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Contents lists available at ScienceDirect

Chemical Engineering Research and Design

j ourna l h omepage: www.elsev ier .com/ locate /cherd

ntensification of Hypericum perforatum L. oilsolation by solvent-free microwave extraction

ohamed Abdelhadia, Alice Meullemiestreb, Antony Gelicusb,icha Hassania, Sid-ahmed Rezzougb,∗

Laboratoire des molécules bio actives et valorisation de la biomasse, Ecole Normale Supérieure BP 92 Kouba Alger,6308, AlgeriaLaSIE, UMR CNRS 7356, Université de La Rochelle - Pôles Sciences et Technologie, Bâtiment Marie Curie,venue Michel Crépeau, 17042 La Rochelle, France

a b s t r a c t

Solvent-free microwave extraction (SFME) of oil from Hypericum perforatum L. and its antioxidant activity were inves-

tigated and compared to a conventional hydrodistillation (HD) technique (Clevenger apparatus). A central composite

design was applied for evaluating the influences of irradiation power, irradiation time and moisture content of the

plant on extraction yield. Under optimal conditions defined by the experimental design, a yield of 0.365 g/100 g extract

was achieved. For SFME extraction process, 69 compounds representing 96.6% of the oil were identified including two

major groups: total oxygenated compounds and sesquiterpenes hydrocarbons accounted up to 66% of oil. Thus, the

compositions in these two groups were also considered as responses. The results showed that irradiation power and

moisture content had a high significant effect on all responses while irradiation time has a significant effect only

on extraction yield. IC50 values were 462.36 and 40,042.87 �g/mL respectively for SFME and HD suggesting that SFME

represents an interesting alternative protocol for isolation of Hypericum perforatum L. oil.

© 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Solvent free microwave extraction; Hypericum perforatum L.; Hydrodistillation; Essential oil; Antioxidant

activity; Response surface methodology

plant materials are enormous and usually have some dis-

. Introduction

he genus Hypericum L. belongs to Hypericaceae family whichncludes approximately 560 species distributed in 6–9 gen-ra according to the 3rd version of botanical classification,stablished by the Angiosperms Phylogeny Group (APG, 2009).ypericum perforatum L. (Hypericaceae) commonly known ast. John’s wort is an herbaceous perennial plant well-knowns medicinal plant since antiquity (Guedes and Franklin, 2012).ost of investigations on Hypericum perforatum L. focused on

he anti-depressant properties of isolated substances (Saddiqet al., 2010) and their anti-inflammatory, anti-microbial andnti-proliferative activities (Bagdonaite et al., 2012). Accord-

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ng to Crockett and Robson (2011), this plant becomes one of

∗ Corresponding author at: Université de La Rochelle - Pôles Science7042 La Rochelle, France. Tel.: +33 546458615; fax: +33 546458241.

E-mail address: sarezzou@univ-lr.fr (S.-a. Rezzoug).ttp://dx.doi.org/10.1016/j.cherd.2014.04.012263-8762/© 2014 The Institution of Chemical Engineers. Published by

the best selling herbal ingredients worldwide. The bioactivecompound classes found in Hypericum perforatum L. includenaphthodianthrone derivatives (hypericin and pseudohyper-icin), acylated phloroglucinol derivatives (hyperforin andadhyperforin), and flavonoids such as quercetin, quercitrin,hyperoside, rutin, kaempferol, biapigenin, and amentoflavone(Orhan et al., 2013). On the other hand, numerous of sci-entific studies published on the antioxidant activities ofHypericum perforatum L. (Altun et al., 2013; Sánchez-Munizet al., 2012) but few ones reported on optimization of extrac-tion of essential oil (Hatami et al., 2012; Aybastıer et al.,2013). The technologies used to extract essential oils from

of Hypericum perforatum L. oil isolation by solvent-free microwave014.04.012

s et Technologie, Bâtiment Marie Curie, Avenue Michel Crépeau,

advantages as high temperature, long processing time as in

Elsevier B.V. All rights reserved.

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Raw material :

Hypericu m per foratum L.

Preparation:

