Trends in Analytical Chemistry 118 (2019) 338e351

Contents lists available at ScienceDirect

Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

An insight into the extraction and fractionation technologies of the

essential oils and bioactive compounds in Rosmarinus officinalis L.:
Past, present and future
Ameena Ali, Bee Lin Chua*, Yin Hui Chow
School of Engineering, Taylor's University, Lakeside Campus, No 1, Jalan Taylor's, 47500, Subang Jaya, Selangor, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Natural products in drug discovery are in high demand. Thus, researchers are driven to investigate
Available online 1 June 2019 existing and new technologies for effective separation and fractionation of bioactive compounds for
industrial applications. Rosmarinus officinalis L. is a medicinal and aromatic herb whose compounds hold
Chemical compounds studied in this article: global interest for their diverse ethno-pharmacological properties in the medicinal field. This study in-
Carnosic acid (PubChem CID: 65126) cludes the brief history, chemical composition and an extensive in-depth analysis of R. officinalis L. into
Carnosol (PubChem CID: 442009)
various extraction technologies (traditional, current and those with promising future prospects). Ac-
Rosmarinic acid (PubChem CID: 5315615)
cording to the reviewed studies, carnosic acid, carnosol and rosmarinic acid are the most investigated
Oleanolic acid (PubChem CID: 10494)
Ursolic acid (PubChem CID: 64945)
compounds because they are the primary contributors of the bioassays with the highest potential in
Camphor (PubChem CID: 2537) natural drugs. For the production of fractions enriched with these compounds, pressurized liquid and
1,8-cineole (PubChem CID: 2758) supercritical (SC) fluid extraction followed by SC antisolvent fractionation are amongst the most
p-coumaric acid (PubChem CID: 637542) extensively investigated technologies. Moreover, this study highlights the exploitation of deep eutectic
Caffeic acid (PubChem CID: 689043) solvent-based aqueous two-phase system for the production of enriched fraction from R. officinalis L.
© 2019 Elsevier B.V. All rights reserved.
Keywords:
Rosmarinus
Extraction
Fractionation
Carnosic acid
Carnosol
Rosmarinic acid

1. Introduction
Commonly known as rosemary, Rosmarinus officinalis L. of the
Lamiaceae family is a native Mediterranean herb with an intense
aromatic odour reminiscent of pine wood. Since ancient times, ar-
omatic plants and herbs, such as rosemary, have been utilised as
food additives and flavouring agents to improve the taste and other
organoleptic properties [1]. Although many members of the family
have been subjected to experimental studies validating their po-
tential in the industrial sectors, including sage, thyme, basil,
oregano, lemon balm and lavender amongst others, thyme and
rosemary hold the main focus, closely followed by sage [2]. The
potential of rosemary is further highlighted by the fact that an
* Corresponding author.
E-mail addresses: amenaali@ymail.com (A. Ali), BeeLin.Chua@taylors.edu.my
(B.L. Chua), YinHui.Chow@taylors.edu.my (Y.H. Chow).
average of 120 studies have been published for this plant alone
since 2010, and such studies are predicted to increase in the future
[3]. Fresh and dried rosemary leaves are currently used on a regular
basis in the culinary field. Abundantly available on a global scale,
the application of this potent herb also extends to the traditional
and folk medicine as rosemary is a rich source of phenolic com-
pounds, which possess anticancer [4], antioxidant [5], anti-
proliferative [6], anti-inflammatory [7], anti-hyperglycaemic and
anti-hyperlipidaemic properties. The essential oil (EO) of rose-
mary has seen its applicability in industrial products ranging from
colognes and bathing essences to components of disinfectants and
insecticides [8]. The potential of R. officinalis L. is limitless as it is an
abundant source of volatile and non-volatile natural compounds.
In a typical post-harvest production chain (Fig. 1), pre-treatment
(drying) is occasionally performed to reduce the moisture content
of plants/herbs and compounds for better preservation. For the
isolation or separation of the desired compounds (i.e. metabolites/
bioactive compounds), they are then extracted from unwanted raw

https://doi.org/10.1016/j.trac.2019.05.040
0165-9936/© 2019 Elsevier B.V. All rights reserved.
 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351 339

Abbreaviation and acronym list PEF Pulse electric field

PLE Pressurised liquid extraction
ASE Accelerated solvent extraction RA Rosmarinic acid
ATPS Aqueous two-phase system RSM Response surface methodology
BAC Bioactive compounds SAF Supercritical antisolvent fractionation
CA Carnosic acid SD Steam distillation
CR Carnosol SC Supercritical
DES Deep eutectic solvent SE Short-path evaporators
EO Essential oil SFE Supercritical fluid extraction
EU European union SFME Solvent-free microwave distillation extraction
GHz Gigahertz SWE Subcritical water extraction
HD Hydro distillation TFE Thin film-evaporators
MAE Microwave assisted extraction UAE Ultrasound-assisted extraction
MAHD Microwave-assisted hydrodistillation UATPS Ultrasound-assisted aqueous two-phase system
MD Molecular distillation extraction
MHDG Microwave hydro diffusion and gravity WHO World health organisation

DisƟllaƟon
EssenƟal Oils
methods

oleoresin

Rosmarinus Pre-treatment ExtracƟon

Assisted methods
officinalis L. technologies technologies

Carnosic acid
Extracts FracƟonaƟon
and carnosol
SupercriƟcal
technology

Rosmarinic
acid

Fig. 1. Typical production chain for natural compounds in for Rosmarinus officinalis L.

