Chemical Engineering Communications

ISSN: 0098-6445 (Print) 1563-5201 (Online) Journal homepage: http://www.tandfonline.com/loi/gcec20

Integrating Microwave Assisted Extraction of

Essential Oils and Polyphenols from Rosemary and
Thyme Leaves

Ioan Calinescu, Ioana Asofiei, Adina Ionuta Gavrila, Adrian Trifan, Daniel
Ighigeanu, Diana Martin, Constantin Matei & Mihaela Buleandra

To cite this article: Ioan Calinescu, Ioana Asofiei, Adina Ionuta Gavrila, Adrian Trifan, Daniel
Ighigeanu, Diana Martin, Constantin Matei & Mihaela Buleandra (2017): Integrating Microwave
Assisted Extraction of Essential Oils and Polyphenols from Rosemary and Thyme Leaves,
Chemical Engineering Communications, DOI: 10.1080/00986445.2017.1328678

To link to this article: http://dx.doi.org/10.1080/00986445.2017.1328678

Accepted author version posted online: 17

May 2017.

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 Integrating Microwave Assisted Extraction of Essential Oils and Polyphenols from
Rosemary and Thyme Leaves

Ioan Calinescu1, Ioana Asofiei1, Adina Ionuta Gavrila1, Adrian Trifan1, Daniel
Ighigeanu2, Diana Martin3, Constantin Matei2, Mihaela Buleandra4
1
Department of Bioresources and Polymer Science, University Politehnica of Bucharest,
Bucharest, Romania, 2National Institute for Lasers, Plasma and Radiation Physics,
Magurele, Ilfov, Romania, 3SC HOFIGAL SA, Bucharest, Romania, 4Department of
Analytical Chemistry, University of Bucharest, Bucharest, Romania

Address correspondence to Adina Ionuta Gavrila, Department of Bioresources and

Polymer Science, University Politehnica of Bucharest, 1-7 Gh. Polizu, 011061,
Bucharest, Romania. E-mail: adinagav@yahoo.com

Abstract

The extraction of essential oils (EOs) and polyphenols from rosemary and thyme has

been done using an integrated process: microwave hydro-diffusion and gravity (MHG)

for EOs and microwave assisted extraction (MAE) for polyphenols. The innovative

installation based on the MHG principle allows uniform microwave irradiation field due

to a mechanical stirring and experiments at low pressure. The results of quantitative

analysis of the EOs extracted by MHG after 10 min were similar with those obtained by

traditional methods (conventional hydro-distillation – CHD and microwave assisted

hydro-distillation – MHD) after 150 and 105 min, respectively. The specific energy for

MHG was 5 to 15 times lower compared to these classical methods. The MHG extraction

of EOs is also an effective method for plant material pretreatment before polyphenols

extraction. Total phenolic content increased from 35 to 55 mg GAE/g DM for rosemary

and from 23 to 38 mg GAE/g DM for thyme.

1
KEYWORDS: essential oil, polyphenols, microwave hydro-diffusion and gravity,

microwave assisted extraction, rosemary, thyme

INTRODUCTION

The Lamiaceae family represents one of the most important sources of spices which

comprise many phytochemicals (Hosni et al., 2013). Among this family, Rosmarinus

officinalis L. and Thymus officinalis L., which are native from the Mediterranean basin,

are commonly used as food flavouring and preservation (Ojeda-Sana et al., 2013; Sui et

al., 2012). They are also known for their antibacterial, antifungal, antioxidant, anti-

inflammatory, anti-carcinogenic, antiviral, anti-mutagenic, antiseptic and insecticidal

properties (Vallverdú-Queralt et al., 2014; El Hadj et al., 2015). These properties are

mainly due to the EOs and polyphenols content of these plants. Due to these biological

activities, the essential oil from rosemary and thyme is used in pharmaceutical, cosmetic

and agricultural industries, in natural therapies and alternative medicine or as constituents

of insecticides and disinfectants (Gavaric et al., 2015).