Cutt ing in pieces

humidificat ion

hydrodistillation SFME

Residual solid Resid ual solid

Essential oil Essential oil

Energy consumption

SEM

Yield

GC-MS

Antioxidant activity

TPC

Essen �al oil extrac� on Assess ment and analys is

the conventional hydrodistillation or the solvent extractionin which a loss of volatile compounds occurs during sol-vent removal (Rodríguez-Rojo et al., 2012). For a few years,the change in attitude has evolved increasingly to a “green”tendency and there has been an increasing demand for newgreener techniques for essential oil extraction. These newgreen processes will be more environmentally friendly witha shorter extraction times, lesser consumption of organic sol-vent and energy, and minor waste and CO2 emissions, whilemaintaining a high quality of extract. Some intensified extrac-tion methods were investigated including microwave (Jiaoet al., 2013) supercritical fluids (Fornari et al., 2012), D.I.C-assisted extraction (Rezzoug, 2009) or ultrasound assistedextraction (Meullemiestre et al., 2014). Currently, applicationof microwave technology-based methods such as solvent freemicrowave extraction (SFME) becomes highly desirable as avalid alternative to conventional methods and this extractiontechnology was the subject of several studies (Chen et al.,2011; Ma et al., 2012). Hypericum perforatum L. from Mediter-ranean area as that from turkey (S erbetci et al., 2012), Tunisia(Hosni et al., 2010), France (Schwob et al., 2002) or Greece (Giotiet al., 2009) was investigated, but to the best of our knowledgeno study reported on Algerian species. In the present work,extraction of essential oil from Algerian Hypericum perforatumL. was optimized using SFME method. A central compos-ite design (CCD) has been developed to assess the effect ofthree independent variables namely microwave irradiationpower, processing time and moisture content of the planton extraction yield and on the composition of isolated oilin oxygenated compounds and sesquiterpenes hydrocarbons.According to the literature, the sum of these two familiesof compounds varies between 70% and 80% for Bruni et al.(2005) and between 49% and 80% for Radusiene et al. (2005).In our study these proportions varied between 56.6% and66.8% respectively for HD and SFME. A mathematical modelpredicting the yield allowed the optimization of extractionprocess. The constituents of essential oil were qualified andquantified using GC–MS. Total phenol content was deter-mined and antioxidant activities were investigated using twomethods including the measurement of scavenging capacityagainst DPPH radical (2,2-diphenyl-1-picrylhydrazyl) and fer-ric reducing antioxidant power assay (FRAP). The results werecompared with those of hydrodistillation as a conventionalextraction method.

2. Materials and methods

2.1. Plant material and chemicals

The Aerial parts of H. perforatum were collected in June2012, in the region of Blida, at 50 km far from Algiers. Theplant samples were identified by the head of the herbariumof the National Institute of Agronomy (INA, El-Harrach-Algiers-Algeria). Moisture content was measured using ahalogen Moisture Analyzer (Ohaus – MB 35) at 105 ◦C andcorresponded to 12.6% db (dry basis). After storage in a refrig-erated room at 4 ◦C, fresh material was employed in allexperiments. 2,2-Diphenyl-1-picrylhydrazyl hydrate (DPPH),anhydrous sodium carbonate, gallic acid, 2,4,6-tripyridyl-s-triazine (TPTZ), Folin–Ciocalteu’s phenol reagent, hydrochloricacid 37%, Ferric chloride 97% (FeCl3), ascorbic acid, were pur-

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chased from Sigma–Aldrich. Methanol, n-hexane reagents andacetic acid were from Fisher scientific.

2.2. Protocol

In the present study, the experimental design was achievedas illustrated in Fig. 1. Extraction of volatile molecules wasperformed by HD and SFME methods. Each HD operation wasperformed three times in order to test reproducible. SFMEtreatments were analyzed and optimized through statisti-cal study. For HD and for SFME in optimized conditions, theantioxidant activity and total phenolic compounds were eval-uated.

2.3. SFME apparatus and procedure

Solvent-free microwave extraction has been performed on aMilestone NEOS microwave station (NEOS microwave labora-tory oven). It is a multimode microwave reactor 2.45 GHz witha maximum delivered power of 1000 W variable in 10 W incre-ments. Temperature was monitored by an external infraredsensor. In a typical procedure 45 g of dry plant materialwere moistened 24 h prior to extraction by spraying distillatewater to reach the fixed moisture content in the experimen-tal design. Then the samples were subjected to microwaveirradiations in oven cavity, initially at ambient temperature,during a fixed processing time. The microwave heating ofthe water contained inside the raw material allows releasingmolecules constituting isolated oil. This oil was then driven bythe generated vapor. A cooling system outside the microwavecavity permitted to condensate the distillate continuously.Condensed water was refluxed to the extraction vessel in orderto provide uniform conditions of temperature and humidity.Isolated essential oil was dried with anhydrous sodium sul-phate and stored at 4 ◦C in the dark until used. Extraction yieldwas calculated according to Eq. (1)

Extraction yield (%) =(

mass of extracted essential oilmass of dry material

)× 100

(1)

of Hypericum perforatum L. oil isolation by solvent-free microwave014.04.012

Fig. 1 – Protocol of extraction and analysis of hypericumperforatum oil.

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Table 1 – Coded levels for independent variables used indeveloping experimental data.

Coded level

− −1 0 1 +˛

Irradiation power (W) 131.8 200 300 400 468.2Processing time (min) 16.6 20 25 30 33.4Moisture content (%) 43.2 50 60 70 76.8

(axial distance) = 4√N, N is the number of experiments of ortho-gonal design, i.e. of the factorial design. In this case = 1.68. The

2

Ca22dmcalT

2

Tvida

x

wav�

otpotcvfceai

Y

aieTos

Fig. 2 – Responses surfaces showing the simultaneouseffect of irradiation power. Irradiation time and moisturecontent of Hypericum perforatum L. on essential oil yield.

three variables were studied in five levels.

.4. Hydrodistillation apparatus and procedure

onventional hydrodistillation apparatus (Clevenger-typepparatus) according to the European Pharmacopeia (European,012) was employed. A quantity of 100 g of H. perforatum for

L of distilled water was used to perform the hydrodistillationuring 240 min from the first drop of distillate fell until the rawaterial has been completely exhausted. The essential oil was

ollected, dried under anhydrous sodium sulphate, and storedt 4 ◦C for further analysis. Each extraction was performed ateast three times, and a standard deviation was calculated.he extraction yield was calculated according to Eq. (1).

