Cent. Eur. J. Chem.

12(8) 2014 829-836

DOI: 10.2478/s11532-014-0532-4

Central European Journal of Chemistry

Microwave assisted extraction of essential

oils from enzymatically pretreated
lavender (Lavandula angustifolia Miller)+
RICCCE 18

Ioan Calinescu1, Adina I. Gavrila1*, Maria Ivopol1,2,

Gabriel C. Ivopol1,2, Mariana Popescu1,2, Nectara Mircioaga1
1
Department of Bioresources and Polymer Science,
Faculty of Applied Chemistry and Materials Science,
University Politehnica of Bucharest, Bucharest 010072, Romania

Hofigal Export-Import S.A., Bucharest 042124, Romania

Received 27 September 2013; Accepted 29 November 2013

Abstract: In
 this study, microwave assisted a hydrodistillation process (MWHD) of essential oils from lavender (Lavandula angustifolia Miller)
was investigated. In order to examine any potential differences in essential oil extraction, the lavender flowers underwent enzymatic
pretreatment.
A 23 factorial design of experiments, combined with statistical methods of data analysis were used to optimize enzymatic
pretreatment and to evaluate the influence of major variables (enzyme concentration, temperature and pH) on the performance of the
microwave assisted extraction.
Under optimal conditions, an extraction yield of 24 mg oil g-1 substrate was achieved (an increase by approximately 25% in
comparison with the classic extraction conditions of conventional hydrodistillation).
The main compounds of the essential oils obtained were analyzed and identified by gas chromatography coupled to mass
spectrometry (GC-MS). Analyzing the data obtained indicated that the content of main compounds (linalool and linalyl acetate - 73%)
was greater than that obtained by conventional extraction (67%).
Keywords: Lavender oil Hydrodistillation Enzymatic pretreatment

Versita Sp. z o.o.

1. Introduction
In the Labiatae family, the genus Lavandula comprises
approximately 30 botanical species and subspecies, of
which three are utilized for their production of essential
oils: L. angustifolia Miller (syn. L. officinalis Choix)
provides essential oil of the highest quality, L. latifolia
Medicus which has the lowest yields, and various forms
of L. hybrida (L. angustifolia L. latifolia) which has the
highest yields but not such a high quality oil [1,2].
Lavender is a perennial Mediterranean shrub that
grows spontaneously in dry chalky soils at altitudes of
400 m to 1800 m [3]. Lavender has been used since
ancient times, for example the Romans scented their
bathwater and laundry water with lavender, from whence

the Latin verb to wash lavare and subsequent words in

English such as laundry, lavage and lavatory. A typical
yield of essential oil from fresh lavender is between 0.8
to 1.5%, or about 10 25 kg of essential oil per hectare
[1].
Essential oil of L. angustifolia Miller is a colourless to
pale yellow liquid, with a fresh grassy, floral fragrance,
and comprises 1 3% of the dry floret spike [4]. Over
300 compounds have been identified from species of
Lavandula, the two main compounds being linalool, and
linalyl acetate (which is present in a greater quantity in L.
angustifolia and L. hybrida).
Both linalool and linalyl acetate are detectable
in the blood five minutes after topical application of
L. angustifolia essential oil, reaching peak levels in

* E-mail: adinagav@yahoo.com
+
The article has been presented at the 18th Romanian International Conference on Chemistry and Chemical Engineering - RICCCE18 - held in
New Montana, Sinaia, Romania on 4-7 September, 2013.

829

Microwave assisted extraction of essential oils from enzymatically

pretreated lavender (Lavandula angustifolia Miller)