Essential oils consist of volatile aroma compounds, which are involved in the defence

mechanism of the plants. The main constituents of the essential oils are terpenes,

sesquiterpenes and numerous oxygenated derivative compounds such as aldehydes,

alcohols, ketones, phenols, acids, esters or ethers (Villanueva Bermejo et al., 2015;

Fornari et al., 2012). The bioactivity of the EOs derives from their chemical composition,

which is influenced by the plant genus and climatic factors (Rota et al., 2008). The main

compounds of thyme EO are thymol and its isomer carvacol, followed by p-cymene,

2
limonene and γ-terpinene (El Bouzidia et al., 2013). On the other hand, the main volatile

constituents of rosemary EO are camphor and 1,8-cineole, accompanied by verbenone, α-

pinene and camphene (Jiang et al., 2011; Derwich et al., 2011).

The rosemary and thyme have already been studied for their antioxidant capacity (Zill-e-

Huma et al., 2011b). The main polyphenols of rosemary are rosmarinic, carnosic and

ursolic acids (Jacotet-Navarro et al., 2015), while for thyme, the main components are

rosmarinic, caffeic, p-hydroxybenzoic and procatechuic acids (Vallverdú-Queralt et al.,

2014) although the content in polyphenols is lower.

Extraction of EOs from plant tissues is performed using several classical methods such as

steam-distillation, hydro-distillation and liquid-solvent extraction (Okoh et al., 2010;

Calinescu et al., 2014a). Although these conventional methods are rather simple, they

present many disadvantages, including large solvent quantities, long extraction times or

high energy consumption (Li et al., 2013; Calinescu et al., 2014b). Considering the

shortcomings of the traditional methods, many researches were conducted for alternative

techniques of the EOs extraction (Bousbia et al., 2009). Within these new approaches,

much attention has been given to MAE, ultrasound-assisted extraction (UAE),

pressurized solvent extraction, supercritical fluid extraction and solvent-free extraction

(Golmakani et al., 2008; García-Risco et al., 2011; Jacotet-Navarro et al., 2016).

Extraction of polyphenols has been also investigated using conventional solvent

extraction (Meziane-Assami et al., 2013), MAE (Mulinacci et al., 2011; Sui et al., 2012;

3
Alupului et al., 2012) or UAE (Rodríguez-Rojo et al., 2012; Albu et al., 2004) etc. Due to

polyphenols low solubility in water the most used solvents are ethanol, methanol

(mixture with water) (Durling et al., 2007) or acetone (Suhaj, 2006).

Microwave technology is a suitable method for the EOs extraction. There are many

configurations using microwave heating, involving microwave steam-distillation, MHD

and MHG (Vian et al. 2008, Cendres et al. 2011). The main advantages of using

microwave technology for the extraction of bioactive compounds are: short extraction

times, more effective heating, lower energy consumption, reduced thermal gradients, etc.

(Farhat et al., 2009, Asofiei et al., 2016). The MHG method was successfully tested for

the EOs extraction from plants (Binello et al 2014; Bousbia et al 2009; Perino et al 2013).

This technique combines the microwave heating with earth gravity (Vian et al 2008).

The principle of MHG consists on the swelling of the plant cell by heating the constituent

water, which determines further the rupture of the cell’s membrane and allows the

compounds of interest to diffuse outside the plant tissue. In the end, the extract drops by

gravity into the collector vessel (Bousbia et al. 2009).

With the development of the ‘‘Green Chemistry’’ concept, environment-friendly

techniques are becoming more and more attractive, therefore, researches aim at

optimizing the eco-friendliest way of extraction (Boukroufa et al., 2015). One promising

method used is to intensify the extraction process; accordingly, Sui et al. (2012) studied

the influence of microwave pretreatment on the EOs extraction and the stability of

4
polyphenols during this stage and in storage, after pretreatment. They concluded that the

polyphenols are stable during microwave pretreatment and following storage.

Our study describes an integrated process for both EOs and polyphenols extraction from

rosemary and thyme by consecutively use of MHG and MAE. The aim of the integrated

process is the use of an innovative experimental equipment based on MHG method for

EOs extraction, process which constitutes, as well, an efficient pretreatment method for

polyphenols extraction.