.5. Experimental design

he relationships between response functions and processariables have been established by using a central compos-te design (CCD) as well as the optimal conditions of theeveloped process. The independent variables were codedccording to Eq. (2):

i = Xi − Xi0

�Xii = 1, 3 (2)

here xi and Xi are respectively the dimensionless and thectual values of the independent variable i, Xi0 is the actualalue of the independent variable i at the central point, andXi is the step change of Xi corresponding to a unit variationf the dimensionless value. Irradiation power (P), processingime (t) and initial moisture content (W) were chosen as inde-endent variables. The selected responses were the total yieldf H. perforatum essential oil and the composition of essen-ial oil in two major groups of components: total oxygenatedompounds and sesquiterpenes hydrocarbons. For the threeariables, the design yielded 18 experiments with eight (23)actorial points, six axial points (− and +˛) to form a centralomposite design and four center points for replications andstimation of the experimental error and to prove the suit-bility of the model. Both the coded and actual values of thendependent variables are listed in Table 1.

= ˇ0 +n∑

i=1

ˇixi +n∑

i=1

ˇiixi2 +

∑i /= j

ˇijxixj (3)

The responses Y are related to the coded independent vari-bles xi, xj according to the second order polynomial expressedn Eq. (3), with ˇ0 the interception coefficient, ˇi the lin-ar terms, ˇii the quadratic terms, ˇij the interaction terms.he Fisher’s test for analysis of variance (ANOVA) performed

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n experimental data permitted to estimate the statisticalignificance of the proposed models. Response surfaces as

represented by Figs. 2–4 were drawn by using the analysis designprocedure of Statgraphics Plus for Windows software (Centurionversion).

2.6. GC–MS identification

The volatile compounds were analyzed by gas chromatog-raphy coupled to mass spectrometry (GC–MS). Analyseswere performed on a GC/MS Varian 3900 chromatographcoupled to a Saturn 2100T mass spectrometer using fused-silica-capillary column. The non-polar column was Elite5MS (30 m × 0.25 mm × 0.25 �m film thickness). GC–MS spec-tra were obtained using the following conditions: He (helium)as carrier gas at flow rate of 1 mL/min; split mode1: 20;1 �L as injected volume; 250 ◦C as injection temperature. Theoven temperature program was 60 ◦C for 5 min increasing at2 ◦C/min toward 250 ◦C and held at 250 ◦C during 10 min theionization mode used was electronic impact at 70 eV. Mostconstituents were identified by comparison of their GC lin-

of Hypericum perforatum L. oil isolation by solvent-free microwave014.04.012

ear retention indices (RI), determined with reference to ahomologous series of C5–C32 n-alkanes. The Identification

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Fig. 3 – Responses surfaces showing the simultaneouseffect of irradiation power. Irradiation time and moisturecontent of Hypericum perforatum L. on the composition ofessential oil in total oxygenated compounds.

Fig. 4 – Responses surfaces showing the simultaneouseffect of irradiation power. Irradiation time and moisturecontent of Hypericum perforatum L. on the composition ofessential oil in sesquiterpenes hydrocarbons.

was confirmed by comparison of the mass spectral with thosestored in the MS database (National Institute of Standards andTechnology NIST08 and Wiley libraries) and also by compari-son with mass spectra from literature data (Adams, 2007). Thepercentage composition was calculated from the summationof peak areas of the total oil.

2.7. Assay for total phenolics

Total phenols in H. perforatum essential oil extracts was deter-mined using spectrophotometric Folin–Ciocalteau methodaccording to the literature methods (Orhan et al., 2013) withsome modifications, using gallic acid as standard. Extract solu-tion (0.5 mL) of diluted samples were added into test tubes

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followed by 2.5 mL of Folin–Ciocalteau reagent (10%, v/v). After4 min, 2 mL of a solution of 20% Na2CO3 was added. All test

tubes with the mixture were caped and shaken for 10 s and puton to incubation in a water bath at 45 ◦C for 5 min. Absorbancewas measured after 30 min at 760 nm (Helios Omega UV/VISThermo Scientific Merk and Co. Spectrophotometer) againstblank sample. The same procedure was repeated for allstandard gallic acid solutions (2–200 �g/mL) and a standardcurve was obtained with Eq. (4) (R2 = 0.99):

Absorbance = 0.01674 × Gallic acid (mg/mL) + 0.05458 (4)

of Hypericum perforatum L. oil isolation by solvent-free microwave014.04.012

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ef

2

2IerLssi3aaw

I

weaw

2FaietpfwabarCaaTsFp4mUasa

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MFoaSfiw

The results were expressed as milligrams of Gallic acidquivalents (GAE) per g of dry extract. The analyses were per-ormed in triplicate and standard deviation was calculated.

.8. Antioxidant activity evaluation

.8.1. DPPH (2.2-diphenyl-1-picryl-hydrazyl) essayn this method, the antioxidant activity of the essential oilxtract is evaluated in term of the capacity to scavenging freeadicals of DPPH formed, according to a method described byue et al. (2010). A solution of 4 mg of the radical DPPH dis-olved in 100 mL of methanol was prepared. Then 3 mL of thisolution was reacted with 1 mL of oil diluted extract (dissolvedn methanol). The mixture was incubated in dark room for0 min at room temperature. The absorbance was measuredt 517 nm with Helios Omega UV/VIS Thermo Scientific Merknd Co. Spectrophotometer. The percentage inhibition activityas calculated by Eq. (5):

% =(

A0 − At

A0

)× 100 (5)

here A0 is the absorbance of the control sample (withoutssential oil) and At the absorbance of the extract with DPPHt 30 min. Ascorbic acid was used as reference and all analysesere run in triplicates and averaged.