19 minutes, and dissipating after 90 minutes. Linalool

is a GABA-ergic monoterpene (GABA - Gamma
aminobutyric acid), and enhances the sedative effects
of GABA-specific anti-epileptics and benzodiazepines
[5].
Internally, essential oil of L. angustifolia also has
antiseptic, mildly antispastic, carminative, cholagogic,
sedative, depurative and diuretic effects. Externally, it
has analgesic and sedative effects, and is useful as an
inhalation for colds and the flu [2,3].
Essential oils, which are isolated by physical means
only, are defined as products obtained from raw plant
materials. The physical methods used are distillation
(steam, steam/water, and water), squeezing (also known
as cold pressing for citrus peel oils), or dry distillation of
natural materials [6]. The volatile compounds have the
property to solubilize in fatty oils and fats so that they
have been called essential oils.
Data in the literature have shown that microwaveassisted extraction is an appropriate alternative to
conventional techniques due to the reduced extraction
time, more effective and selective heating, simplified
equipment, and a higher purity of the final product [6,7].
The reduced extraction time allows for the preservation
of more thermolabile components in the final product [3].
Another alternative method to improve the release
of essential oil is that of enzymatic pre-treatment
in order to hydrolyze the essential oil cells. Due to
their high specificity and efficiency, enzymes are
useful for the extraction of essential oil. The use of
appropriate enzymes for essential oil extraction from
a few condiments and the like such as garlic, cumin,
pepper, mustard, chilli and citrus peel has been
reported [8-10]. They have presented that the pretreatment of these materials results in an important
increase in the yields of oil and their major components
[10,11].
The aim of this paper is to obtain essential oils from
L. angustifolia Miller (lavender) with the highest possible
yield by using microwave (MW) assisted hydrodistillation
processes on lavender that has undergone enzymatic
pretreatment as well as to examine any potential
differences in essential oil composition resulting from
enzymatic pre-treatment.

2. Experimental procedure
2.1. Plant material and chemicals

As raw material, fresh lavender floret spikes were used,

from flowers harvested at the beginning of July 2013 at
Hofigal S.A. in Bucharest and were dosed in samples
of 50 g.
830

Cellulase (Carezyme Premium 5000T) was

purchased from Novozyme, Denmark. Di-sodium
hydrogen phosphate 2-hydrate (Na2HPO42H2O) was
obtained from Merck and citric acid was purchased from
Aldrich.

2.2. Enzymatic pre-treatment

Lavender flowers (50 g) were mixed with 500 mL

pretreatment solution (a buffer solution containing
0.2 M Na2HPO42H2O and 0.1 M citric acid solutions in
different proportions) to keep the pH (2.6, 4.6 and 6.6)
during enzymatic pretreatment. Cellulase (0.1, 0.2 and
0.3 g Carezyme Premium 5000T) has been dissolved in
200 mL of buffer solution and 300 mL of ultrapure water
was added. The mixtures were stirred in an open vessel
for 1 h at the desired temperature (40, 50 and 60C)
then subjected to microwave assisted hydrodistillation
(MWHD) for essential oil extraction. Control samples
without any treatment were directly subjected to MWHD.

2.3. MWHD apparatus and procedure

The essential oils were extracted using microwaveassisted hydrodistillation using a multimode microwave
reactor (Plazmatronika, Poland). The hydrodistillation
(HD) was carried out by concurrently bubbling nitrogen
gas at 5 L h-1 to avoid oxidation of the components.
During the experiments, time, temperature, pressure
and power were controlled via an operating console.
Steam produced in the reactor carrying the lavender
essential oil was directed to a modified Neo Clevenger
trap with a 5 mL graduated tube. The separated oils
were kept at 4C and analyzed by GC-MS. A schematic
diagram of the MWHD apparatus used for essential oil
extraction is shown in Fig. 1.

2.4. Methods of determination

The compounds extracted were further analized by

GC-MS. The analyses were undertaken with the help
of a Thermo Electron Corporation Focus GC gas
chromatograph with a Macrogol 20 000 R column
(film thickness 0.25 m); l = 60 m; = 0.25 mm.
The mobile phase used was helium with a debit of
1.5 mL min-1, while the sample injection volume was
0.2 L. A Thermo Electron Corporation DSQII mass
spectrometer was used for detection. Identification of
the samples analysed gas chromatographically was
carried out by comparing the sampled spectral peaks
with spectra from a Wiley database.
The lavender flowers were examined by means
of a High Resolution Scanning Electron Microscope
(HRSEM), FEI Inspect F 50 (field emission gun). A 5 kV
voltage was used and fracture surfaces were examined,
after a gold-spatter coating was applied.

I. Calinescu et al.

Table 1.

Pretreatment process variables, central point, variation interval and levels considered.