In all studied literature which refers at MHG method, there is not presented any

laboratory installation equipped with a stirring system of plant material that allow a

uniform microwave heating (Vian et al., 2008; Bousbia et al., 2009a; Bousbia et al.,

2009b; Zill-e-Huma et al., 2009; Farhat et al., 2010; Cendres et al., 2011; Perino-Issartier

et al., 2011; Zill-e-Huma et al., 2011a; Zill-e-Huma et al., 2011b; Cendres et al., 2012;

Al Bittar et al., 2013; Li et al., 2013; Perino-Issartier et al., 2013; Binello et al., 2014;

Cendres et al., 2014; Pérez et al., 2014; Boukroufa et al., 2015; Khan et al., 2016; López-

Hortas et al., 2016; Perino et al., 2016). The only pilot installation which is provided with

a rotating PTFE drum system for a uniform microwave heating of samples up to 75 L of

vegetal material is described by Perino et al. 2016. Our equipment is a laboratory scale

one with a small loading of plant material (approximatively 100 g). In order to prove the

necessity of the sample stirring, a series of experiments were carried out. Those studies

have shown that the absence of a stirring system led to a non-uniform heating of vegetal

material. The dielectric properties of some aromatic plants were determined by Navarette

5
et al 2011 and Galan et al 2017. Considering these properties, the microwave penetration

depth into the plant material was calculated and the obtained value was the order of

several millimetres (Tang, J. 2005). Since the penetration depth value is much lower than

the radius of the cylindrical vessel, a stirring system is required. Compared with the

equipment cited in the literature (equipment of well renowned manufacturers), our

installation was designed and built with a stirring system of vegetal material which

provides a uniform microwave heating and consequently a better yield of the extracted

products.

MATERIALS AND METHODS

Plant Material

Fresh rosemary and thyme (stems and leaves) were harvested at the beginning of October

2015 at Hofigal S.A. in Bucharest. The vegetable material was dosed in samples of 100 g

and kept at 4 ºC. The content of water of fresh rosemary and thyme was 65.2% and

71.5% respectively.

Chemicals

The solvents used were analytical grade and were purchased from VWR International

(Darmstadt, Germany). Folin–Ciocalteu and sodium carbonate Na2CO3 were purchased

from Sigma Aldrich (St. Louis, USA).

Protocol Treatment

6
All the experiments were carried out using 100 g of fresh plant material (rosemary or

thyme leaves and stems) sliced into 1-2 cm pieces. The essential oil extraction was

carried out by MHG. The residue sample resulted from MHG extraction was treated with

a solution of 50% ethanol in water (residual water obtained after MHG process) in order

to determine the total phenolic content (TPC). The performed protocol is presented in

Figure 1.

Microwave Extraction Equipment

An innovative experimental installation was specially designed and built for fresh plants

treatment by MHG (Figure 2).

As shown in Figure 2, the innovation of the equipment, compared with classical MHG

apparatus, consists of a stirring system and the ability to work at low pressures. The

treatment leads to separation of the intracellular water and EOs. The equipment has the

following characteristics: treatment reactor capacity 100-500 g, continuous setting of

microwave power between 0-700 W, continuous plant stirring (in order to allow a

uniform microwave irradiation and to avoid overheating and local degradation),

temperature monitoring and recording with an infrared device, condensation of resulted

vapours and EOs separation under gravity, normal or low pressure possibility for the

experiments.

Essential Oil Extraction Procedure

7
The EOs extraction was performed using three methods: CHD, MHD and MHG. For

CHD and MHD, 400 mL of distilled water were added to 100 g of fresh plant material.

The protocols for both conventional methods were described in our previous paper

(Calinescu et al., 2014b).

Microwave Hydro-Diffusion and Gravity

The extraction of the EOs from fresh rosemary and thyme was performed using the

innovative experimental installation presented in Figure 2.

The extraction conditions have been chosen considering the principle that the MAE is

more efficient when the extraction time is shorter and the microwave specific power is

higher (Li et al 2013). Other studies in the literature reported the extraction of natural

products at a specific absorption rate of 1 W/g of plant (Vian et al 2008; Bousbia et al

2009, etc.). Since our equipment is provided with a stirring system the extraction of

essential oils can be performed at higher specific power which further led to better

results. In Table I are presented the preliminary tests for establishing the appropriate

extraction conditions.