materials (i.e. insoluble cellular marc). Lastly, the extracted com-
pounds are subjected to either fractionation/purification on the
basis of the desired application prior to its occasional encapsulation
to improve their stability against environmental stimuli [9]. The
extraction process plays a vital role in the industry as extraction is
the most crucial first step in separating compounds with high
commercial value. Solvent extraction is the most common form of
extraction/separation process that has been extensively exploited
extensively for the separation of organic and inorganic compounds
[10]. Extraction processes are governed and affected by many fac-
tors such as internal chemical properties (bonding) and external
physical properties (temperature and pressure). In addition,
extraction processes can account for 40%e70% of the overall capital
and operating costs [11]. Thus, proper application can be beneficial
from an economic perspective. Furthermore, several stages of
extraction are often implemented to reach a certain level of purity,
which is followed by occasional fractionation [12]. Thus, devising
an efficient process with appropriate key design and operating
parameters that do not compromise product quality is imperative.
Such an efficient process can be achieved by carefully and sys-
tematically reviewing old and ongoing technologies being investi-
gated to identify the highs, lows and gaps together with the
potentials that can provide good outcomes.
The phytochemistry and biological activities of R. officinalis L.
[3,13] have been extensively studied, but no organised review is
available regarding the extraction technologies that can affect the
final quality of R. officinalis L. compounds. Due to the growing
interest in the medicinal properties of R. officinalis L., reviewing the
current trends that can best preserve the therapeutic properties of
this herb is crucial. Therefore, the current study reviews the pri-
mary contributors of the pharmacological properties of rosemary.
Moreover, this paper systematically presents a literature review of
published works on investigated extraction and fractionation
technologies, their advantages, limitations and future prospects.
Furthermore, this research highlights the novel approaches that
can be potentially used in the future.
2. Research method and criteria
The databases of PubMed, ScienceDirect and Scopus were
searched for published work since 1990 with the keywords of
‘Rosemary,’ ‘Rosmarinus officinalis L.,’ ‘extraction,’ ‘separation,’
‘fractionation’ and ‘purification.’ The search was solely restricted to
English language with an emphasis on peer-reviewed journals but
not limited to EO, phenolic compounds and/or isolated compounds.
The literature search was confined from 1990 to December 2018.
Reported studies, case studies, related reviews and books were
explored for the current review.
3. Brief history of R. officinalis L.
Rosemary is originally derived from the Latin words ‘ros-roris’
and ‘marinus,’ which translate to ‘dew of the sea’ [14]. Rosemary is
native to temperate Mediterranean regions, such as Italy, but is
340 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351
widespread in European, American and Asian countries [2,3]. Since
its discovery, rosemary has become a part of several cultures and
traditional medicine for curing gastrointestinal, skin and respira-
tory disorders amongst others [15]. Furthermore, from traditional
practices to modern applications, rosemary has spread into orna-
mental, cosmetic, culinary and other industries.
Rosemary or ‘Rosmarinus L.’ is a diverse group of flowering
plants belonging to the Lamiaceae family that spans over 236
genera and 7200 species globally [16]. Rosemary is an evergreen
dense bushy shrub that can reach up to 1.0 m in height with up-
right stems and whitisheblue flowers [17]. Its characteristically
curved, leathery, linear and aromatic leaves are 1.0e2.5 cm in
length, 4.0 cm in width, 1e3 mm thick with the upper and lower
surface coloured green and grey, respectively [14]. The rosemary
genus consists of a diverse species that includes R. officinalis, R.
eriocalyx, R. lavandulaceus, R. tomentosus and R. laxiflorus [15].
Amongst them, R. officinalis L. is the most exploited species in the
research community due to its valuable EO and bioactive phenolic
compounds (BACs) that are present in almost all parts (i.e. leaves,
stems, flowers and roots) of the plant.
4. Chemical composition of volatile and non-volatile
compounds in R. officinalis L.
The compounds that are responsible for the pharmacological
properties in R. officinalis L. can be grouped into the following
categories: flavonoids, di and tripenoids, monoterpenes, sesqui-
terpenes, alcohol, ester, ketone, hydrocinnamic derivatives and
other minor ones [15]. The major components of the volatile frac-
tion (EO) are the terpenes, whereas the non-volatile fraction
(extract) consists of flavonoids, certain non-volatile terpenoids and
phenolic acids. Phytochemical studies have indicated that the EO
composition in rosemary leaves ranges from 1.8% to 2.5% [17,18].
Physical characteristics imply that oil is colourless to pale yellow
with a characteristic flavour and taste [14]. Based on the chemical
composition from various regions (Table 1), the predominant con-
stituents are 1,8-cineole, a-pinene, camphor, borneol and limonene
with their concentrations varying due to differences in cultivator,
cultivation cycle and plant ageing [18e21].
To date, the major phenolic acids that have been reported in
rosemary's non-volatile fraction (extract) include rosmarinic,
chlorogenic, r-coumaric and caffeic acid [22]. Together with the
phenolic acids, the extract also contains a high amount of di and
triterpenoids, including carnosic acid (CA), oleanolic acid, betulinic
acid, ursolic acid, rosmanol and carnosol (CR) amongst others [3].
Other noted polyphenols are apigenin, luteolin and diosmin [20].
Amongst these listed compounds in the extract fraction, CA, CR and
rosmarinic acid (RA) are the most investigated compounds. Ac-
cording to a review study, 30% of the reported research involving
any form of biological activity from rosemary are attributed to CA,
which is closely followed by 17% CR, 12% RA and 6% ursolic acid [3].
These attributed percentages are primarily due to the fact that these
compounds are majorly responsible for the medicinal properties in
the extract. Therefore, these compounds have been exposed quite
extensively and exclusively in the research community.
The volatile and non-volatile composition of rosemary varies
with climate changes and plant age [21,23,24]. In addition, the
concentration of compounds is not equally distributed in all parts of
the rosemary plant as CA and CR are majorly found in the photo-
synthetic tissues of the plant, closely followed by sepals and petals
[25]. This is true in case of volatile compounds as well [26].
Moreover, various fractions are affected by processing technologies
and the degradation of these compounds can lead to loss of potent
medicinal value [5,27]. A proper extraction and fractionation
technology must be implemented at appropriate conditions
(temperature, time and solvent) to effectively isolate the com-
pounds without compromising their quality. Thus, the following
sections involve an in-depth understanding of the impact of various
extraction/fractionation technologies (Fig. 2) and respective pa-
rameters implemented over the years.
5. Extraction of the EOs and BACs of R. officinalis L.:
Traditional and novel extraction techniques
The extraction of EOs and BACs is essential for their separation
from undesired components to ensure that their bioactivities are
well preserved in many subsequent applications. To meet con-
sumer demand, research has shifted its focus onto the application
of natural compounds in medicine. According to World Health
Organization (WHO), the widespread interest in natural medicine is
exponentially increasing and will continue to do so provided the
soaring health care costs and rapidly rising chronic diseases [28].
Approximately 70% of the newly approved drugs for treating cancer
and other infectious diseases are based on natural compounds.
Moreover, extraction process can be an expensive affair. These
factors have driven the process industries to develop, integrate and
optimise an extraction process that can effectively and efficiently
separate natural compounds of interest. Therefore, the various
technologies currently being investigated and those with potential
need to be investigated.
Liquid-liquid and solid-liquid extraction methods have been
extensively utilised in the past, with the latter being more promi-
nent than the former. The most common classical extraction
techniques include maceration, Soxhlet extraction, steam distilla-
tion (SD) (separate) and hydro distillation (HD) (combined) [29].
The industry has moved on from techniques, such as maceration
and soxhlet extraction, as they suffer from several disadvantages,
including long extraction times, thermal decomposition of ther-
molabile compounds, low selectivity and solubility, high energy
and solvent consumption, contamination and loss of solutes [29].
These days, maceration and soxhlet are treated as reference
methods by researchers to demonstrate the efficiencies of the
newly developed methods or improved techniques.
To overcome the major challenges of conventional extraction
methods together with the preventing decomposition of natural
compounds, several new and promising non-conventional, non-
thermal ‘green’ extraction methods have been introduced and
investigated. They are ultrasound-assisted extraction (UAE),
microwave-assisted extraction (MAE), supercritical fluid extraction
(SFE), pressurized liquid extraction (PLE)/accelerated solvent extrac-
tion (ASE), pulse electric field (PEF) extraction, enzyme-assisted
extraction and molecular distillation (MD) [30]. Extraction effi-
ciency is not only affected by the type of method but also influenced
by the properties of the solute and solvent, such as temperature,
particle size, polarity and solvent concentration, pH, plant material
(climate and geography) and acidity [30]. Therefore, a great deal of
consideration is necessary when selecting appropriate extraction
methods and conditions in terms of economy and efficiency. Con-
ventional and non-conventional extraction processes that have been
utilised for the extraction of EOs and BACs from R. officinalis L. are
discussed in the consequent sections and presented in Table 2.
5.1. Steam and hydro-distillation
In any distillation method, the plant material is typically packed
in a still compartment. In SD method, water is added to a separate
compartment and boiled, and the resulting steam is passed through
the plant material. Alternatively, direct steam can be introduced to
the plant for extraction. In HD, water is added to the plant material
and boiled concurrently. In both cases, the steam, which contains
 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351 341

Table 1
Volatile composition of Rosmarinus officinalis L. from different regions.

Volatile compound Concentration (% relative peak areas)

India Italy Lebanon

Cevoli Lunigiana Shewiah Ballouneh Sehaileh

1,8-cineole 20.1 7.28 55.3 23.4 20.6 25.1

a-pinene 11.8 26.05 11.5 39 23.3 29.2
Camphor 26.8 10.06 8.14 2.1 3.7 3.1
Borneol 2.6 5.97 3.0 1.7 4.5 2.6
Bornyl acetate 1.1 ND 1.1 4.1 2.8
Verbenone 6.2 8.21 ND
b-pinene 2.6 0.38 6.52 3.9 2.1 2.7
Limonene 3.6 4.58 ND 3.6 2.3 3.4
Myrcene 1.9 3.13 1.67 1.5 1.1 1.5
Caryophyllene oxide ND 0.1 0.3 0.2
Tricyclene 0.2 ND
Camphene 5.3 5.6 2.57 4.4 2.7 2.8
b-Caryophyllene 1.4 ND 0.15 ND
a-Terpinene 0.5 0.36 0.63 0.6 0.4 0.5
Verbenol ND 0.4 0.7 0.5
Terpinene-4-ol ND 0.8 1.8 1.2
Geranoil 0.1 ND
Thymol ND 0.39 ND
Sabinene 0.2 0.96 ND 0.8 0.4 0.5
a-Terpineol 2.3 0.47 3.34 5.6 11.0 6.8
r-Cymene 1.9 1.23 0.86 1.4 0.9 1.1
a-Phellandrene 0.3 ND 0.2 0.1 0.2
Linalool 1.6 2.84 0.38 1.9 4.9 3.2
Eugenol 0.5 ND
Methyl Eugenol 0.1 ND
Terpinolene 0.5 ND 0.9 0.7 1.0
Carvacrol 0.1 ND
Cis-sabinene hydrate 0.4
Linalyl acetate 0.2
a-Humulene 0.4 ND 0.3 0.6 0.5
Citronellol 0.5 ND ND
d-Cadinene 0.3 0.4 0.2
g-Cadinene 0.2 ND
a-Cedrene 0.88
Geranoil 0.99 2.6 5.3 3.4
b-Phellandrene ND 2.16 1.7 4.7 4.3
g-Terpinene 0.98 1.3 0.9 1.4
Isopinocamphone ND 0.2 0.7 0.4
b-Terpineol 0.2 0.7 0.4
Geranyl acetate 0.3 0.6 0.4
a-muurolene 0.3 0.6 0.5

[21,26,100] ND e Not detected.