.8.2. Ferric reducing antioxidant power assay (FRAP)e (III) reduction is used as an indicator of electron-donatingctivity, which is an important mechanism of phenolic antiox-dant action. The FRAP procedure described by Dobravalskytet al. (2012) is based on the reduction of a ferric-tripyridylriazine (TPTZ) complex to its ferrous colored form in theresence of antioxidants. An intense blue color complex wasormed when ferric tripyridyl triazine (Fe3+-TPTZ) complexas reduced to the ferrous (Fe2+) form and the absorption

t 593 nm was recorded. Thus in reduction of Fe3+-TPTZy antioxidants, blue colored Fe2+-TPTZ is formed and thisntioxidant power can be referred to as reducing ability. FRAPeagent was prepared as a mixture of three solutions A, B and

mixed in proportion of 10:1:1 (v/v/v) respectively, where A, Bnd C solutions were: A: Acetate buffer (0.32 g sodium acetatend 1.6 mL acetic acid make up to 100 mL) (pH 3.6); B: 10 mMPTZ solution (0.0312 g) in 40 mM HCl (100 mL); C: 20 mM FeCl3olution (0.324 g ferric chloride in 100 mL distilled water). TheRAP reagent was prepared fresh daily and warmed to 37 ◦Crior to use. A total of 0.15 mL samples extract were added to.5 mL of the FRAP reagent and mixed well. The absorbance iseasured at 593 nm using spectrophotometers (Helios OmegaV/VIS Thermo Scientific Merk and Co.) after 4 min. Samplesre measured in three replicates. Standard curve of iron (II)olution (100, 400, 600, 800 and 1500 ppm (�g/mL) is preparednd analyzed.

.9. Scanning electronic microscopy

icrostructures were observed using an environmental SEMEI/Philips Quanta 200 FEG (Field Effect Gun). Samples werebserved under 1.00 mbar of water vapour pressure withoutny metallic coverage. The accelerating voltage used is 20 kV.econdary electrons (SE) images were obtained with a large-

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eld detector (LFD) and back scattered electrons (BSE) images,ith a solid state detector (SSD).

3. Results and discussion

3.1. Regression coefficients and fitting the models

The complete design matrix together with the values of exper-imental yield and oil composition in two major studied groupsof components namely total oxygenated compounds andsesquiterpenes hydrocarbons are given in Table 2. A regres-sion analysis was carried out to fit mathematical models tothe experimental data aiming at an optimal region for thestudied responses. The predicted models can be describedby Table 3 in term of coded values. The significance of eachcoefficient was determined using Fisher test (F-value) and theprobability p (p-value) in Table 4, which displays the vari-ance analysis of the system (ANOVA). Corresponding variableswould be more significant if absolute F-value becomes greaterand p-value becomes smaller. For the yield of H. perforatumessential oil, it can be seen that the linear terms are stronglysignificant (p < 0.05) as well as the quadratic effect of mois-ture content and the interaction between irradiation powerand moisture content. For the composition of essential oilin total oxygenated compounds, irradiation power and mois-ture content are positively significant as well as the quadraticeffect of irradiation time indicating a degradation of theoxygenated compounds for a prolonged processing time. Con-cerning sesquiterpenes hydrocabons, the same linear effectswere significant but with an opposite sign that obtained foroxygenated compounds as well as an interaction between irra-diation power and processing time that will be discussed inSection 3.2. In general way the results suggest that chang-ing in irradiation power, moisture content and in a lesserextent irradiation time had a highly significant effect on theyield of isolated oil and on its composition. The quality ofthe models developed was evaluated based on the correlationcoefficient R2 and also on the lack-of fit value. From ANOVA(Table 4), it can be seen that R2 were systematically close to90% and p-value of lack-of-fit systematically higher than 0.05(non-significant) suggesting that the predicted models reason-ably represent the observed values. Thus the responses weresufficiently explained by the models.

3.2. Response surfaces analysis

Three-dimensional profiles of multiple non-linear regressionmodels were employed to illustrate the linear and quadraticeffects as well as the interaction effects between irradiationpower, irradiation time and moisture content on the extrac-tion yield and its composition in total oxygenated compoundsand sesquitepenes hydrocarbons. Fig. 2 highlights the extrac-tion yield behavior in function of two variables. In each plot,the third one is fixed at its central value (“0”). The most influ-ential effects are the linear terms of moisture content (x3)of H. perforatum and irradiation power (x1): the yield increaselinearly as irradiation power increase and decreased linearlywith moisture content (Fig. 2a). This decreasing was alsopointed out by Li et al. (2012) as a consequence to a hydrol-ysis of some volatile components under a high proportionof water. The same linear effect has been noticed for irra-diation time (x2) but with a less predominant influence asindicated by p-values of ANOVA analysis. The moisture con-tent also exerted a quadratic effect clearly showed in Fig. 2b

of Hypericum perforatum L. oil isolation by solvent-free microwave014.04.012

and c in which the yield systematically decreased until acertain value after which an increase was observed. Fig. 2c

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Table 2 – Experimental data and yield of hypericum perforatum L. essential oil with different combinations of irradiationpower (x1), irradiation time (x2) and initial moisture content of (x3) used in the randomized central composite design.