Considering the preliminary tests, the following experiments were carried out at 360 W

(set from microwave power supply) for 9 to 13 min. For each experiment, maximum

temperature was about 106 – 108 ºC. Before extraction, in order to improve the EOs

yield, distilled water was pulverized on the vegetable material (about 24 g for rosemary

8
and 25 g for thyme). This amount of water is absorbed by the fresh plant material. The

cooling agent temperature was 5 °C for all experiments.

Polyphenols Extraction Procedure

The vegetable materials resulted from the MHG extraction of EOs were used to extract

the polyphenols by MAE, using the same microwave system (Biotage Initiator) for both

type of plant materials. A MAE without the pretreatment of plants was also performed.

The experiments were carried out in triplicate, using a 20:1 (v:w) ratio of solvent to plant,

at a temperature of 60 °C and a stirring rate of 900 rpm for 450 s. The solvent used is a

mixture of 50% ethanol in water (residual water obtained after MHG process or distilled

water for untreated fresh plant material). After the extraction, the mixture was centrifuged

at 3000 rpm for 5 min at room temperature and the supernatant was collected and fresh

analysed every time. This procedure was determined to be optimal for polyphenols

extraction from sea buckthorn leaves (Asofiei et al., 2016).

GC-MS Analysis of the Major Compounds of the Eos

The GS-MS equipment used for qualitative analysis of the EOs was a Thermo Electron

GS-MS system that consists of a gas chromatograph and anion trap mass spectrometer, as

previously detailed in Calinescu et al. (2014b).

Determination of Total Phenolic Content

The total phenolic content of extracts was determined calorimetrically using the Folin-

Ciocalteu method according to ISO 14502-1 with minor modifications. The fresh extracts

9
were diluted 125 times with distilled water. Further, 0.5 mL of diluted extract was mixed

with 5 mL of 10% Folin-Ciocalteu reagent and stirred for 5 min to perform the reaction.

Next, 1.5 mL of 20% Na2CO3·10H2O and 3 mL of distilled water were added. Before

analysis, the samples were kept for 60 min in the dark at room temperature. The

absorbance was measured at 760 nm using a Shimadzu UV mini-1240 UV/Visible

Scanning Spectrophotometer, 115 VAC. The samples were analysed in duplicates. The

results were quantified as milligram of gallic acid equivalents per 1 gram of dry matter

(mg GAE/g DM) using a standard curve corresponding to 1-5 mg/mL gallic acid solution.

RESULTS AND DISCUSSION

The MHG experiments were compared with the classical extraction methods (CHD and

MHD). During the experimental work, for the entire extraction interval, the temperature

profile was recorded. The specific power and specific energy were calculated for each

extraction, using the following equations:

Psupplied
Pspecific (1)
mpm

Psupplied t
Especific (2)
mEO

Where: Pspecific - specific power, W/g of plant; Psupplied – power supplied to the system, W;

mpm – plant material weight, g; Especific - specific energy, J/g of EO; t – extraction time, s;

mEO – EOs weight, g.

The amount of each EOs compound for all experiments was calculated using the

equation:

10
 Ccompound
mcompound mEO 1000 (3)
100

Where: mcompound – the amount of each EOs compound, mg; mEO – EOs weight, g (see

Tables I and II); Ccompound – concentration of each EOs compound, % (see Tables V and

VI). Further, using this equation, the amount of the compounds with boiling points below

or higher than 200 ºC was calculated by summing up the amount of each corresponding

compound.

Quantitative Results of Eos From Rosemary and Thyme

The results obtained for EOs extraction from rosemary and thyme leaves and stems are

detailed in Tables II and III. The experimental conditions were chosen in order to

improve the extraction of the bioactive compounds (sub-atmospheric pressure and

addition of water). Thus, as shown in these tables, the amount of the EOs for both plants

is dependent on the type of extraction method: CHD, MHD or MHG (approximately 200

– 300 mg EOs/100 g of plant for thyme and 700 – 800 mg EOs/100 g of plant for

rosemary). The difference between these three methods consists in a shorter extraction

time for MHG (approximately 10 min with a lower specific energy) compared to 105 for

MHD and 150 min for CHD, respectively.