the dissolved oil, is condensed through indirect contact with water.
The condensed mixture is separated into oil and water components
in a different container, usually through decantation due to their
difference in densities [30]. These methods are still used in small-
scale extractions for various plants because of their simplicity,
wide applicability and low cost. Commercial-scale production with
SD still being used as the Clevenger type is recommended by the
European Pharmacopoeia for the recovery of EOs [31].
Although SD and HD follow a similar principle, SD is a superior
method. In a comparative study, the yield of EOs retained by SD
(1.2%) was 63% higher than by HD (0.44%) [27]. In addition,
approximately 80% of the volatiles (monoterpenes, sesquiterpenes
and oxygenated monoterpenes) were recovered within 10 min of
SD in contrast to HD, which took approximately 30 min. A similar
comparative analysis by other researchers also came to the same
conclusion that SD is a superior method for processing rosemary
[32]. The reduced volatile fraction in HD can be explained by the
hydrolysis reactions caused by the combined action of water and
high temperature that results in compound degradation. This
degradation can be avoided in SD as the compounds are not in
direct contact with water, thereby producing high-quality EOs.
Nevertheless, researchers always aim to improve the efficiency of
the process and exhaust the extraction of EOs from the plant in a
single step. Therefore, microwave technology is integrated with
distillation methods.
Microwave-assisted hydrodistillation (MAHD) is another unique
method that combines the mechanism of HD and MAE for
extracting of EOs from plant sources. The volumetric heating of
microwave causes the swelling and stretching of the glandular
trichomes causing them to burst and release oils. The evaporation
of the oil-water emulsion occurs, and this emulsion is condensed
and separated via distillation [33]. MAHD has obtained similar
yields at a reduced extraction duration of 15.5 min compared with
UAE, HD and SFE, which took 15 min, 30 min and 1 h, respectively.
Even with similar yields, the combination of volumetric heating
with distillation method in MAHD resulted in fast solubilisation of
volatiles. In addition, the efficiency MAHD over other distillation
methods has been demonstrated in terms of reduced time, energy
and cost without compromising the quality [34].
5.2. Solvent-free microwave distillation extraction (SFME) and
microwave hydro diffusion and gravity (MHDG)
The integration of microwave into extraction technologies, such
as MAHD and MAE, highly improves extraction efficiency. Two of
the novel integrations of microwave with other extraction
342 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351

Essen al Oil (EOs)

Rosmarinus
Bioac ve
officinalis L.
Compounds (BACs)

Pre-treatment

Milling Grinding Deoiling Pre-treatment

(drying)

Sun Oven Vacuum Microwave

drying drying oven drying drying

Ambient Solar Vacuum microwave

drying drying drying
Supercri cal
an solvent
frac ona on

Extrac on Frac ona on

Molecular
dis lla on
Steam Microwave Ultrasound Supercri cal Pressurized liquid/
Macera on Soxhlet subcri cal water,
dis lla on hydrodiffusion assisted fluid
and gravity extrac on microwave accelerated solvent
extrac on extrac on

Microwave Microwave- Solvent free Dispersive liquid-

Supercri cal
Hydrodis lla on assisted assisted hydro microwave liquid
fluid extrac on
extrac on dis lla on dis lla on microextrac on

Fig. 2. Compiled pre-treatment, extraction and fractionation technologies investiged for Rosmarinus officinalis L.

