenes (terpenic alcohols and phytosterols),fatty acids and Vitamin E.©2005Elsevier B.V .All rights reserved.
Keywords:Piper gaudichaudianum Kunth;Terpenes;Fatty acids;Vitamin E;Pressurized liquid extraction;Soxhlet;Ultrasound-assisted extraction
1.Introduction
The first step in the qualitative and quantitative analysis of medicinal plant constituents is the extraction and it is an important step in studies involving the discovery of active compounds of plant materials.Ideally,an extraction proce-dure should be exhaustive with respect to the constituents to be analyzed,rapid,simple,inexpensive and –for routine analysis –amenable to automation [1–3].
Usually,the traditional techniques require long extraction time and have low efficiency.Moreover,many natural prod-ucts are thermally unstable and could be degraded during the extraction if the temperature be increased.
∗
Corresponding author.Tel.:+555133369284;fax:+555133167304.E-mail addresses:jenifer.saffi@.br (J.Saffi),elina@ufrgs.br (E.B.Caram˜a o).Traditionally the extraction of terpenes (terpenic alcohols and phytosterols),fatty acids and Vitamin E is performed by maceration,hydrodistillation and soxhlet extraction [4–8].These techniques are often time-consuming and require large volumes of organic solvent.The use of large volumes of solvent implies in additional costs,due to the fee associated with the purchasing and disposal of toxic solvents,and environmental hazard.In the last decade,alternative extraction techniques that reduce the volume of solvent and the time of extraction have been considered.For example,some of the newer techniques are ultrasound-assisted extraction (USE),pressurized liquid extraction,microwave extraction and supercritical fluid extraction [9–12].
Pressurized liquid extraction operates at high pressures and temperature above point of the boiling point of the organic solvent.The use of higher temperature increases the
0021-9673/$–see front matter ©2005Elsevier B.V .All rights reserved.doi:10.1016/j.chroma.2005.07.113
116V.F.P´e res et al./J.Chromatogr.A1105(2006)115–118
ability of solvent to solubilize the analyte,decreases the vis-cosity of liquid solvents,allowing better penetration of the solvent into the matrix.The use of higher pressure facilitates the extraction of the analytes from samples by improving the solvent accessibility to the analytes that is trapped in the matrix pores.
The use of PLE decreases significantly the total time of treatment and in addition,this extraction method can be more effective and selective by changing some parameters like tem-perature,time,cycles and solvent[13–17].
Several species of the Piperaceae family have been stud-ied with respect to the presence of compounds with sig-nificant biological and pharmacological activities[18–23]. Piper gaudichaudianum Kunt is the species more abun-dant in Brazil,occurring from Northwest to South Brazil. This plant is known by Pariparoba,Paripaioba,Muta,Iab-orandi or Jaborandi.Their leaves are used in popular medicine as anti inflammatory and in the relief of toothache [24–29].
The goal of this work is to compare classical extraction techniques of plants(Soxhlet)with unconventional solid liq-uid extraction method(Ultrasound-assisted extraction and pressurized liquid extraction)of leaves from Piper gau-dichaudianum Kunth with respect of amount of extracted material and chemical composition of the extracts.It was used petroleum ether and ethanol in the extractions for ver-ifying the effect of the solvent polarity in the process.The analysis of the extracts was made by gas chromatography with mass spectrometric detection(GC–MS).
2.Experimental
2.1.Plant material
The leaves of Piper gaudichaudianum Kunth were col-lected in the municipal district of Riozinho-RS,in a delimited area of Rain forest,in April of2003.The plant was identified by the specialist botanic Dr.Daniel Ruschel(Bio-sciences Institute,Department of Botanical,UFRGS,RS,Brazil),and the voucher specimen(ICN:128412)was deposited in the herbarium of UFRGS.The leaves were dry at35◦C until constant weight,and stored in dark bags to protect them from humidity and light.