As shown in Tables II and III, the addition of water and a slight decrease of pressure lead

to a higher amount of EOs for both plants. When distilled water was pulverized on the

fresh plant material, the amount of the EOs was higher than the extractions without the

addition of water. The water content of fresh plant materials (71.5% for thyme and 65.2%

for rosemary) is not high enough to entrain all the EOs constituents, especially those

11
having relatively high boiling points (see Tables V and VI). As expected, the water

addition led to an increased amount of EOs components, especially those with boiling

points higher than 200 ºC (66% for thyme and 22% for rosemary). Reducing the pressure

to 0.7 atm favours an increased amount of the compounds with boiling points below 200

ºC (with 65% for thyme and 32% for rosemary, respectively). The reduction of pressure

favours the evaporation rate of the water inside the solid material, thus increasing the

cells membrane degradation. Therefore, the EOs compounds with lower molecular

masses (higher mobility due to the internal energy) are extracted faster than the

compounds with higher molecular masses. Consequently, for the same extraction time,

the concentration of the former will be slightly higher.

An advantage of this extraction equipment is the consumption of energy; the specific

energy is lower compared with MHD and CHD methods. Although the specific power for

MHG is few times higher than for classical methods (MHD and CHD), the specific

energy is about 5 to 15 fold lower. This is owed to the extraction times which are smaller

for MHG method.

Table IV shows the calculated values of vapour pressure for a mixture of water, α-pinene

(a more volatile constituent of EOs) and thymol (a less volatile constituent of EOs). The

influence of added water and reduced pressure on the amount of thyme EOs is higher

than for rosemary EOs due to the smaller content of the EOs found in thyme leaves, 300

mg/100g of plant for thyme compared with 800 mg/100 g of plant for rosemary.

12
Qualitative Results of Eos from Rosemary and Thyme

Thyme Essential Oils Results

The volatile compounds of thyme EOs samples obtained by MHG, MHD and CHD were

analysed and identified by GC-MS. For all methods, around 30 compounds were

identified by GC-MS analysis. The results are shown in Table V. The main constituents

of thyme EOs are thymol, γ-terpinene and p-cymene. These compounds represent

approximately 75% of the total amount of resulted EOs. As shown in Table V, in terms of

the EOs composition, there is only a slight difference between the three extraction

methods. However, the addition of water led to an increase of the percentage of the

extracted compounds (heavier compounds especially, thymol for example). The

composition of EOs for the extraction at reduced pressure is similar with that obtained by

classical methods (see exp. no. 3).

Rosemary Essential Oils Results

As in the case of thyme, the rosemary EOs samples, carried out using the MHG, MHD

and CHD methods, were subjected to GC-MS analysis and 23 components were

identified for all extraction methods. The results are shown in Table VI. The main

components of rosemary EOs are camphor, eucalyptol, α-pinene, verbenone and

camphene. These four constituents represent approximately 72% of the total amount of

resulted EOs. As shown in Table VI, there is only a small difference between MHG,

MHD and CHD samples. These results show a similar behaviour as in the case of thyme.

The highest percentage of the main components was achieved for the extraction in the

presence of water and low pressure (75.13%).

13
Total Phenolic Content of Rosemary and Thyme

The second part of this study is dedicated to polyphenols extraction from plant material.

Thus, the dried plant residue resulted from MHG extraction of EOs is further used for

polyphenols extraction by MAE. Besides the advantage of extracting EOs in a very short

time, the MHG can be considered a pretreatment method of vegetable materials.

In order to determine the efficiency of MHG pretreatment of rosemary and thyme leaves

before polyphenols extraction, the MAE of untreated vegetable material was performed.

The TPC of the pretreated leaves extracts of rosemary and thyme was determined. The

results are presented in Tables VII and VIII. It can be noticed that rosemary and thyme

leaves contain a significant amount of polyphenols. The polyphenols concentrations of

extract obtained from pretreated rosemary and thyme leaves are higher than those

obtained from untreated leaves (approximatively 40% higher than untreated leaves

extracts for thyme and approximatively 36% for rosemary respectively). The microwave

pretreatment of vegetable material before MAE causes the degradation of the cell wall,

thus releasing more easily the polyphenolic compounds.