techniques are SFME and MHDG. In SFME, extraction is imple-
mented without adding solvents (organic or water). The internal
heating of the in situ water in the interior part of the plant ruptures
the oil bodies and oleiferous [35]. The released EO is evaporated
with the in situ water, which is then cooled and collected in a
separated vessel, whereas the excess water is recycled back into the
extraction vessel. MHDG combines the volumetric heating mech-
anism for the disruption of plant cells (glandular trichomes) to
favour the release of oil constituents from within. The mixture of
steam (in situ water) and EOs is collected on a spiral condenser out
of the reactor by gravity. On the basis of their densities, water and
EOs are separated in a different ‘Florentine flask’ vessel by decan-
tation [36]. Both methods do not use organic solvents, thus
reducing waste and environmental impact. SFME is an upgraded
distillation method, whereas MHDG is a completely unique
method. MHDG does not use organic solvents, such as MAE.
Furthermore, no water distillation or evaporation occurs in MHDG,
similar to MAHD and SFME. MHDG solely relies on internal heating
by microwave for extraction from which MHDG consumes little
energy.
The literature indicates that the advantages of SFME and MHDG
over SD and HD in terms of time, energy and cost savings. In one
study, MHDG produced an EO yield of 0.33% under 15 min, whereas
HD took more than 180 min to achieve the same goal [37]. More-
over, the MHDG-extracted oil contained 8.6% of camphor, which is
one of the major oxygenated monoterpenes in rosemary, whereas
HD-extracted oil contained 7.82% camphor. Similarly, SMFE pro-
duced EO yields of 0.54% within 30 min, which was comparable to
HD's 0.57% after two hours [38]. Substantially high-oxygenated
compounds were detected in SFME extracts. Such oxygenated
compounds correspond to high-quality EO. Both methods avoid
thermal and hydrolytic degradations that arise from the use of high
temperature with/without water, thus producing high-quality
extract in a short time without using solvents. Unlike SD and HD
that rely on the slow diffusion for extraction, the cell wall disrup-
tion contributed by microwave effect allows for fast solvent mass
transfer and overall extraction, thereby proving the superiority of
SFME and MHDG.
Microwave techniques are often overshadowed by uneven
heating. The energy primarily absorbed by polar (solvent/water)
molecules are often unevenly distributed in natural sources.
Therefore, an agitation (stirring) system is integrated into MHDG to
induce uniform heating during extraction [39]. The integrated
system of MHDG-extracted EOs within 10 min was similar to those
of HD and MAHD within 150 and 105 min, respectively. In addition,
the specific energy consumption was reduced by 5e15 times
compared with HD and MAHD. The same study considered MHDG
as a form of pre-treatment that improved the MAE extraction of
phenolic content by 36%. Likewise, SFME was applied as a pre-
treatment method to remove the EOs, and SFME enhanced the
maceration, as well as UAE and MAE extractions of BACs by two to
four factors [5]. Moreover, both methods were proven efficient for
other natural sources, such as MHDG for lavender [40] while SMFE
for thyme and lemon balm [41]. Therefore, concluding which
method is superior is difficult. However, all distillation methods, as
well as SFME and MHDG, are often sought for extracting EOs due to
the ease of extraction/solubilisation of volatiles by water. However,
solvent-assisted extraction techniques, such as UAE and MAE, are
preferred for non-volatile compounds (BACs) as they utilise organic
or non-polar solvents, which are more efficient [10]. Therefore, the
most heavily studied assisted technologies for recovering BACs are
discussed in the following sections.
 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351
Table 2
Compilation of the various extraction technologies and conditions for Rosmarinus officinalis L.
Extraction method studied Bioactive compounds
SD EO
MAHD Essential oil (EO)
MHDG Essential oil (EO)
SFE EO
SFME EO
Maceration CA, RA and carnosol
SWE CA, RA, carnosol, rosmanol,
flavonoids, total
antioxidant activity
PLE CA, RA, carnosol, total
antioxidant activity
UAE and MAE CA, RA, carnosol, flavonoids,
total phenols, total
terpenoids
Ionic liquid based CA, RA and EO
microwave assisted
simultaneous extraction
Ionic liquid based
ultrasound assisted
simultaneous extraction
Two-step SFE Extraction yield, TPC, total
antioxidant activity, CA and
CR
SAF TPC, TEAC antioxidant
activity, CA, RA and CR
MD EO
Best extraction conditions
Solvent: Water vapour, temperature: 100
C
Solvent: water: time: 15.5 min, power: 900 W
Power: 1 W/g for 500 g of sample, time: 15 min,
solvent free
Solvent: CO2, pressure: 220 bar, temperature:
40
C, time: 3 h
Microwave power: 150 W for 150 g of sample,
time: 30 min, temperature: 100
C, pressure:
atmospheric pressure
Solvent: 70% ethanol, Solid-liquid ratio: 1:5 g/
mL, time: 55 min
Solvent: water, temperature: below 100
C for
high polar compounds, 200
C for CA and similar
compounds, time: 15e30 min
Solvent: water (high to medium polarity
compounds), ethanol (medium to low polarity
compounds), temperature: 100e200
C, time:
20 min, Solid-liquid ratio: 1:11 g/mL
For UAE, frequency: 19.5 kHz, power: 140 W
For MAE, multimode under N2 (2 MPa) at 100
C
Solid-liquid ratio: 1:10 g/mL and time: 10 min
for both methods
Solvents: Acetone (for general recovery of CA
and RA), n-hexane (specific for CA)
For CA and RA
Solvent: 1.0 M 1-octyl-3-methylimidazolium
bromide, Solid-liquid ratio: 1:12 g/mL, time:
15 min, power: 700 W
For EO
Solvent: Water, Solid-liquid ratio: 1:10 g/mL,
time: 4 h, power: 700 W
Solvent: 1.0 M 1-octyl-3-methylimidazolium
bromide, Solid-liquid ratio: 1:20 g/mL, time:
30 min, power: 220 W
Step 1
Solvent: Neat CO2, time: 60 min, pressure:
300 bar, temperature: 40
C
Step 2
Solvent: CO2 with 7% ethanol, time: 120 min,
pressure: 150 bar, temperature: 40
C
Stage 1 (Raffinate)
Solvent: CO2, Feed: 20% (v/v) water, Pressure:
300 bar, Feed flow ratio: 0.025 extract/SC- CO2,
temperature: 40
C
Stage 2 (Extract)
Solvent: CO2, Feed: 50% (v/v) water, Pressure:
100 bar, Feed flow ratio: 0.025 extract/SC- CO2,
temperature: 40
C
Stage 1 and 2
Pressure: 0.078 bar, Evaporation temperature:
12
C, Feed flow: 0.02 mL/s, Condenser
temperature: 2
C, Motor speed: 200 rpm
Stage 3
Pressure: 0.065 bar, Evaporation temperature:
12
C, Feed flow: 0.024 mL/s, Condenser
temperature: 2
C, Motor speed: 200 rpm
343
Conclusions Ref
The extraction yield efficiency of EO was [27]
significantly improved in terms of reduced duration
as 80% of EOs was recovered by SD within 10 min
whereas HD took 30 min for same results
This approach produced similar results to HD and [33]
slightly lower quantified volatile fraction than UAE.
However, MAHD was demonstrated to be a cleaner
method with reduced extraction period and higher
repeatability
This new method achieved the same yield as HD [37]
with 180 min. The proposed method was of
significantly low cost as well as reduced carbon
dioxide emission and energy consumption
compared to HD
The yields of EO was optimised by manipulating the [83]
pressure and temperature conditions of SFE
Pilot-scale study had similar results to lab-scale [38]
study in terms of EO's yields. The power
consumption and extraction time were drastically
reduced when compared to HD. Overall, this study
demonstrated the applicability of SFME in the
industry
High concentration of the three compounds was [44]
obtained in the final extract. Additionally, the final
extract contained higher antioxidant properties
than the raw material
This method is highly effective for both polar and [58]
non-polar compounds as the polarity properties of
water can be manipulated by altering the
temperature
PLE provided the best results on the basis of [65]
individual compounds and antioxidant activity.
However, good results for different polarity
compounds were obtained separately
This study investigated multi-stage extraction with [88]
different solvent each time. Overall, it was
concluded that acetone, in general, produced the
best quality, whereas, n-hexane resulted in highest
CA among all the solvents investigated
The proposed two-step method of microwave pre- [90]
treatment followed by MAE with IL resulted in
simultaneous extraction of highest yield of RA and
CA with reduced time. However, the higest yield of
EOs were obtained with MHD
The extraction efficiency of RA was improved by [8]
more than 40% by using ILs paried with UAE in
comparison to 80% ethanol with maceration, heat
flux, soxhlet, UAE and stirring extraction. CA,
however, was highest with 80% ethanol despite the
extraction method implemented rather than the IL.
The yields of CA and CR at the said conditions [68]
produced highly enriched fractions at step 2 after
removal of waxes and oleoresins in step 1. Even
though, the yields of CA and CR were similar to
single step SFE, the two-step SFE has the additional
advantage of reduced extraction time
By optimising the conditions through response [6]
surface methodology (RSM), the raffinate fraction
was enriched with RA by 2.7 fold. The extract was
enriched with CA and CR by 2 fold. Complete
fractionation was not achieved as CA and CR were
still detected in the raffinate fraction, however, it
was noted that this was the maximum separation
achieved in literature till date
Through the manipulation of the operating [75]
conditions, the concentrations of EOs in residue and
distillate fractions can be varied
344 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351
5.3. Microwave-assisted extraction (MAE)
MAE is a non-thermal extraction technique that also relies on
the mechanism of volumetric heating for extracting desired com-
pounds from natural sources. MAE is based on the physical mech-
anisms of dipole rotation/reorientation and ionic polarisation. In
the presence of an electric field generated by microwave radiation,
the orderly aligned polar molecules (water) begin to violently
vibrate to the rapid alternating current [42]. The dipoles (positive
and negative) of the polar molecules continuously aligning them-
selves with the constantly alternating electric field at high fre-
quency. For a common microwave frequency of 2.45 GHz, the
electric field changes direction 49000 times per second making the
dipoles move with them. Such continuous collision motion pro-
duces a considerable amount of frictional heat that results in the
heating and evaporation of microscopic traces of moisture inside
the plant sources. Tremendous pressure built inside the plant's cell
wall causes it to rupture from the inside. As a result, the constitu-
ents from inside the plant material explode into the extracting
media. Additionally, the disruption of hydrogen bonds promote the
solvent penetration into the plant matrix, leading to faster extrac-
tion [30].
Similarly to another assisted extraction method UAE, the
application of MAE on rosemary's BACs and EOs has been investi-
gated by several researchers [5,43,44]. However, the choice of
assisted extraction method remains debatable as each method has
its own advantages and undesirable consequences. Overexposure
to ultrasound in UAE may lead to structural alteration and disin-
tegration of bioactive compounds, resulting in reduced bioactivity
[45]. The heat energy reflux due to microwave effect in MAE results
in thermal degradation of phenolic compounds [46].
Due to the long-term effects, the stability of CA and RA were
tested by exposing the pure compounds to high-power ultrasound
at 200 W for 30 min [8]. Recoveries of more than 90% demonstrated
the compound stability in ultrasound. On the otherhand, degra-
dation of CA under reflux microwave was reported, although RA
was thermo-resistant [43]. Furthermore, researchers do not advise
MAE for rosemary because the internal volumetric heating in-