Coded variable level Experimental responses dataa

Run x1 x2 x3 Yield 1 2

1 −1 −1 −1 0.0222 38.61 49.712 1 −1 −1 0.0177 43.02 48.923 −1 1 −1 0.0560 36.28 61.914 1 1 −1 0.2039 37.48 23.595 1 1 1 0.0442 63.61 14.306 −1 −1 1 0.0215 39.67 57.847 −1 1 1 0.0127 57.64 37.698 1 −1 1 0.0215 63.21 27.369 0 − 0 0.0095 64.90 29.92

10 0 0 + 0.0115 55.51 32.5511 0 0 − 0.1561 41.69 54.9712 + 0 0 0.1193 51.92 42.0613 0 + 0 0.0242 43.35 52.8114 − 0 0 0.0122 25.86 71.5115 0 0 0 0.0160 43.44 50.9316 0 0 0 0.0185 51.48 46.3117 0 0 0 0.0177 44.20 53.8418 0 0 0 0.0172 46.21 52.12

Mean absolute error for replications 0.0009 2.13 2.79Classical hydrodistillation procedure 0.0821 0.56 5.49

1: total oxygenated compounds; 2: sesquitepenes hydrocarbons. The yield is expressed in g/100 (d.m) and the groups of components areexpressed in relative percentage of GC–MS area.

describes the interactive effect of irradiation time and mois-ture content. For an irradiation power fixed at 300 W, theyield of essential oil increased from 0.025 to 0.114 g/100 gd.m for moisture content fixed at its low value (50%) anddecreased from 0.012 to 0.001 g/100 g d.m for a moisture con-tent fixed at its high value (70%). Figs. 3 and 4 displayedthe simultaneous effects of the three variables on the totaloxygenated compounds and sesquiterpenes hydrocarbons.It should be noted that an inverse trends were observedfor the effects of irradiation power and moisture contentof Hypericum perforatum L.: increasing of irradiation poweror moisture content induced an increment in oxygenatedcompounds and decreasing of sesquiterpenes hydrocarbons.These results clearly indicate that the microwave polarization

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and the water solubility of the components influence extractcomposition as it will be discussed in the next section. For the

Table 3 – Regression coefficients of the second-order polynomia

Regression coefficients Response var

Yield

ˇ0 0.0146

ˇ1 0.0519

ˇ2 0.0379

ˇ3 −0.0648

ˇ11 0.0317

ˇ12 0.0459

ˇ13 −0.0279

ˇ22 −0.0028

ˇ23 −0.0514

ˇ33 0.0444

1: total oxygenated compounds; 2: sesquiterpenes hydrocarbons. The fitte

Y = ˇ0 + ˇ1x1 + ˇ2x2 + ˇ3x3 + ˇ11x21 + ˇ22x2

2 + ˇ33x23 + ˇ12x1x2 + ˇ13x1x3 + ˇ23x2

where x1, x2 and x3 are the coded values of respectively irradiation powebefore processing.

two groups of components irradiation time was not a criticalvariable.

3.3. SFME versus HD

The optimal conditions selected by the software were thehigher values of irradiation power (468 W) and irradiation time(33 min) and the lowest value moisture content (43%). Underthese conditions, the software indicates an optimum yieldof 0.405 g/100 g d.m while the experimental yield was about0.365 g/100 g d.m. This difference can be due to a loss of essen-tial oil on the flask test walls. It should noted that the yieldwas largely enhanced compared to hydrodistillation methodfor which the maximal yield of 0.08 g/100 g d.m was obtained

of Hypericum perforatum L. oil isolation by solvent-free microwave014.04.012

after 240 min. Similar yields (from 0.058 to 0.092 g/100 g d.m)were obtained by Schwob et al. (2004) for hydrodistillation

l equations.

iables

1 2

46.27 51.1011.57 −20.87−3.75 −1.1413.45 −12.39−5.15 1.25−5.21 −7.60

5.98 −3.685.62 −9.656.75 −5.032.04 −7.95

d models are given by the following Eq.:

x3.

r, irradiation time and moisture content of Hypericum perforatum L.

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Table 4 – Analysis of variance showing the effect of thethree independent variables as linear, quadratic andcross products terms on the studied responses.

Source DF Sum of squares F-Ratio p-Value

Extraction yieldx1 1 9.22 × 10−3 13.41 0.0064**

x2 1 4.90 × 10−3 7.13 0.0284*

x3 1 1.43 × 10−2 20.90 0.0018**

x21 1 3.18 × 10−3 4.63 0.0635

x1x2 1 4.23 × 10−3 6.15 0.0381*

x1x3 1 1.56 × 10−3 2.27 0.1704x2

2 1 2.54 × 10−5 0.04 0.8522x2x3 1 5.30 × 10−3 7.71 0.0241*

x23 1 6.25 × 10−3 9.10 0.0167*

Lack-of-fit 5 1.09 × 10−3 2.24 0.1728Pure error 2 2.53 × 10−5

R2 0.958Total oxygenated compoundsx1 1 457.073 34.71 0.0098**

x2 1 48.2231 3.66 0.1516x3 1 618.221 46.94 0.0064**

x21 1 83.9595 6.37 0.0858

x1x2 1 54.3925 4.13 0.1351x1x3 1 71.6405 5.44 0.1019x2

2 1 99.8817 5.71 0.0438*

x2x3 1 86.4613 6.56 0.0831x2

3 1 9.24453 0.70 0.4636Lack-of-fit 5 327.656 4.98 0.1485Pure error 2 39.5104R2 0.911Sesquiterpenes hydrocarbonsx1 1 1487.28 144.61 0.0012**

x2 1 4.4897 0.44 0.5560x3 1 524.267 50.97 0.0057**

x21 1 4.95888 0.48 0.5374

x1x2 1 115.748 11.25 0.0439*

x1x3 1 27.1953 2.64 0.2024x2

2 1 294.775 28.66 0.0128*

x2x3 1 50.5515 4.92 0.1134x2

3 1 200.258 19.47 0.0216**

Lack-of-fit 5 956.713 2.60 0.1455Pure error 2 30.855R2 0.897

∗ p < 0.05.∗∗ p < 0.01.