Since the solubility of polyphenols in water is low, their concentration in the residual

water resulted from MHG extraction of EOs is small (approximatively 1.2 mg GAE/g

DM for thyme and only 0.04 mg GAE/g DM for rosemary). However, in order to

increase the efficiency of polyphenols extraction, it is recommended to use as extraction

solvent a solution of ethanol and residual water from MHG.

14
 CONCLUSIONS

The aim of this work was to extract EOs from fresh rosemary and thyme using an

innovative experimental installation. This equipment is based on the MHG principle and

provides a uniform microwave irradiation, due to the mixing provided by a stirring

system. The equipment permits working at low pressures and addition of extra water

before or during the extraction. For comparison reasons, the experiments were also

performed using the MHD and CHD methods. For both plants, the EOs yields obtained

using the innovative MHG equipment were similar with those obtained by classical

methods, but in significant shorter time: from approximatively 10 min to 105 min

extraction time for MHD and 150 min extraction time for CHD, respectively. In addition,

the specific energy for MHG compared with MHD and CHD is about of 5 to 15 times

lower for both plant materials. A way to improve the extraction yield was to increases the

water availability for vegetable material by pulverizing the plant material with distilled

water (about 25 mL for 100 g of plant). Reducing the pressure below 1 atm is also a

feature of MHG equipment to increase the yields. The influence of added water and

reduced pressure is more pronounced when the EOs content of plant is lower (a higher

influence for thyme compared with rosemary).

The MHG used for EOs extraction was also an efficient pretreatment of vegetable

material before polyphenols extraction. The pretreatment of rosemary and thyme leaves

led to an efficiency of polyphenols extraction from 35 to 55 mg GAE/g DM for rosemary

and from 23 to 38 mg GAE/g DM for thyme.

15
Further researches are required to investigate the choice of the most appropriate

technology for scale up and industrialization.

ACKNOWLEDGMENTS

The authors acknowledge the financial support received from the Unit Executive for

Funding Higher Education, Research Development and Innovation, Action Joint Applied

Research Projects, PN-II-PT-PCCA-2013-4, project: “Eco-friendly process for extraction

of valuable compounds from plants – ECOVALUEPLANT”, financed by contract:

172/2014.

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24
Table I. Preliminary tests to determine the extraction conditions (all the experiments

were performed by MHG method at a pressure of 1 atm)

Exp. Extraction EO [mg/100 g Specific power Specific

time [s] plant] [W/g plant] energy [KJ/g

EO]

Thyme

a 2480 178±23 1.00 1.39

1
b 612 184±25 3.60 1.20

c 437 152±18 5.00 1.43

Rosemary

d 2400 670±30 1.00 0.36

2
e 579 691±32 3.60 0.30

f 413 592±28 5.00 0.35

1
exp. b correspond to exp. 1 from Table II
2
exp. e correspond to exp. 6 from Table III

25
Table II. Extraction conditions and results for thyme essential oil

Exp Metho Adde Pressur Extractio EO Specifi Specifi Amount of

. d d e [atm] n timea [mg/10 c c compounds,

water [s] 0g power energy [mg/100 g

[g] plant] [W/g [KJ/g plant]

plant] EO] b.p. b.p.

< >

200º 200º

C C

1 MHG - 1 612 184±25 3.60 1.2 78.9 97.6

2 MHG 25.3 1 686 262±23 2.87 0.94 89.8 162.4

3 MHG 24.5 0.7 768 313±22 2.89 0.88 148.9 153.0

4 MHD 400 1 6300 299±17 0.43b 4.5 129.0 156.8

5 CHD 400 1 9000 305±19 0.76b 11.2 132.8 159.5

a
The extraction time means the time until the temperature of 106-108 °C was achieved
b
The specific powers for MHD and CHD (exp. 4 and 5) were calculated based on total

mass weight (plant and water)

26
Table III. Extraction conditions and results for rosemary essential oil

Exp Metho Adde Pressur Extractio EO Specifi Specifi Amount of

. d d e [atm] n timea [mg/10 c c compounds,

water [s] 0g power energy [mg/100 g

[g] plant] [W/g [KJ/g plant]

plant] EO] b.p. b.p.