2.2.Equipments
The GC–MS system consisted of a Shimadzu,model QP5050-A.The pressurized liquid extraction was per-formed with an ASE TM300equipment(Dionex,Sunny-vale,CA,USA).Ultrasonic extractions were performed with an ultrasound cleaning bath–Fisatom Scientific-FS14H (Frequency of40KHz,nominal power90W and24cm×14cm×10cm internal dimensions water bath).Soxhlet extractions were performed with a120ml siphon system (Vidrolabor).2.3.Chemicals
All the solvents(purchased from Merck)were bi-distilled and stored in darkflasks.Anhydrous sodium sul-fate(Sigma–Aldrich)was of analytical grade and treated at 300◦C before use.Some standards such as nerolidol,palmitic acid,methyl palmitate,methyl linoleate,methyl stearate, methyl lignocerate,phytol,squalene,Vitamin E,stigmasterol and␤-sitosterol were acquired from the Sigma–Aldrich. 2.4.Gas chromatographic procedure
All the extracts were analyzed by GC–MS on a Shi-madzu model QP5050-A with a split-splitless injector (split mode ratio1:20)at280◦C and the interface at 300◦C.An OV-5(Ohio Valley Company)(cross-linked5% phenyl–methyl–silicone,30m×0.25mm×0.25␮m)col-umn was employed.The oven temperature was programmed to start at150◦C,heated to210◦C at5◦C/min and heated to 300◦C at10◦C/min,staying8min in this temperature.All of the extracts were identically treated and analyzed using equal conditions.The analyses of the extracts were performed by injecting1␮L of each extract directly into the GC–MS.The extracts with ethanol were previously derivatized with BF3 in methanol.This procedure prevents the loss of not volatile compounds(like acids and high molecular weight alcohols) in the injector port.Quantification of terpenes(terpenic alco-hols and phytosterols),fatty acids,and Vitamin E by GC–MS was made by comparing the areas of their chromatographic peak versus the perylene(internal standard)area with the areas of standards(when the fatty acid standard was not avail-able,quantifications were made based on the response of the standards having the same molecular weight as the target compound).
2.5.Extraction procedures
2.5.1.Soxhlet extraction procedure(SE)
30.0g and200ml of solvent were refluxed by48h using Soxhlet apparatus.Petroleum ether and ethanol were used for comparison purposes.The extracts werefiltered and concen-trated under reduced pressure until dryness.Three hundred microliters of a100ppm internal standard(perylene)solu-tion(in ethyl acetate)were added to each extract before the chromatographic analysis.The experiment was performed in triplicate.
2.5.2.Ultrasonic extraction(USE)procedure
Three grams of dried leaves from Piper gaudichaudianum Kunth were extracted with90mL of the same above solvents in an ultrasonic apparatus.The temperature of the bath was controlled at30◦C by changing the water at each30min.
Particle size(ground leaves)and extraction time(60min for ethanol and180min for pentane)were previously opti-mized for this technique[30].
V.F.P´e res et al./J.Chromatogr.A1105(2006)115–118117
The extracts werefiltered,concentrated under reduced pressure until dryness and received300␮L of100ppm inter-nal standard solution before the analyses.The experiment was also performed in triplicate.
2.5.
3.Pressurized liquid extraction(PLE)procedure
A weighed amount(3g)of the sample was placed into the sample cell(100mL)and the cell was loaded into the carousel of the ASE TM300system.Extractions were per-formed byfilling the cell with the solvent before heating, using approximately80mL of solvent in each experiment. The same solvents of the previous experiments(SE and USE) were used.The extraction pressure(1500psi)was imposed by the ASE300apparatus.Particle size(ground leaves), extraction temperature(85◦C for ethanol and150◦C for pen-tane)and extraction time(10min for both solvents)were previously optimized for this technique[31].Each extraction was performed with one cycle of extraction and maintain-ing5min in the static extraction mode.The extracts were concentrated under reduced pressure until dryness.To the extract,300␮L of100ppm internal standard(perylene)were added for quantification.The experiment was performed in triplicate.
3.Results and discussion
3.1.Mass yield comparison
Table1shows the comparison of mass yield(g of extract/100g of sample)for the three techniques and both solvents,in the best conditions.