Independent
variables factors)

Variation levels: dimensionless (D) and natural (N)

Variation
interval

Notation

Enzyme amount,
mg g-1 biomass

-1

+1

x1

pH

-1

2.6

4.6

+1

6.6

x2

-1

40

50

+1

60

10

x3

Temperature, C
o

Table 2.

Experimental matrix and values obtained for the essential oil yield.

Number of
trials

Factor level combinations

x0

x1

x2

x3

x1x2

x1x3

x2x3

x1x2x3

Response, Y
Essential oil yield

-1

-1

-1

-1

21.684

-1

-1

-1

-1

22.54

-1

-1

-1

-1

23.678

-1

-1

-1

-1

20.202

-1

-1

-1

-1

21.5

-1

-1

-1

-1

20.162

-1

-1

-1

-1

21.402

21.324

19.942

10

20.762

11

20.2

12

20.4

3. Results and discussion

3.1. Experimental design and statistical analysis

A 23 factorial design of experiments, combined with

statistical methods of data analysis were used for
optimizing the enzyme preatreatment of lavender
before obtaining the essential oil via microwave assisted
hydrodistillation. In this test, eight experiments and four
replicates at the central point were used to obtain the
polynomial model:
Y = b0 + b1x1 + b2x2 + b3x3 +
+ b12x1x2 + b13x1x3 + b23x2x3 + b123x1x2x3

Figure 1. Microwave-assisted hydrodistillation aparatuss.

(1)

The optimization criterion will be the quantity of

essential oil obtained (mg oil per g plant).
The variables of the pretreatment process are:
quantity of enzyme, pH and temperature.
The preliminary experiments and data from the
literature have led to the selection of the central
point and the variation interval for these variables
831

Microwave assisted extraction of essential oils from enzymatically

pretreated lavender (Lavandula angustifolia Miller)

Table 3.

Coefficients of the polynomial model.

b1

b2

b3

b12

b13

b23

b123

-0.5045

0.09

-0.4645

-0.384

0.1505

0.176

0.699

b0
21.14967

Table 4.

Variances and F factors associated to each term of the polynomial model.

S2b0
466.0663

S2b1

S2b2

S2b3

S2b12

S2b13

S2b23

S2b123

2.036162

0.0648

1.726082

1.179648

0.181202

0.247808

3.908808

S2rez= 0.119635
Calculated F

Confidence
level

Tabulated
F

90%

5.54

17.01983

0.541649

14.42794

9.860419

1.514628

2.071373

32.67287

Yes

Yes

No

Yes

Yes

No

No

Yes

Yes

Yes

No

Yes

No

No

No

Yes

95%

10.1

Yes

No

No

No

No

No

No

No

99%

34.1

3895.747

(see Table 1). The experimental matrix will have the

form presented in Table 2.
Vector b of the proposed linear model can be
calculated with the following relation [13]:
b = (XT*X)-1*(XT*Y)

(2)

which in the case of an experimental orthogonal matrix

(that is, the matrix produced XT*X has only diagonal
elements other than zero) leads to the following relations:

(6)

The testing of two variances is part of an

F-distribution, F is defined as:

(7)

where F is distributed as the F-distribution with n1-1 and

n2-1 degrees of freedom;
The tabulated F-values [12] correspond to 1 degree
x0 j y j
j =1
(3) of freedom for the numerator and 3 degrees of freedom
b0 =
,
k = 23 = 8
k
for the denominator, are 5.54; 10.1 and 34.1 for the
90%, 95% and 99% confidence levels respectively.

k
The variances that correspond to each term in the

xij y j
polynomial model as well as the calculated F factor

j =1
3
bi =
,
k =2 =8
(4)
values are presented in Table 4.
k
For the 90%, 95% and 99% confidence levels, the
Applying these relations (Reactions 3 and 4) coefficients that can be retained are those for which the
the values presented in Table 3 are obtained for the calculated value of factor F is greater than the tabulated
F values (see Table 4).
coefficients of the polynomial model.
Thus we obtain the following models:
Testing the significance of each coefficient is
For 90% confidence level
necessary. To this end, the variance of the data obtained
Y=21.14967 - 0.5045x1 at the central point (S2rez) shall be determined. It is
considered that this dispersion is due to random errors.
- 0.4645x3 - 0.384x1x2 + 0.699x1x2x3
(8)
The variances generated by each term of the proposed
For 95% confidence level
polynomial model are determined via the following
Y=21.14967 - 0.5045x1 - 0.4645x3 + 0.699x1x2x3 (9)
relations [13]:
k
The adequacy test is then carried out so as to
( y j )2
confirm that the chosen model (with a 95% confidence
j =1