creases solvent (ethanol) temperature leads to its vaporisation.
Therefore, using an additional refrigeration column with reflux was
encouraged to tackle this arising issue [5]. The thermo-sensitivity of
certain compounds demonstrates that UAE is a more suitable
extraction method than MAE for rosemary as further elaborated in
consequent sections.
5.4. Ultrasound-assisted extraction (UAE)
The application of ultrasound for extracting of several metabo-
lites at a lab scale began in the 1950s. UAE relies on the effect of
acoustic cavitation and its resulting mechanical and thermal effects
for extraction. During sonication, ultrasound waves propagate
longitudinally through an extracting medium, creating alternate
regions of rarefaction (expansion) and compression [47]. Due to the
alternating pressure, regions of negative pressure or the ‘cavities/
micro-bubbles’ of dissolved gases are generated and then absorb
energy from the sound waves. With each passing cycle, the bubbles
expand and grow as a result of large surface areas and high diffu-
sion of dissolved gases called rectified diffusion. A critical stage is
reached when the provided ultrasonic energy is not ample to
maintain the vapour pressure inside the bubbles, causing them to
implode. Shock waves are generated in the medium that induces
shear forces and turbulence, which enhances the mass transfer of
the system (extraction) [48].
Over the years, researchers have proposed various underlying
physical mechanisms of the UAE method. Sonoporation,
fragmentation, ultrasound capillary effect, erosion, local shear
stress, micro-jet formation and detexturation are listed in review
articles [48]. These effects contribute to cell wall destruction and
particle size reduction. Such results improve the reaction rate
without affecting the structure, properties and function of the
desired extracting material [49]. Due to its ease of operation and
maintenance, the application of UAE for the extraction of natural
compounds is common because UAE is recognised as the easiest
approach to be integrated into the industry [50]. With multiple
pilot-scale studies proving their viability in the industry, large-scale
high-power ultrasound reactors from 500 W (analytical purpose) to
16 KW (industrial application) of reactor volume ranging from 30 L
to 1000 L with processing capabilities of 5 L/min to 500 L/min are
commercially available from companies, REUS (France) and
Hielscher (Germany) [48].
A pioneering work revealed that UAE to produce higher CA yield
than maceration [51]. This result is expected because the structural
changes from ultrasound lead to enhanced mass transfer. By
contrast, maceration relies on the slow softening of intact cell walls
for solvent diffusion and penetration to solubilise the compounds
inside the plant cell [29]. In addition, the maximum extraction yield
with sonication from a probe (18.8%) and reactor system (18.1%) is
more effective than a bath system (13.1%) at 40
C with 90% ethanol
[51]. Ultrasound probe and reactor is more effective in inducing
acoustic effects because it produces more uniform energy, higher
power and direct delivery of ultrasound than bath systems [48].
UAE with de-oiling pre-treatment via SMFE and milling improves
the yield, phenolic content, quantified CA and RA by more than two
factors when compared with maceration and UAE [5].
The highest yields of RA (15.4 mg/g) and ursolic acid (15.8 mg/g)
were reported in UAE extracts, whereas the maceration extracts
recorded a maximum oleanolic acid of 12.2% mg/g [52]. This phe-
nomenon suggests that the extraction parameters also have
prominent effects on the extraction efficiency rather than the
method itself. For instance, RA in UAE extracts was the highest with
70% ethanol, whereas ursolic acid in UAE extracts was at the
maximum with 90% ethanol. The importance of using an appro-
priate solvent during extractions is further discussed in Section 6.1.
The feasibility of UAE on the industrial level has been investi-
gated for two different batch extraction scale-ups from 0.1 L to 20 L
and 125 L by vertically submerging six transducers into the
extracting vessel and operating at 40 Hz and 35
C [53]. High yields
of CA (22 mg/g) and RA (1.7 mg/g) obtained at 125 L in comparison
to CA (21.5 mg/g) and RA (1.1 mg/g) obtained at 25 L after 60 min of
extraction demonstrated the method's effectiveness. No pilot study
has been conducted on rosemary, but reviews and studies on other
natural sources have indicated UAE to be a viable economic option
for large-scale production in terms of reduced duration, cost and
power consumption [48]. Nevertheless, the issue of sustainability is
still attached due to the use of organic solvents. Thus, researchers
are reluctant to label these assisted methods ‘green,’ unlike the next
two methods discussed in the subsequent sections.
5.5. Pressurized liquid extraction (PLE)
Apart from MAE and UAE, another type of assisted extraction
method that is typically investigated is PLE. Initially described and
proposed as a method for sample preparation, PLE follows the
concept of high pressure and temperature to retain solvents in a
liquid state for extraction [54]. In PLE, the plant material is packed
in the extraction cell in between cellulose filter paper and sand
layers that are pumped with the solvent. After a certain duration,
the cell is flushed with nitrogen at controlled pressure and tem-
perature to solubilise the components. The extract is filtered and
collected after the final purge [55]. This method has proven that the
 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351
combination of high pressure and temperature improves solubility
and mass transfer rate, as well as reduces solvent viscosity and
surface tension, thus enhancing extraction speed and yield [56]. At
present, this method has several names, including ASE and
subcritical water extraction (SWE) amongst others. PLE is consid-
ered a green extraction technique due to the low volume of solvent
used and short extraction times. Moreover, it can be even greener
when using water, in this case it's called PHWE (pressurized hot
water extraction) or SWE (superheated or subcritical water
extraction)". PLE provides an additional benefit over the previous
two assisted methods as water is non-toxic, non-flammable, cheap,
environmentally friendly and recyclable.
The application of PLE for extracting BACs from rosemary has
been widely studied [57,58]. PLE is the most common method for
this herb alongside SFE [59]. An optimisation work indicated that
the highest antioxidant activity (56%) together with maximum RA
(15.23 mg/g) and CA (10.99 mg/g) was obtained using PLE coupled
with 56% methanol at 129
C [59]. Moreover, the same study
revealed that temperature influences the extraction of BACs. Sec-
tion 5.1 discusses such an extraction. Extracting CA and CR through
PLE with hexane as solvent is promising because hexane has high
selectivity for lipophilic compounds, such as terpenoids [60]. The
major advantage of PLE lies in the use of deoxygenated water that
allows the usage of high temperature for the enhanced extraction
of CA and CR. Many studies suggest that both compounds are more
prone to degradation (oxidation) in the presence of oxygen than
high temperature by itself [58,59]. However, PLE is not recognised
by WHO for producing rosemary extracts. Its limitations in com-
parison to SFE are further highlighted in Section 5.6.
5.6. Supercritical fluid extraction (SFE)
SFE is implemented in single- or multi-stage extraction and/or
occasionally fractionation. SFE uses solvent at supercritical (SC)
state where the solvent does not possess any singular gas or liquid
properties. Beyond the critical point at a characteristic temperature
and pressure, the solvent has gas-like properties of viscosity, sur-
face tension and diffusion with liquid-like density and solvating
capacity [61]. A typical SFE extraction system consists of an
extraction vessel, CO2 tank and pump, heat exchanger, gauge,
control valves, controller and instrumentation, and yield trap, and
occasionally co-solvent vessel, pump and separators [61]. The plant
sample is placed inside the extraction vessel amongst layers of glass
beads. The sample is allowed to reach optimum temperature before
being injected with highly pressurized and compressed CO2 for a
desired period. Following the extraction, the valves are released to
directly obtain the sample or to be transferred to subsequent sep-
arators for further fractionation [62].
The main advantage and its limitation lie in the use of the sol-
vent, CO2 and the equipment to a certain extent. SC CO2 has a high
diffusion coefficient with low surface tension and viscosity; it is
also non-toxic, reusable and recyclable with properties that are
achievable by manipulating pressure and temperature, unlike
liquid/organic solvents [30]. SFE's additional advantages include
reduced extraction period, easy removal of solvent through
depressurization and effective in small-scale extractions compared
with conventional methods. The inefficiency of CO2 lies in its
inability to solubilise polar compounds because most targeted
bioactive compounds that are pharmacologically active are polar in
nature [63].
The major volatile composition (monoterpene, sesquiterpene
and oxygenated aliphatics) obtained from all SFE, HD, UAE, SD and
MAHD methods are similar, but the lowest extraction yield is re-
ported for SFE due to the poor selectivity and non-polar nature of
CO2 [33]. Moreover, the yields of CA (94.84 mg/g) and CR (6.81 mg/
345
g) in SFE extracts are half of those of CA (161.49 mg/g) and CR
(13.64 mg/g) in PLE extract [60]. This finding further explains why
the overall TEAC (Trolox equivalent antioxidant activity) of PLE
(0.721 mmol trolox/g) is significantly higher than SFE extracts
(0.578 mmol trolox/g). PLE is performed at 100
C and 100 bar with
hexane, whereas SFE is implemented at 40
C and 300 bar with CO2.
Moreover, a comparative study has indicated that the antioxidant
activity of SFE extract (37.55 mg trolox/g) is 14 times better than SD
(2.65 mg trolox/g) and HD (2.10 mg trolox/g), but the yields for
distillation methods are higher [32]. Inconsistencies between the
phenolic content and antioxidant activity of rosemary were noted
before because only few selected bioactive compounds are active
[3]. Major bioactive compounds, such as CA, CR and RA, were not
quantified in the previous study. Therefore, why such a high anti-
oxidant activity (14 times) was obtained is unclear. Nevertheless,
the authors claimed that the seasonal variation can influence vol-
atile composition.
CO2 is inefficient for its non-selectivity towards polar com-
pounds. The yield of CA with SFE can be improved by influencing
the properties of CO2 by manipulating temperature and applying
high pressure [64]. Therefore, organic solvents are still added into
the system as co-solvents (modifiers) to improve the overall effi-
ciency and compete with other existing methods. The extraction
yield of SFE (3.1%) was doubled (6.5%) simply by adding a modifier
(7% ethanol) whilst keeping other parameters constant (40
C at
100e150 bar for 300 min) [65]. The extraction of major compounds
(CA, CR, p-coumaric acid and caffeic acid) were improved, except
for RA. The inclusion of organic solvent, namely, ethanol, allows for
the good solubilisation and extraction of natural compounds, which
are polar in nature. Moreover, CA, CR, p-coumaric acid and caffeic
acid have moderate polarity that closely resemble ethanol, unlike
RA, which is polar. Thus, the extraction of other compounds is
improved by the modifier, unlike RA.
Similar to the two assisted methods previously mentioned, the
selection between SFE and PLE is debatable due to the solvent
system. PLE at 100
C and 100 bar is more efficient for high polar
compounds, such as RA and caffeic acid, due to its usage of water.
On the contrary, SFE at 100