DF: degree of freedom.

oGopirepaetotmtscs

nolic groups and to avoid their contribution in side reactions

f Hypericum perforatum L. from south-east of France and bylisic et al. (2008) for supercritical carbon dioxide extractionf Serbian specie (∼0.05 g/100 g d.m). For both processes, twohases were observed in extraction process. The first one

s represented by an increasing line until the temperatureeaches 100 ◦C and thus achieves the distillation of the firstssential oil droplet. In the second part, the extraction tem-erature is approximately equal to water boiling temperaturet atmospheric pressure. However, the most important differ-nces was observed between the both extraction methods, ishe ability of SFME process to quickly raise the temperaturef the sample to 100 ◦C. This rapid increasing of extractionemperature gives to acceleration of extraction rates under

icrowaves and could be due to a synergy combination ofhe two transfer phenomena mass and heat acting in theame way. This could be explained by the fact that in thease of HD, mass transfer occurs from the inside to the out-

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ide whilst heat transfer occurs from the outside to the inside.

For microwave extraction, the two transport phenomena arein the same direction from the inside to the outside of thepeel, which facilitates oil diffusion (Farhat et al., 2011). Forhydrodistillation and SFME oils about 61–69 components wererespectively identified among which non-terpenes, monoter-penes and sesquiterpenes compounds. By focusing on the twostudied groups (oxygenated and sesquiterpenes compounds),it can be seen from Table 5 that sesquiterpenes hydrocar-bons are present with a larger percentage (in g/100 g extractor %) in essential oil obtained by hydrodistillation (42.93%)than that obtained by SFME method (19.01%). Sesquiterpenesare effectively cited by the literature as the predominantcomponents in hydrodistilled Hypricum perforatum L. essen-tial oil (Schwob et al., 2002; Chatzopoulou et al., 2009). Aninverse trend is observed for essential oil obtained by SFMEwhere the predominant group is that of oxygenated com-pounds (37.62%) compared to hydrodistillation (23.90%). Orioet al. (2012) have linked this phenomenon to microwaveenergy effect which differs for these two different aromaticcompounds. For the more polar compounds (oxygenatedcompounds), the more easily the microwaves irradiationsare absorbed, the better the interaction between electro-magnetic wave and matter is established and more polararomatic components are obtained, conversely to sesquiter-penes hydrocarbons. Electromagnetic interactions are alsocited as possible cause to presence of more oxygenated com-pounds in SFME extracts: organic compounds that have a highdipolar moment as many oxygenated compounds interactmore vigorously with microwaves and can be extracted moreeasily. It should be noted that the oxygenated compoundsare most valuable in the essential oils; they are character-istic of a strong odor and are known to play a key role inthe antioxidant activity. Among the oxygenated compounds,two ketones (Z-�-Damascone and Z-�-Damascone) seems tobe predominant in SFME extracts and probably have also apart in antioxidant activity as it was remarked by Li et al.(2012). Moreover, as indicated by Table 5, many componentswere identified in SFME and HD oils. A part of these com-pounds were identified by some authors (Bruni et al., 2005;Radusiene et al., 2005) among which �-pinene, Germacrene-D, �-humulene, �-cadinene, viridiflorol, �-cadinol or myrcene.The relative percentages were also close to those obtainedin our study as indicated in the review Guedes and Franklin(2012) on the composition of essential oil from Hypericumspecies.

3.4. Assay for total phenolics

Total phenol content of the essential oil of H. perforatumwas calculated according to Eq. (4) as gallic acid equiva-lent (mg g−1 extract) and is based on absorbance value ofthe polar sub fraction of essential oil extract reacting withFolin–Ciocalteu reagent with the absorbance value of thestandard curve of gallic acid. Total penolics for SFME extrac-tion method was 21.32 ± 0.02 mg GAE g−1 extract and only4.44 ± 0.02 mg GAE g−1 extract for HD method. These resultsclearly shown that the application of microwaves leads torecover phenolic compounds with a higher concentration incomparison with methods based on hydrodistillation. ThusSFME is an efficient technology to save the structure of phe-

of Hypericum perforatum L. oil isolation by solvent-free microwave014.04.012

(Donelian et al., 2009).

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Table 5 – Chemical composition of Hypericum perforatumL. essential oils obtained by GC–MS.