(5) level) and the values of its coefficients are correct. To
Sb20 =
k
that end, the following terms are determined:
k

832

I. Calinescu et al.

yj

21.684

21.420

y- y
y j y j yj y

y j y- yy

yj- y

0.122

0.264

-0.142

22.540

21.809

0.978

0.731

0.247

23.678

22.818

2.116

0.860

1.256

20.202

20.411

-1.360

-0.209

-1.151

21.500

21.889

-0.062

-0.389

0.327

20.162

19.482

-1.400

0.680

-2.080

21.402

20.491

-0.160

0.911

-1.071

21.324

20.880

-0.238

0.444

-0.682

1.0

A model is that much better as SPreg is large with regards

to SPrez while the value of the correlation coefficient is
closer to 1. For the example above the determination
of the correlation coefficient R is presented in
Table 5. The correlation coefficient has a very good
value: R2 = 0.966119
Furthermore, a procedure is developed in order to
explore the experimental domain outside of the limits
established via the factorial program. We shall take into
account the mathematical model with a 95% confidence
level.
The following procedure is followed [16]:
- The optimal placement on the coordinates with the
greatest slope value is established;
- The placement of the other coordinates is
calculated in relation to the ratio of the slopes;
- The coordinates of the new test point are
determined;
- The theoretical (from the model) and practical
values are determined for new test point;
- If the trend given by the practical value is the same
as the trend of the theoretical value, a new point can be
calculated on the direction of the maximal slope, if for
example, the exploration does not finish or the factorial
experiment is repeated from the previous best value
point; compared to the first factorial experiment, much
smaller variation intervals shall be utilized as well as a
second-order model equation.
In Table 6 and Fig. 2, the values of the experimental
points along the optimal direction established by the
factorial program are presented. At test points 1, 2, 3,
and 4, experimental determinations were undertaken.
The values obtained demonstrate that the chosen

0.5

SPrez

SPreg

3.0303

9.0279

0.0

21.15 20.3

22.2
22.3
22.5
22.6
22.9
24.5
23.4 22.5

-0.5
-1.0
-1.5

1.0

0.5

-2-.20.0

-1
.5

0.0

-0.
5

-1
.0

Enz
ime

-0
.5

-1.
0

-1.5

1.
0
Figure 2.

pH

Temperature

(10)

SPam
9.3445

Re
al
va
lu
es

where:
y - is the average of all the experimental observations
(yj)
y - are the resultant y values calculated with the model
j
obtained
The correlation coefficient R can be calculated using
the Eq. 10.

y^ j

yj

Exp no.

va
lu
es

Sum of the
squared
deviations
of the
regression
minus the
average
SPreg

0.
5

SPam

Sum of the
squared
deviations
of the
observations
minus the
regression
SPrez

Determination of the correlation coefficient, for the model

with a 95% level of confidence.

Th
eo
re
tic
al

Sum of the
squared deviations
of the observations
minus the average

Table 5.

0.
0

-2.
0

Exploration of the experimental domain outside of

the initial domain of the variation of the variables.
Experimental points and theoretical and practical values
of the essential oil yield.

direction is correct. This direction can be followed up to

point 3, following this direction further does not lead to
an improvement in the results.
The best yield for the in essential oil extraction was
obtained using mild pretreatment conditions: a small
amount of enzyme (1 mg g-1) and a temperature of
only 37C. These parameters are consistent with the
small thickness of the cells walls which incorporate the
essential oil.

3.2 Composition of lavender essential oils

The essential oil of lavender obtained by MWHD in

different conditions was analysed by GC-MS in order
to determine the chemical composition. The data
obtained as a result of this analysis are presented in
Table 7.
833

Microwave assisted extraction of essential oils from enzymatically

pretreated lavender (Lavandula angustifolia Miller)

Table 6.

Exploration of the experimental domain outside of the initial factorial experiment.