C and 100e150 bar with CO2 and 7%
ethanol is effective for moderate polar compounds, such as, CA, CR
and p-coumaric acid [65]. Since CA and CR are used as quality in-
dicators of rosemary extract amongst other compounds, SFE with a
modifier is a recognised and accepted method by the European
Union (EU) and WHO to produce rosemary extract, unlike PLE [66].
Furthermore, the greenness of the methods can be assessed
through life cycle assessment, a vital tool used for assessing the
environmental impact of a process, such as global warming or
carbon footprint, throughout the entire cycle. A life cycle assess-
ment on pilot-scale PLE and SFE concluded the later to be more
environmentally friendly on the basis of multiple categories
including global warming, ozone layer depletion, human toxicity,
fresh water ecotoxicity, marine aquatic ecotoxicity and terrestrial
ecotoxicity [67]. Although, it has to be mentioned that the assess-
ment, based on obtaining 10 g of rosemary extract, included the
primary extraction unit (SFE and PLE) and subsidiary equipments
such as oven, pumps, freeze dryer and rotarvapor only, while
excluding all upstream and downstream operations and equip-
ments. While both processes had minimal impact due to use of
green solvents, they still contributed to environmental impact with
the major factor being the amount of CO2 generated from electricity
production. However, it must be highlighted that the contribution
of CO2 in global warming or carbon footprint of PLE that was three
fold than that of SFE. Similar results were also obtained for human
toxicity, marine aquatic ecotoxicity as well as terrestrial ecotoxicity.
Taking all factors into consideration, it is no surprise that SFE is
favoured over PLE.
346 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351
Due to the standard sets by authorities, researchers are inclined
to exhaust the usage of SC technology to produce enriched fractions
of BACs, following its extraction to improve its purity. Therefore,
fractionation technology is implemented by SC CO2, and is dis-
cussed in the following section.
5.7. Supercritical antisolvent fractionation (SAF)
Fractionation is defined as a separation process where a mixture
is segregated into multiple fractions to enhance the content of
target compounds [12]. The primary purpose is to produce fractions
enriched with active constituents and remove unwanted com-
pounds to produce semi-purified products. Therefore, rosemary
extracts are often subjected to fractionation technologies that cater
towards producing CA and CR-enriched fractions to meet the re-
quirements set by EU [66]. Fractionation can be achieved in two
ways. The first method is through a multi-stage SFE process with
two consequent separators operated at different conditions to
manipulate the solubility of compounds.
In a two-step sequential SFE process applied other authors, the
waxes and oleoresin fraction were removed using neat CO2 at
300 bar and 40
C at the first stage, whereas CA and CR remained
soluble. Therefore, CA- and CR-enriched fractions (10 times more)
were precipitated and obtained in the second stage using CO2 with
7% ethanol at 100e150 bar and 40
Ce50
C [65,68]. Fractionation is
achievable as the solubility of compounds is manipulated according
to their molecular weights in different temperature and pressure
levels (further explained in Section 5.1). By removing the oleoresins,
a CA-enriched fraction (10 times more) was obtained. In addition, the
fractionation of the oleoresin was further enhanced by 20% with an
increase in pressure from 150 bar to 300 bar in the first stage at 40
C.
The second method to obtain enriched fractions of targeted
compounds is by using SAF. SAF is a unique fractionation method
that uses subcritical carbon dioxide fluid technology. A polar liquid
mixture (extract with solvent) is brought into continuous contact
with CO2 at moderate temperature and pressure inside a chamber.
The high-pressure CO2, which acts as an antisolvent, decreases the
solubility of high polar (water and hydrophilic compounds) com-
pounds, whereas the moderate polar compounds remain dissolved.
A fast supersaturation of the insoluble high polar compounds takes
place, causing them to precipitate at the bottom of the first sepa-
rator that is collected as the raffinate. The soluble components are
transferred to a different chamber/separator of reduced pressure
that precipitates the moderate polar compounds in the second
fraction called extract [69]. Through SAF, production of enriched
fractions and removal of impurities can be simultaneously achieved
in multiple stages.
In an extended work, SAF was used to simultaneously achieve
fractionation and encapsulation of two separate fractions contain-
ing CA and RA [70]. However, the recovery of the compounds was
low, with losses of more than 50%. Note that purification was not
achieved due to the limitations of the equipment (vessel and filter
devices). The fractionation of RA, CA and CR was achieved a few
years later where the raffinate was enriched with RA by 2.7 times,
whereas the extract fraction was enriched with CA and CR by two
times [6]. The raffinate fraction contained a certain amount of CA
and CR, whereas the extract fraction did not quantify any RA.
Pressure, temperature and ethanol contents were identified as the
driving factors in a pure SC-CO2eEtOHeH2O system, and the in-
crease in the two latter parameters improved CA solubility [71]. On
the basis of mole fraction equilibrium data for the ternary system of
SC-CO2eEtOHeH2O, low pressure, low feed/SC-CO2 flow rate ratio
and high water content resulted in the maximum separation of RA
from CA and CR [6,72]. Thus, solvent polarity is an important factor
even during fractionation of compounds. The application of SC
technology in consecutive stages (SFE-SAF) can further improve the
process with the enhanced solubility of compounds [72].
5.8. Molecular distillation (MD)
MD is another separation technique that uses the concept of
distillation (partial evaporation) to achieve fractionation under
vacuum at temperatures below its boiling point [73]. MD is suitable
for thermosensitive compounds because the application of low
temperature and vacuum condition avoids degradation through
oxidation. In an MD process system, the extracted material is intro-
duced into a unit consisting of an evaporator, condenser, rotor and
vacuum pump [30]. The feed is agitated with the rotor wiper system
under high vacuum conditions. By tuning the parametric conditions
of evaporator temperature, pressure, wiper speed and flow rate,
volatile (lighter) and heavy components are separated into distillate
and residue fractions, respectively [30]. The primary difference be-
tween any distillation and MD is that the distance travelled by the
evaporated molecules from the evaporator to the condenser is
shorter than the mean free path (i.e. the average distance travelled by
evaporated molecule without colliding with one another) [74]. In
industrial MD systems, two types of evaporators exist at present:
thin film-evaporators (TFE) and short-path evaporators (SE). The
difference lies in the positioning of the evaporators where in SE, the
condenser is placed in the centre of the evaporation unit, whereas in
TFE, the condenser is placed outside the unit [30].
In this study, a three-stage TFE MD was performed on rosemary
EO to purify volatile compounds. The residue was subjected to
consequent two additional stages of MD for further purification
[75]. Volatile and light fractions (a-pinene and myrcene) were
separated in the early stages as they distillate at 78 bar, whereas the
heavy fraction (camphor and 1.8 cineole) was removed as residues
at latter stages at 65 bar. In an extended work, a four-stage MD with
harsh conditions of 0.06 bare0.0075 bar was investigated [76]. A
similar observation was made with light fractions being distilled
first and heavy fractions being distilled later at harsher conditions
than 0.06 bare0.0075 bar. By introducing severe vacuum condi-
tions in stages, the boiling point of the heavy compounds can be
manipulated, thus, facilitating fractionation. So far, no research has
focused on the application of MD on rosemary for BAC fraction-
ation. One possible reason can be the use of water that can lead to
the hydrolytic degradation of CA. Nevertheless, the application of
MD is listed as one of the acceptable separation methods for the
BACs by EU [66].
6. Influence of extraction parameters on the EOs and
bioactive compounds in R. officinalis L.
The extraction parameters that influence the recovery of natural
compounds are the solvent type, solvent concentration, solvent-to-
solute ratio, temperature and time [29]. However, parameters, such
as temperature and pressure for SFE, ultrasound power and fre-
quency for UAE, microwave power for MAE and solvent toxicity
properties for PLE, are specific towards for these method them-
selves. From the current literature review, the process parameters
with the most impact were temperature, solvent type and pressure
(SFE) because they heavily influenced the recovery of EOs, CA, CR,
RA and other compounds. Therefore, these parameters are dis-
cussed in the following sections.
6.1. Effect of temperature and pressure on the recovery of
compounds
Certain compounds are thermolabile, hence, high processing
temperatures can lead to their degradation and property loss [10].
 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351
The compounds in rosemary are no exception to this phenomenon.
In this study, RA degradation was reported when the applied
temperature exceeded 129
C in ASE [59]. The result corresponded
to an increase in caffeic acid concentration because RA is an ester of
the latter compound and 3, 4-dihydroxyphenyllactic acid. However,
despite the degradation, the antioxidant properties improved
beyond 129
C. A similar study reported that caffeic acid is a more
potent antioxidant than RA. Melanoidin production from Maillard
reactions was observed to enhance simultaneously. Several studies
have indicated that melanoidins produced from Maillard reactions
possess high antioxidant, antimicrobial and antibiotic potencies
[77]. However, Maillard reactions produce potentially harmful
compounds (mutagens, carcinogens) [77]. Therefore, such reactions
must be avoided by not using temperatures above 129
C.
CA is more thermosensitive than RA because CA drastically de-
grades beyond 50
C to other diterpenes (CR) [78]. Stability studies
found that CR may further degrade to produce other components,
such as rosmanol, epi and iso-rosmanol in a chain reaction through
stability studies [79]. Losing these compounds results in decreased
bioactivity. Thus, applying low temperatures or non-thermal pro-
cesses during their extraction is more favorable [25]. Moreover,
fresh rosemary leaves should be directly subjected to extraction as
the drying temperature and time may inversely affect the com-
pounds and their consequent activities [80].
The importance of temperature does not end with the stability
of compounds. Temperature in conjunction with pressure can in-
fluence solvent properties during the extraction with SFE because
both parameters manipulate solvent viscosity and density in the SC
state [64]. At mild temperature and high pressure, the viscosity of
CO2 spikes with increasing pressure that is a gas-like behaviour that
allows for easy movement of particles [81]. Likewise, the density of
CO2 increases, making it behave similarly to liquid solvents, thus
allowing for easy penetration into the porous sample matrix and
improved molecular interactions. Overall, this behaviour enhances
solvating power and the solubility of solutes in the solvent. This
enhancement can be a direct result of the compression effect
because an increase in pressure diminishes the mean distance be-
tween the solvent and solute, thereby improving their interactions