These results show that PLE produced the higher yield in the extraction and considerable differences were confirmed in the solvent extractive capacity,as expected by their prop-erties.Ethanol was the only solvent that extracts all the studied compounds,and for this reason it was chosen as Table1
Mass yield(%)of extraction of leaves the Piper gaudichaudianum Kunth using three extraction procedures and two extractor solvents
Technique Mass yield(%)Time b Temperature(◦C) Yield SD%a
Petroleum ether extraction
USE 1.900.07180min30 PLE 3.840.2210min85 SE 2.790.1248h–
Ethanol extraction
USE 4.360.1260min30 PLE25.520.9520min150 SE20.340.2048h–
a Percent standard deviation.
b Average time consuming of the procedure,non-including the concentra-tion step.
the best extractor solvent.Some compounds(palmitc and stearic acids,stigmasterol,Vitamin E and squalene)were better extracted by petroleum ether.The advantage of PLE is increased if it is considered the reduction in the extraction time and in the solvent consumption.
3.2.Chemical composition of the extracts
Table2shows the comparison of the amount of individ-ual compounds in each extract obtained for each solvent and extraction technique.It is possible to note that the major com-pounds were nerolidol,phytol and palmitic acid.
The major concentration of nerolidol was achieved in the soxhlet extraction with petroleum ether while the major con-centration of palmitic acid was found in the PLE extract with petroleum ether.Phytol appears in higher amount in the PLE ethanolic extract.
Sonication with ethanol was the better technique to extract linoleic and9,12,15-octadecatrienoic acids.Ligno-ceric acid was better extracted by PLE with ethanol.The other compounds monitored(squalene,Vitamin E,stearic
Table2
Chemical composition of pentane and ethanol extracts obtained using three extraction procedures
Compounds Concentration(mg of the compound/kg of sample)
Petroleum ether extracts Ethanol extracts
SE USE ASE®SE USE ASE®
Yield SD%a Yield SD%Yield SD%Yield SD%Yield SD%Yield SD% Nerolidol3435.62 1.79271.18 2.012144.51 2.9085.32 1.8460.96 1.2080.07 1.29 Palmitic acid2853.910.851359.24 1.503793.93 3.10688.30 1.062353.42 2.01711.83 1.05 Linoleic acid––––––108.940.19385.10 1.72124.050.02 9,12,15-Octadecatrienoic acid––––––236.89 1.78809.09 2.72295.50 1.62 Phytol254.98 1.6982.000.41309.140.981935.98 2.101622.20 2.012081.27 2.08 Stearic acid956.560.15767.45 1.521234.54 1.371649.03 1.981545.920.031777.04 1.05 Lignoceric acid––––––65.880.0553.560.0174.540.03 Squalene80.160.05 4.450.0298.12 1.8925.830.1220.510.1230.740.01 Vitamin E432.310.1468.840.16576.04 2.49290.72 1.29223.29 1.52346.74 1.39 Stigmasterol963.980.25219.43 1.071205.31 3.01183.610.03107.07 1.02232.04 1.04␤-Sitosterol106.920.6246.510.04194.28 1.8958.920.0126.030.0876.970.05 a Percent standard deviation.
118V.F.P´e res et al./J.Chromatogr.A1105(2006)115–118
acid,stigmasterol and␤-sitosterol)appear in higher amount in the PLE extract with petroleum ether.
It is also interesting to note that linoleic acid,9,12,15-octadecatrienoic acid and lignoceric acid was detected only in the ethanolic extracts.
We think that the interaction between unsaturated acids with polar solvents(like ethanol)is greater than with not polar solvents(like petroleum ether).On the other hand,the amount of lignoceric acid in the sample was very low(by comparing with the other fatty acids)and this can explain its extraction only when ethanol was used.
The ultrasonic waves are more effective in the retreat of these acids starting from the organic matrix,probably because they promote larger agitation(cavitation)in the system,pro-moting the more effective breaking of the interactions.These more effective interactions could be those between the double bond and the material of the plant.
4.Conclusion
The utilization of PLE has proven to be a much simpler and more effective than the conventional USE and SE extraction methods for obtaining active compounds from plants.The PLE can be carried out at higher temperature and pressure with economy in time.
Comparison of these extraction methods revealed that they produce extracts with similar qualitative characteristics,how-ever quantitative differences.
GC–MS and the utilization of certified standard allowed the identification and quantification of many compounds(ter-penes,fatty acid,phytosterols and Vitamin E)present in extracts of Piper gaudichaudianum Kunth.Some of these compounds already have been reported in genus Piper,but most of them are reported for thefirst time in this species. Acknowledgements
The authors thank to FAPERGS,CNPq and FINEP for the support of this work.
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