Independent variables
Amount of
enzyme

pH

Temperature

Base level
(central point)

Zj0

4.6

50

Variation unit

zj

10

Dimensionless slope

bj

-0.5045

0.09

-0.4645

Do1

Do2

Do3

Doi / Domax

-1.00

0.18

-0.92

4.96

40.8

Optimal dimensionless
placement (Do)

-1.2

0.216

-1.104

Test point 2 (natural)

1.6

5.032

38.96

Optimal dimensionless
placement (Do)

-1.50

0.27

-1.38

Test point 3 (natural)

1.00

5.14

36.19

Optimal dimensionless
placement (Do)

-1.75

0.315

-1.61

Test point 4 (natural)

0.5

5.23

33.9

Optimal dimensionless
placement (Do)
Test point 1 (natural)

Optimization criterion
(essential oil yield)
Calculated
value

Determined
value

22.2

22.3

22.5

22.6

22.94

24.5

23.4

21.3

Test point 2

Test point 3

Figure 3. SEM images of the lavender flowers after the extraction of their essential oil: after MWHD, without enzymatic pre-treatment (A) and after

MWHD, with enzymatic pretreatment (B).

The analyzed lavender oils show a relatively similar

composition whether the plants have been enzymatically
pretreated or have not undergone MWHD, or whether
the essential oil has been obtained by conventionally
heated HD. If we analyze the linalool and linalyl acetate
content, we note that MWHD assures a greater content
of these two compounds in comparison to conventional
HD alone, 71.7% versus 67.7%. Likewise, the utilisation
of enzymatic pretreatment results in an even higher
834

average concentration of linalool + linalyl acetate of

73.37%. This can perhaps be explained by the fact
that microwave irradiation accelerates the extraction
process, without producing considerable modifications
in the essential oil composition, which is in accordance
with data reported in the literature [13]. The use of
enzymes shortens the time required to release essential
oils from the plant, which explains the increased
concentration in linalool plus linalyl acetate.

I. Calinescu et al.

Table 7.

Chemical composition of lavender oils obtained by MWHD.

Compound

CAS no.

Retention
time,
min

Concentrations, %

Central
point

Factorial Experiment

MWHD
without
enzyme

Conventional
HD

Myrcene

123-35-3

9.28

0.6

0.58

0.63

0.71

Cineole
(Eucalyptol)