< >

200º 200º

C C

6 MHG - 1 579 691±32 3.60 0.3 273.6 347.3

7 MHG 23.8 1 518 762±31 2.90 0.24 270.6 423.5

8 MHG 23.4 0.7 781 825±37 2.91 0.34 357.8 398.2

9 MHD 400 1 6300 769±22 0.43 b 1.76 355.1 329.7

10 CHD 400 1 9000 837±24 0.76 b 4.08 371.8 372.9

a
The extraction time means the time until the temperature of 106-108 °C was achieved
b
The specific powers for MHD and CHD (exp. 9 and 10) were calculated based on total

mass weight: plant and water.

27
Table IV. Vapour pressure of a water - α-pinene - thymol mixture at 95 ºC (total pressure

= 1 atm) and 85.7 ºC (total pressure = 0.7 atm) (webbok.nist.gov).

Temperature Total Vapour Vapour Vapour Mole Mole

[oC] pressure pressure of pressure of pressure of fraction fraction

[mmHg] water, α-pinene, thymol, of α- of

[mmHg] [mmHg] [mmHg] pinene thymol

95 760.3 633.6 121.2 5.5 0.1605 0.0073

85.7 533.8 444.9 85.5 3.4 0.1612 0.0064

28
Table V. Chemical composition of thyme EO extracted by MHG, MHD and CHD

RT Compound CAS Boiling Composition, [%]

point, Exp. no.

[ºC] 1 2 3 4 5

9.23 α-Pinene 80-56-8 155 0.52 0.38 0.99 0.62 0.68

9.49 Camphene 79-92-5 159 0.30 0.31 0.58 0.46 0.64

9.78 Sabinene 3387-41-5 163 0.06 0.05 0.08 0.07 -

10.07 β-Pinene 127-91-3 165 1.73 1.42 2.15 2.04 2.08

10.38 α-Phellandrene 99-83-2 171 0.21 0.19 0.33 0.29 0.27

10.59 α-Terpinene 99-86-5 173 1.50 1.41 2.05 1.96 2.06

10.63 p-Cymene 99-87-6 177 13.90 8.62 15.48 13.98 14.37

10.81 Eucalyptol 470-82-6 176 2.43 1.04 2.61 1.72 1.18

11.25 γ-Terpinene 99-85-4 183 18.55 17.92 20.78 19.62 19.75

11.36 trans-Sabinene 17699-16-0 201 1.38 1.07 0.77 0.58 0.62

hydrate

11.77 α-Terpinolene 586-62-9 187 2.14 1.73 1.66 1.74 1.82

11.85 Linalool 78-70-6 198 0.18 0.14 0.09 0.07 0.08

12.51 Camphor 76-22-2 204 0.70 0.09 1.28 1.47 0.94

12.93 Borneol 507-70-0 213 0.70 1.88 0.86 1.25 1.13

13.09 Terpinen-4-ol 562-74-3 209 0.23 0.23 0.22 0.31 0.36

13.24 α-Terpineol 98-55-5 219 0.37 0.21 0.32 0.29 0.21

13.43 Verbenone 1196-01-6 227 - - 0.22 0.33 0.11

13.79 Thymol methyl 1076-56-8 214 0.12 0.30 0.12 0.87 0.34

29
 ether

13.95 Isothymol methyl 31574-44-4 - 0.13 - 0.43 0.50

ether

14.50 Thymol 89-83-8 232 41.73 52.02 39.43 41.28 42.16

14.64 Carvacrol 499-75-2 236 2.66 2.79 2.43 2.25 2.26

15.36 Thymol acetate 528-79-0 241 0.21 0.08 0.14 0.10 0.08

16.75 β-(E)- 87-44-5 254 2.78 2.93 2.47 2.38 2.17

Caryophyllene

17.16 α-Curcumene 644-30-4 275 0.11 0.12 0.11 0.11 0.10

17.34 Germacrene D 23986-74-5 236 0.06 0.05 0.08 0.07 0.06

17.46 β-Curcumene - 273 1.51 0.63 0.45 0.68 0.98

17.64 γ-Cadinene 39029-41-9 271 1.35 0.18 0.45 0.22 0.44

17.80 δ-Cadinene 523-47-7 279 0.44 0.19 0.16 0.21 0.40

17.86 Caryophyllene 1139-30-6 279 0.01 0.08 0.12 0.08 -

oxide

18.56 γ-Eudesmol 1209-71-8 301 0.07 0.08 - 0.09 0.08

Total main 74.17 78.57 75.69 74.88 76.29

components

For experimental conditions, see Table 1.