for enhanced extraction [64]. In addition, SC CO2 has almost zero
surface tension for which no hindrance is present during the
penetration of the solvent into the microporous structure of sample
matrix. The highest global yields of EOs are obtained at high
pressure (220e300 bar) and moderate temperature (40
Ce50
C)
[82,83]. In terms of individual compounds, this condition is true for
volatile (camphor and 1,8-cineole) and non-volatile (CA and CR)
compounds, except for RA [64,84].
The addition of modifier into the SFE system affects the solu-
bility of non-volatile compounds in SC CO2. Several studies have
reported the enhanced solubility of CA and CR in CO2 with 7%
ethanol at 100e150 bar at 40
C [65,68]. Moreover, researchers
exploited the tuning of the solubility of compounds to achieve
fractionation. In a two-step sequential SFE process, the waxes and
oleoresin fraction was removed at 300 bar and 40
C at the first
stage. At a reduced pressure of 100e150 bar and 40
Ce50
C, CA and
CR fractions were precipitated and obtained in the second stage
[65,68]. A similar approach was undertaken by other studies to
achieve the SC fractionation of enriched fractions from the crude
extract obtained from other methods [6,78]. The use of high pres-
sure is desirable because it helps to manipulate compound solu-
bility. However, further investigation is necessary to understand
the behaviour when other parameters are involved.
In a detailed solubility investigation, the solubility of solid CA
was reported to increase with high pressure and low temperature
in CO2 þ ethanol system on the basis of thermodynamic modelling
and physical properties (solid volume, sublimation pressure and
347
critical properties). However, the threshold limit is not identified.
On the basis of GCA-EoS model, an in-depth analysis leads to
inconclusive results due to uncertainties in the calculation of the
hard sphere diameter, which is a crucial parameter in determining
solid solubility [71]. No such reported works are available for other
major components in rosemary. Thus, further investigation and
optimisation of these parameters can provide insights into the
solubility of CA and other components of rosemary in
CO2 þ ethanol system.
6.2. Effect of solvent system in the recovery of compounds
Solvent selection drives all assisted technologies. Bioactive
compounds have high affinity for protic polar solvents, which are
compounds that contain a hydroxyl or an amine group [85].
Double hydrogen bonds are formed by donating a proton (Hþ) to
the hydroxyl groups of bioactive compounds and solvents [85].
Therefore, the affinity of phenolic compounds towards certain
solvents is specific because of properties, such as polarity. Each
phenolic structure and functional group vary, leading to divergent
interactions with solvents. Therefore, the bond formation of
different phenolics with the solvents vary depending on how
closely they resemble each other's structures and polarities. The
highest concentration of RA is detected in the water extracts of
Orthosiphon stamineus leaves [86]. The reason is that the presence
of four hydroxyl groups and a carboxylic group forms double
hydrogen bonds with high polar water molecules. Several re-
searchers [5,44,52,65] have suggested the same case for RA when
extracted from rosemary. Likewise, the same researchers have
affirmed that CA, CR and other phenolic acids in rosemary (ursolic
and oleanolic acid) are efficient with medium polar organic sol-
vents. One study has concluded that PLE with water is more
effective for high-to-medium polarity compounds, whereas PLE
with ethanol is more suitable for less polar compounds [65].
Additionally, solvent solution drives the selection of extraction
method. Due to high polarity and low solubility in SC CO2, even at
high pressures and with a modifier, maximum RA, chlorogenic acid
and caffeic acid are extracted with PLE and water, whereas
threshold CA, CR and r-coumaric acid are obtained with SFE, CO2
and 7% ethanol [65].
RA is efficiently extracted with methanol, whereas ethanol is
preferred by CA [53]. The polarity depends on several factors, such
as the number of hydroxyl groups and bond chain length. Organic
compounds have hydrophobic and hydrophilic components. As the
carbon chain increases, the hydrophobic fragment proportionally
increases, thereby decreasing its overall polarity. Thus, methanol is
considered more polar than ethanol [87]. Moreover, RA has a high
affinity for methanol, similar to CA towards ethanol. Moreover,
solvent type can be a crucial element during fractionation. During
SAF, a high amount of water favours the fractionation of RA in the
raffinate phase, whereas a high quantity of ethanol favours the
separation of CA in the extract phase [6]. Additionally, the sensi-
tivity of CA in water rapidly degrades to CR by the radicals gener-
ated from water [88], thus leading to reduced bioactivity [5].
According to EU, the solvent acceptable for the extraction and
separation of CA and CR from rosemary is either ethanol or the
mixture of ethanol and acetone, followed by hexane [66]. Polar and
non-polar solvents are preferred by CA due to its phenolic and non-
volatile terpene nature. On the contrary, RA is only selective to-
wards high polar solvents [5,43,89]. The combination of a green
solvent, such as ionic liquid (1-octyl-3-methylimidazolium bro-
mide) with UAE and MAE, has also shown potential in extracting CA
and RA [8,90]. However, considerations were made due to cost
limitations.
348 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351
Dried and
powdered
rosemary
leaves
Ultrasound
Solvent 1 (i.e.
DES)
Solvent 2 (i.e.
hexane)
Fig. 3. Possible application of UATPS with DES to achieve simultaneous extraction and fractionation.
7. Future prospects of the extraction technologies for
R. officinalis L.
The largest trends in the current research community for
extraction technologies are efficiency and sustainability. Therefore,
the present study develops methods that are easy to operate and do
not require complicated processing/equipment to facilitate indus-
trial integration. Thus, one technology that can be explored for the
simultaneous extraction of diverse polar compounds from rose-
mary is the aqueous two-phase system (ATPS). ATPS has all the
advantages of conventional and latest techniques, such as low cost,
continuous operation, environment-friendly and efficient in puri-
fication/fractionation, which have been demonstrated in lab and
pilot scale studies [91,92]. An review has highlighted the oppor-
tunities and challenges of ATPS because the transition of promising
technologies from lab to commercial scale can often be difficult and
expensive [93]. The large-scale adoption of ATPS is achievable by
implementing appropriate materials (solvents), operating condi-
tions, employing phase separation techniques (gravitational and
centrifugal field) and removing ATPS from desired products
(filtration and precipitation).
ATPS is formed by combining two immiscible hydrophilic
components in an aqueous solution above critical concentrations
[94]. Primarily, ATPS was reserved for the bio-industry because
ATPS is widely used for the separation and purification of proteins,
DNA and antibodies [93]. The characteristic feature of water as the
primary content and low interfacial tension amongst conjugated
phases allows for gentle separation of biomolecules and stabilisa-
tion of their structure and activities by the polymers [91]. However,
the application of ATPS has extended to the extraction of high-value
products from natural sources [95]. ATPS composed of ethanol
(alcohol), and salt is a effective solvent system due to the affinity of
natural compounds towards organic solvents [94]. Hence, the
simultaneous extraction of high- and low-polarity targeting com-
pounds from rosemary to separate phases can be easily achieved by
using the ATPS dual solvent system (ethanol and salt). Moreover,
the need for further fractionation stages that require additional
equipment, high energy consumption and economic consideration
Top phase enriched
with high polar
compounds such as
RA and other
phenolic acids
Extrac on and
separa on/
frac ona on in a
single stage
Bo om phase
enriched with CA
and CR
can be avoided. Furthermore, the integration of assisted technolo-
gies like ultrasound can further accentuate the recovery/isolation of
natural compounds at low temperature [96]. Therefore, the appli-
cation of ultrasound-assisted aqueous two-phase system extraction
(UATPS) can be a potential technologies that can be explored for
extraction of BACs from rosemary.
Green solvents, such as deep eutectic solvents (DES), can be
investigated to make the process sustainable, particularly the latter,
as illustrated in Fig. 3. DES are eutectic mixtures of two or more
Lowry or Bronsted-Lowry acids and bases that have lower melting
points than their original constituents [97]. DES are usually pre-
pared from the complexation of a quaternary ammonium halide
salt (hydrogen bond acceptor) and hydrogen bond donor [98]. One
of the most attractive features of DES is the tunability of solvent
properties used as extracting solvents. The extraction efficiency of
the major compounds depends on physical properties, such as
density, viscosity, polarity and miscibility. Tailor-made DES with
varying physical properties can be prepared by the manipulation of
starting materials and their ratios to enhance the extractability of
the desired compounds from source material (plant matrix) [97].
Thus, the development of DES, which can extract the major com-
pounds from rosemary, can provide a viable green alternative to
organic solvents. DES have seen its applicability for the extraction
of RA from other sources such as Salvia miltiorrhiza [99], but its
applicability to other major components, such as CA and CR from
rosemary, have yet to be included, leaving a whole new field to be
explored.
8. Conclusion
Rosemary and its bioactive compounds have attracted the
attention of researchers, professionals and consumers for their
health benefits. Therefore, the current research was conducted to
provide an updated review of various extraction technologies for
effectively and efficiently preserving and extracting compounds.
With the development of novel and modified traditional technol-
ogies, extracting plant compounds in small and large quantities
according to the desired applications is possible. This literature
 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351
review concludes that the major pharmacologically active com-
pounds in the volatile fraction are 1,8-cineole, a-pinene and
camphor, whereas those in the non-volatile (extract) fraction are
CA, carnosol and rosmarinic acid.
The implementation of extraction technologies is endless
because they stretch from traditional (maceration/Soxhlet/micro-
wave distillation) to developed methods (ultrasound assisted/
pressurized liquid/SFE). In this study, a trend was observed
amongst the technologies wherein method selection depended on
the desired compound. Distillation methods are catered to EO,
whereas assisted and SC extraction methods were employed to
extract fractions. In addition, fractionation steps were implemented
to produce enriched fractions of CA, CR and RA by removing oleo-
resins. The study concluded that a technology needs to be devel-
oped for simultaneous extraction and fractionation of different
polarity compounds at low temperatures with high efficiency.
Extended lab and pilot scale studies must also be conducted to
demonstrate the applicability of such a technology for the com-
pounds of rosemary in the industry.
Acknowledgements
The authors would like to acknowledge Taylor’s University
Lakeside for providing financial support under the postgraduate
research assistance grant (TRGS/MFS/1/2017/SOE/008).
References
[1] M. Viuda-Martos, Y.R. Navajas, E.S. Zapata, J. Fern
andez-Lo
pez, J.A. Pe