470-82-6

11.24

0.91

0.95

1.06

0.85

1.04

0.98

0.99

0.83

0.73

1.33

2.55

Pinene

80-56-8

13.47

5.42

5.47

5.35

4.34

4.71

5.15

5.13

5.14

4.27

4.84

6.63

-3-Carene

13466-78-9

14.16

2.41

3.04

2.85

2.18

2.5

2.88

2.6

2.42

2.04

2.55

2.17

Octenol
acetate

37366-04-4

18.98

1.03

0.87

0.97

0.81

0.91

0.9

0.86

0.87

0.87

0.97

0.74

Linalool

78-70-6

23.82

42.31

43.27

40.67

43.9

42.75

42.52

43.09

44.75

42.56

38.55

33.62

Linalyl
acetate

115-95-7

24.02

27.76

27.82

32.18

30.84

33.31

30.7

31.82

29.26

33.98

33.16

34.11

Caryophylene

87-44-5

24.67

2.77

2.24

3.23

2.74

2.27

2.19

2.32

2.12

2.67

2.9

4.33

Terpinen-4-ol

562-74-3

24.92

4.31

3.71

3.02

3.25

3.13

3.38

3.17

3.28

3.23

3.01

2.07

Lavandulyl
acetate

25905-14-0

25.15

1.11

0.93

1.01

0.88

1.05

0.9

0.86

0.95

1.13

3.48

-Farnesene

18794-84-8

26.6

0.63

2.42

507-70-0

27.1

0.96

0.83

0.89

0.83

0.83

0.86

0.85

0.73

0.75

0.98

Borneol
Terpineol

8000-41-7

27.2

5.09

5.05

3.79

4.1

3.5

4.06

3.54

4.16

3.31

4.38

3.18

Germacrene

28387-44-2

27.36

0.59

0.89

Geranyl
acetate

105-87-3

27.85

0.91

0.91

0.84

0.92

0.64

0.78

0.71

0.86

0.73

1.07

Lavandulol

1845-51-8

28.53

1.71

1.66

1.65

1.77

1.15

1.45

1.36

1.67

1.32

1.91

1.20

Geraniol

106-24-1

29.44

0.78

0.77

0.61

1.01

Nerol

106-25-2

30.47

2.51

2.49

1.65

1.81

1.66

1.92

1.41

1.76

1.37

1.79

Caryophyllene
oxide

1139-30-6

33.06

0.84

0.8

0.56

0.7

0.64

0.58

0.85

70.07

71.09

72.85

74.74

76.06

73.22

74.91

74.01

76.54

71.71

67.73

Linalool +
Linalyl acetate

3.3. SEM analysis

In lavender, the spherical essential oil containing cells

are situated on the exterior surface of the calyx and are
known as glandular trichomes (hairs). Each essential
oil-containing cell can differ in the compounds it
secretes, and in the way that it secretes them, with
the extracted essential oil of L. angustifolia being an
average sum of the plants individual essential oilcontaining cells [14].
As a result of the extraction process, the lavender
flower calyces undergo various physical modifications,
for example all of the trichomes reduce in volume
following their loss of essential oil (Fig. 3A).
In the case of lavender that has undergone enzymatic
pretreatment, after MWHD, the glandular trichomes have
a different appearance. In this case, after the extraction
of the essential oil, the trichomes appear as scraps of
cuticle (Fig. 3B). This can be explained by the rupture

of the exterior cuticle (membrane) of the trichomes by

hydrolysis under the action of the cellulases and due to
the generation of higher internal pressures as a result
of microwave irradiation. Thus, the results confirm that
in the presence of adequate amounts of enzyme the
extraction yield of essential oil increases (see Table 2).

4. Conclusions
The process of obtaining essential oil from lavender
flowers by using microwave assisted hydrodistillation
and an enzymatic pretreatment was studied. A faster
extraction was achieved in enzymatically pretreated
microwave irradiated samples, with consequently less
chance for oxidation and less time spent at extraction
temperatures for the volatile compounds present in the
essential oil obtained.
835

Microwave assisted extraction of essential oils from enzymatically

pretreated lavender (Lavandula angustifolia Miller)

The effects of enzymatic pretreatment and the

influence of major variables (enzyme concentration,
temperature and pH) on the performance of the
microwave assisted extraction were investigated.
Pretreatment with enzymes may lead to a slight
increase in essential oil yield, if it is carried out
in appropriate conditions. These conditions were
established by using a 23 factorial experimental
program type in order to study the influence of the
enzymatic pretreatment parameters upon the essential
oil yield from lavender flowers. The coefficients of the
polynomial model obtained were tested by the F-test.
Thus, it was observed that for pretreatment, the
quantity of enzyme and temperature were especially
important (direct influences), as well as the pH by a
term of interaction.
The adequacy of the model was determined,
obtaining a correlation coefficient with a very good value.
The model was further used for determining the maximal
slope. Using this direction, new experimental points
were determined that were tested. The pretreatment
conditions were determined for the highest yield of
essential oil, by comparing the initial central point of the
experiment with the best experimental point obtained in
the factorial experiment.

The best pretreatment conditions were found to be

a reduced quantity of enzyme (1 mg g-1 of plant) and
a reduced temperature (37C). In these conditions, the
essential oil yield increases by approximately 15% in
comparison to the initial conditions (from the centre of
the factorial experiment) and by approximately 25%
in comparison with the classic extraction conditions
(conventional hydrodistillation). The influence of
enzymatic pretreatment should be more evident in plant
material where the essential oils are less superficially
located and more difficult to distill out conventionally.
In order to study the differences between extraction
processes (with or without enzymatic processing)
and microscopic changes of materials, a scanning
electron microscope (SEM) analysis was performed.
Furthermore, chemical compositions of the extracted
oils were analyzed using gas chromatography coupled
to mass spectrometry (GC-MS).
GC-MS analyses of the essential oil samples
obtained from plants exposed to different pretreatment
conditions show a relatively similar composition. The
valuable linalool + linalyl acetate content increases
in the following order: Conventional HD < MWHD
without enzyme pretreatment < MWHD with enzyme
pretreatment.

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