30
Table VI. Chemical composition of rosemary EO extracted by MHG, MHD and CHD

RT Compound CAS Boiling Composition, [%]

point, Exp. no.

[ºC] 6 7 8 9 10

9.23 α-Pinene 80-56- 155 5.56 3.96 9.01 9.45 8.29

9.49 Camphene 79-92- 159 3.57 2.33 4.84 6.77 5.60

9.78 β-Pinene 127- 165 2.47 2.79 2.65 2.33 2.47

91-3

9.96 α-Phellandrene 99-83- 171 1.76 1.15 2.05 2.27 2.55

10.81 Eucalyptol 470- 176 21.39 20.49 21.02 21.56 21.33

82-6

11.25 γ-Terpinene 99-85- 183 1.03 0.86 0.79 0.82 1.11

11.36 trans-Sabinene hydrate 17699- 201 0.20 0.15 0.15 0.13 0.18

16-0

11.66 α-Terpinolene 586- 187 0.07 0.08 0.08 0.08 0.07

62-9

11.77 Linalool 78-70- 198 3.55 3.71 2.79 2.77 2.81

12.51 Camphor 76-22- 204 37.41 40.79 33.90 29.93 31.81

31
 2

12.93 Borneol 507- 213 1.87 2.28 1.53 2.00 1.61

70-0

13.09 Terpinen-4-ol 562- 209 0.72 0.87 0.66 0.68 0.71

74-3

13.24 α-Terpineol 98-55- 219 1.74 2.13 1.76 1.75 1.77

13.43 Verbenone 80-57- 227 3.21 3.62 6.36 5.49 5.92

14.64 Bornyl acetate 76-49- 228 1.25 1.04 0.68 0.79 0.90

15.92 α-Copaene 3856- 246 0.16 0.20 0.09 0.10 0.13

25-5

16.15 Methyleugenol 93-15- 254 0.20 0.25 0.18 0.11 0.09

16.75 β-(E)-Caryophyllene 87-44- 254 2.65 3.06 2.21 1.28 1.13

17.15 Humulene 6753- 166 0.48 0.59 0.42 0.26 0.23

98-6

17.64 γ-Cadinene 39029- 271 0.04 0.05 - 0.16 -

41-9

17.80 δ-Cadinene 483- 279 0.14 0.19 0.13 0.13 0.09

76-1

32
17.86 Caryophyllene oxide 1139- 279 0.34 0.47 0.32 0.14 0.13

30-6

18.56 γ-Eudesmol 1209- 301 0.04 0.06 0.04 0.06 0.04

71-8

Total main constituents 71.15 71.18 75.13 73.19 72.96

For experimental conditions see table 2.

33
Table VII. Extraction conditions and TPC results for thyme

Exp. Pretreatment Added Pretreatment Pretreatment TPC

Method water [g] pressure [atm] time [s] [mgGAE/g

DM]

1 MHG - 1 612 38.28

2 MHG 25.3 1 686 36.21

3 MHG 24.5 0.7 768 32.36

5 No - 1 - 23.12

34
Table VIII. Extraction conditions and TPC results for rosemary

Exp. Pretreatment Added Pretreatment Pretreatment TPC

Method water [g] pressure [atm] time [s] [mgGAE/g

DM]

6 MHG - 1 579 55.5

7 MHG 23.8 1 518 43.39

8 MHG 23.4 0.7 781 48.43

5 No - 1 - 35.29

35
Figure 1. Protocol treatment of rosemary or thyme

36
Figure 2. The innovative experimental installation using MHG

37