Compounds RI SFME HD

Octane, 2-methyl- 859 7.46 8.14Nonane 900 0.9 1.1�-Pinene 933 8.95 13.65Nonane. 3-methyl- 964 1.27 1.34�-Pinene 979 0.65 1.35Myrcene 993 0.51 0.38P-Cymene 1026 0.13Limonene 1028 0.17 0.36�-Phellandrene 1035 0.19 0.33E-�-Ocimene 1044 0.21 1.3Decane. 2-methyl- 1054 2.33 1.51Undecane 1092 0.65 1.91Linalool 1104 – 0.13Nonanal 1106 0.08 0.09�-Campholenal 1130 0.11 0.15trans-verbenol 1146 0.12 0.15Z-Isocitral 1155 0.06 –�-Terpineol 1183 0.08 0.042-Allyl phenol 1203 0.003 –Dihydro carveol 1207 0.07 0.584-Methylene-isophorone 1222 0.1 0.112-Methyldodecane 1262 0.64 0.54n-Tridecane 1300 0.12 –�-Elemene 1333 0.11 0.02�-Longipinene 1349 0.23 0.62Z-�-Damascone 1354 9.14 –�-Ylangene 1370 0.1 0.72Z-�-Damascone 1374 9.93 –�-Copaene 1376 – 0.45�-Bourbonene 1383 – 0.08�-Elemene 1393 0.02 0.39Sibirene 1400 0.21 0.7(+)-�-Funerbene 1408 0.05 0.19�-Caryophyllene 1418 3.03 3.61�-Copaene 1427 0.09 0.17(+)-Aromadendrene 1437 0.05 0.33�-Himachalene 1447 1.1 3.64�-Humulene 1453 – 0.28(E)-�-farnesene 1456 0.97 0.85allo-Aromadendrene 1463 0.11 0.389-epi-E-Caryophyllene 1468 0.2 –�-Himachalene 1473 0.17 0.88�-Amorphene 1476 0.28 0.44Germacrene-D 1481 0.45 0.68�-Selinene 1486 6.17 8.81�-Selinene 1494 4.64 16.98�-Himachalene 1498 0.29 1.11�-Farnesene 1505 0.08 0.43�-Cadinene 1510 0.28 0.34�-Cadinene 1517 0.38 0.83Silphiperfol-5-en-3-ol B 1533 – 0.26�-Calacorene 1541 – 0.11E-Nerolidol 1562 0.37 0.553-Hexanyl-benzoate 1576 – 0.26(+) spathulenol 1578 2.43 2.65Viridiflorol 1613 0.17 0.78�-cadinol 1637 0.72 1.68Himachalol 1646 0.43 0.9�-cadinol 1652 1.16 2.2314-hydroxy-Z-Caryophyllene 1666 0.07 –n-Tetradecanol 1671 3.15 11.9�-Bisabolol 1683 0.07 0.27trans-Longipinocarveol 1699 0.08 –Benzyl Benzoate 1772 0.19 0.01Pentanoic acid eugenyl ester 1812 2.6 –Hexahydrofarnesyl acetone 1828 0.07 0.4Z.Z octadecadienoic acid ethyl ester 1864 1.06 –Hexadecanol 1883 – 0.18

– Table 5 (Continued)

Compounds RI SFME HD

1-Heptadecanol 1886 1.28 –Kaurene 1936 1.08 –Phyllocladene 2003 2.6 –n-Heneicosane 2092 4.22 –E-phytol 2124 0.38 0.18n-Pentacosane 2510 2.21 0.25n-Heptacosane 2696 1.13 0.33n-Octacosane 2794 1.59 –n-Nonacosane 2903 6.64 0.73Total identified% 96.61 99.78Extraction time – min 33 240Yield % (g/100 g d.m) 0.365 0.082Total oxygenated compounds 37.62 23.90Sesquiterpenes hydrocarbons 19.01 42.93

RI: retention index calculated on a varian Elite-5MS column.%: relative area percentage (peak area relative to the total peak).

3.5. Antioxidant activity assays

3.5.1. Free radical scavenging activity (DPPH)A freshly DPPH prepared solution gave a deep purple colorwhich generally fades when an antioxidant is present inthe medium. This transformation is spectrophotometricallymeasured and the disappearance of the purple color mon-itored at 517 nm. The radical-scavenging properties of theextracts obtained by HD and SFME methods as well as ascor-bic acid as reference antioxidant compound are given in Fig. 5.All extracts exhibited concentration-dependant DPPH radicalscavenging activity. The extract isolated by the conventionalmethods (HD) revealed moderate antioxidant activities in thisassay system compared to SFME with inhibition percentagesof 58% and 85% respectively. From this figure, the concen-tration of sample required to scavenge 50% of DPPH (IC50)can be determined. It appears that SFME extract showeda lowest IC50 (462.36 ± 0.15 �g/mL) compared to IC50 of HDextract (40,042.87 ± 0.23 �g/mL) indicating that SFME extractwas largely more effective than HD extract against DPPHradical. Many authors obtained à similar classification by com-paring microwave extracts and conventional methods extractsaccording to their antioxidant activity (Li et al., 2012; Donelianet al., 2009; Zill-e-Huma Abert-Vian et al., 2011). Our resultscan be favorably compared with those of Altun et al. (2013)for methanolic extracts of Turkish specie of H. perforatum.The authors obtained 65.22% of inhibition for an extract con-centration of 500 �g/mL while our results for “solvent free”microwave extraction was 50% inhibition for 462.36 �g/mL.However, it is worth noting that the antioxidant activity islower than that of ascorbic acid reference for which IC50 valuewas 96.11 ± 0.08 �g/mL.

3.5.2. Ferric reducing antioxidant power assay (FRAP)Due to the complexity of antioxidative mechanism, an individ-ual assay is inappropriate to generalize the total antioxidantactivity. Thereby a complementary evaluation was carried out:the reducing power assay (FRAP). In this assay, the presenceof antioxidant in the extracts results in a reduction of ferric-tripyridyl triazine complex to its ferrous form. The reductioncapacity of a compound may serve as a significant indica-tor of its potential antioxidant activity; a higher absorbance

of Hypericum perforatum L. oil isolation by solvent-free microwave014.04.012

indicated a higher ferric reducing power. For example, at a con-centration 614 �g/mL, the absorbance was about 0.521 ± 0.002

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Fig. 5 – DPPH scavenging ability of HD (a) and SFME (b)extracts from H. perforatum and ascorbic acid (c) as positivecontrol. Results are mean of three experiments. Extractiontime for HD and SFME were 33 and 240 min respectively.The standard mean errors (n = 3) were 0.17 (a), 0.10 (b) and0.12 (c).

fctcrRSaioiohi

Fig. 6 – SEM images of Hypericum perforatum L. leaf: (A)untreated; (B) after extraction by HD; (C) after extraction bySFME.

H. perforatum samples were examined by SEM for an evalu-

or SFME while it was only 0.012 ± 0.001 for HD extraction pro-ess. However, in the two cases the reducing power was lowerhan that of reference FeCl2 (absorbance of 1.5 for 600 �g/mLoncentration). Based on absorbance results, the obtained cor-elations between DPPH and FRAP were y = −0.3234x + 0.2392;2 = 0.971 and y = −5.8822x + 5.1175; R2 = 0.944 respectively forFME and HD, by considering “y” as FRAP absorbance and “x”s DPPH absorbance. These high and negative correlations aren agreement with the fact that a low value of absorbancef the reactant mixture indicates a high sequestrant capac-

ty of free radicals (Inhibition %) and confirm the consistencyf DPPH and FRAP assays. The results also indicated that theigher value of TPC for SFME correspond to a lower absorbance

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n DPPH assays and a higher absorbance in FRAP assays,

indicating that the phenolic compounds contribute to antiox-idant activity.

3.6. Microstructural changes after extraction

of Hypericum perforatum L. oil isolation by solvent-free microwave014.04.012

ation of the microstructural alteration induced by SFME and

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HD extractions compared to untreated sample. As shown inFig. 6A, the external surface of untreated sample was smoothand contains many folds. After extraction by HD (Fig. 6B) someruptures and perforations appeared on the leaf surface and thefolds were still present while after extraction by SFME (Fig. 6C),perforations and creation of canals was clearly observed andthe surface appeared completely disrupted indicating that thestrain induced by a rapid rise in temperature in SFME extrac-tion and subsequent change in the surface tension of theglandular wall, causing it to crumble or rupture more readily.

3.7. Energy consumption and environmental impact

The reduced cost of essential oil extraction is clearly advan-tageous for SFME method in terms of energy and time.HD method required 240 min to reach a maximum yieldof 0.08% while 33 min were sufficient to reach a maximumyield of 0.365% for SFME. The quantity of water to evapo-rate was 2 kg for HD and about 30 g for SFME (in definedoptimal conditions). Then energy required for performing HDand SFME extraction methods are respectively 4 kWh and0.26 kWh. Regarding the environmental impact, the calcu-lated quantity of carbon dioxide rejected in the atmospherefor HD was 3564 g CO2, largely higher than that of SFME(232 g CO2). These calculations were preformed accordingto literature provided by the French Nuclear Energy Soci-ety (http://www.sfen.org/fr/societe/developpement/edf.htm):to obtain 1 kWh from fuel 891 g of CO2 is rejected in atmo-sphere during combustion. Filly et al. (2014) tested the SFMEtechnique at a pilot plant scale of 150 L capacity, for extractionof essential oil from aromatic herbs. The authors indicate thatmicrowaves have wide-ranging large scale commercial appli-cations as processing technology and can provide high returnson capital investment.

4. Conclusion

This is the first report on the utilization of solvent-freemicrowave technology (SFME) for extraction of essential oilfrom Hypericum perforatum species and more specifically Alge-rian one. Response surface methodology was successfullyimplemented for optimization of extraction yield and for eval-uation of simultaneous effects of three independent variables(irradiation power, irradiation time and moisture content)on two major groups of compounds: total oxygenated com-ponents and sesquiterpenes hydrocarbons. By comparisonwith HD method, SFME contained a higher proportion ofoxygenated compounds. The antioxidant activities of theresulting essential oils have been evaluated by DPPH andreducing power test. Based on the results, we conclude thatSFME represents a valuable alternative to traditional HDextraction process for an efficient extraction of essentialoil from Hypericum perforatum L. despite a lower antioxidantactivity than that of synthetic compounds as ascorbic acid.Moreover, the results indicate that essential oil of Hypericumperforatum L. extracted by SFME could be considered as a nat-ural source of phenolic compounds. Thus our study showsthat SFME extraction technique is effective and involves anenvironmentally friendly approach, with a reduced consumeof energy and reagents. The next step could be the potential

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applicability of SFME technique at a pilot plant scale as indi-cated in conclusion section. At laboratory scale, this work can

be extended through the study of the variability of the resultswith the harvesting season.

Acknowledgements

The authors thank Ms. Egle Coforto from “Centre Commund’Analyses” of university of La Rochelle for her assistance inscanning electron microscopy.

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