rez-


Alvarez, Antioxidant activity of essential oils of five spice plants widely used
in a Mediterranean diet, Flavour Fragrance J. 25 (2010) 13e19.
[2] C.M. Uritu, C.T. Mihai, G.-D. Stanciu, G. Dodi, Teodora Alexa-Stratulat, A. Luca,
M.-M. Leon-Constantin, R. Stefanescu, V. Bild, S. Melnic, B.I. Tamba, Medicinal
plants of the family Lamiaceae in pain therapy: a review, Pain Res. Manag.
2018 (2018) 1e44.
[3] J.M. Andrade, C. Faustino, C. Garcia, D. Ladeiras, C.P. Reis, P. Rijo, Rosmarinus
officinalis L.: an update review of its phytochemistry and biological activity,
Futur. Sci. OA. 4 (2018) FSO283.
[4] J. Moore, M. Yousef, E. Tsiani, Anticancer effects of rosemary (Rosmarinus
officinalis L.) extract and rosemary extract polyphenols, Nutrients 8 (2016)
731.
[5] S. Rodríguez-Rojo, A. Visentina, D. Maestri, M.J. Cocero, Assisted extraction of
rosemary antioxidants with green solvents, J. Food Eng. 109 (2012) 98e103.
[6] A.P. Sa
nchez-Camargo, J.A. Mendiola, A. Valde
s, M. Castro-Puyana, V. García-
Can~ as, A. Cifuentes, M. Herrero, E. Ib
~ ez, Supercritical antisolvent frac-
an
tionation of rosemary extracts obtained by pressurized liquid extraction to
enhance their antiproliferative activity, J. Supercrit. Fluids 107 (2016)
581e589.
[7] I. Takaki, L.E. Bersani-Amado, A. Vendruscolo, S.M. Sartoretto, S. Diniz,
C. Bersani-Amado, R. Cuman, Anti-inflammatory and antinociceptive effects
of Rosmarinus officinalis L. essential oil in experimental animal models,
J. Med. Food 11 (2009) 741e746.
[8] G. Zu, R. Zhang, L. Yang, C. Ma, Y. Zu, W. Wang, C. Zhao, Ultrasound-assisted
extraction of carnosic acid and rosmarinic acid using ionic liquid solution
from Rosmarinus officinalis, Int. J. Mol. Sci. 13 (2012) 11027e11043.
[9] A. Kar, Pharmacognosy and Pharmacobiotechnology, New Age International
Pvt Ltd., 2008.
[10] J. Dai, R.J. Mumper, Plant phenolics: extraction, analysis and their antioxidant
and anticancer properties, Molecules 15 (2010) 7313e7352. https://doi.org/
10.3390/molecules15107313.
[11] J.L. Humphrey, G.E. Keller, Separation Process Technology, McGraw-Hill,
1997.
[12] WHO, WHO guidelines on good herbal processing practices for herbal
medicines, WHO Tech. Rep. Ser. 1 (2018). No. 1010:Annex.
[13] G. Nieto, G. Ros, J. Castillo, Antioxidant and antimicrobial properties of
rosemary (Rosmarinus officinalis, L.): A review, Medicines 5 (2018) 98.
[14] A. Begum, S. Sandhya, S.S. Ali, K.R. Vinod, S. Reddy, D. Banji, An in-depth
review on the medicinal flora Rosmarinus officinalis (Lamiaceae), Acta Sci.
Pol. Technol. Aliment. 12 (2013) 61e73.
[15] R. Ribeiro-Santos, D. Carvalho-Costa, C. Cavaleiro, H.S. Costa,
T.G. Albuquerque, M.C. Castilho, F. Ramos, N.R. Melo, A. Sanches-Silva,
A novel insight on an ancient aromatic plant: the rosemary (Rosmarinus
officinalis L.), Trends Food Sci. Technol. 45 (2015) 355e368.
[16] R.R. Raja, Medicinally potential plants of Labiatae (Lamiaceae) family: an
overview, Res. J. Med. Plant 6 (2012) 203e213.
349
[17] M.R. Al-sereti, K.M. Abu-Amer, P. Sen, Pharmacology of rosemary (Rosmar-
inus officinalis Linn.) and its therapeutic potentials, Indian J. Exp. Biol. 37
(1999) 124e130.
[18] M. Ozcan, J. Chalchat, Chemical composition and antifungal activity of
rosemary (Rosmarinus officinalis L.) oil from Turkey, Int. J. Food Sci. Nutr. 59
(2008) 691e698.

Calín-Sa
[19] A.
nchez, A. Szumny, A. Figiel, K. Jałoszyn
ski, M. Adamski,


A.A. Carbonell-Barrachina, Effects of vacuum level and microwave power on
rosemary volatile composition during vacuumemicrowave drying, J. Food
Eng. 103 (2011) 219e227.
[20] P. Mena, M. Cirlini, M. Tassotti, K.A. Herrlinger, C.D. Asta, D. Del Roi, Phyto-
chemical profiling of flavonoids, phenolic acids, terpenoids, and volatile
fraction of a rosemary (Rosmarinus officinalis L.) extract, Molecules 21 (2016)
1576.
[21] Y. Diab, L. Auezova, H. Chebib, J.-C. Chalchat, G. Figueredo, Chemical
composition of lebanese rosemary (Rosmarinus officinalis L.) essential oil as a
function of the geographical region and the harvest time, J. Essent. Oil Res. 14
(2002) 449e452.
[22] S. Oksana, B. Marian, R. Mahendra, S.H. Bo, Plant phenolic compounds for
food, pharmaceutical and cosmetiсs production, J. Med. Plants Res. 6 (2012)
2526e2539.
[23] A. Szumny, A. Figiel, A. Gutie


rrez-Ortíz, A.C.-B. Angel, Composition of rose-
mary essential oil (Rosmarinus officinalis) as affected by drying method,
J. Food Eng. 97 (2010) 253e260.
[24] S. Birti

c, P. Dussort, F.-X. Pierre, A.C. Bily, M. Roller, Carnosic acid, Phyto-
chemistry 115 (2015) 9e19.
[25] M.J. del Ban ~ o, J. Lorente, J. Castillo, O. Benavente-García, J.A. del Río,
A. Ortun ~ o, K.-W. Quirin, D. Gerard, Phenolic diterpenes, flavones, and ros-
marinic acid distribution during the development of leaves, flowers, stems,
and roots of Rosmarinus officinalis. Antioxidant activity, J. Agric. Food Chem.
51 (2003) 4247e4253.
[26] G. Flamini, P.L. Cion, I. Morelli, M. Macchia, L. Ceccarini, Main agronomic-
productive characteristics of two ecotypes of Rosmarinus officinalis L. and
chemical composition of their essential oils, J. Agric. Food Chem. 50 (2002)
3512e3517.
[27] C. Boutekedjiret, F. Bentahar, R. Belabbes, J.-M.B. Bessie re, Extraction of
rosemary essential oil by steam distillation and hydrodistillation, Flavour
Fragrance J. 18 (2003) 481e484.
[28] World Health Organization, WHO Traditional Medicine Strategy: 2014-2023,
2013.
[29] N. Azwanida, A review on the extraction methods use in medicinal plants,
principle, strength and limitation, Med. Aromatic Plants 4 (2015) 1e6.
[30] M. Selvamuthukumaran, J. Shi, Recent advances in extraction of antioxidants
from plant by-products processing industries, Food Qual. Saf. 1 (2017)
61e81.
[31] P. Tongnuanchan, S. Benjakul, Essential oils: extraction, bioactivities, and
their uses for food preservation, J. Food Sci. 79 (2014) R1231eR1249.
[32] L.A. Conde-Herna
Guerrero-Beltra

ndez, J.R. Espinosa-Victoria, A. Trejo, J.A.
n,
CO2-supercritical extraction, hydrodistillation and steam distillation of
essential oil of rosemary (Rosmarinus officinalis), J. Food Eng. 200 (2017)
81e86.
[33] M. Lo Presti, S. Ragusa, A. Trozzi, P. Dugo, F. Visinoni, A. Fazio, G. Dugo,
L. Mondello, A comparison between different techniques for the isolation of
rosemary essential oil, J. Sep. Sci. 28 (2005) 273e280.
[34] S. Moradi, A. Fazlali, H. Hamedi, Microwave-assisted hydro-distillation of
essential oil from rosemary: comparison with traditional distillation, Avi-
cenna J. Med. Biotechnol. 10 (2018) 22e28.
[35] A. Proctor (Editor), Alternatives to Conventional Food Processing, RSC Pub-
lishing, 2011.
[36] M.A. Vian, X. Fernandez, F. Visinoni, F. Chemat, Microwave hydrodiffusion
and gravity, a new technique for extraction of essential oils, J. Chromatogr., A
1190 (2008) 14e17.
[37] N. Bousbia, M. Vian, M.A. Ferhat, E. Petitcolas, B.Y. Meklati, F. Chemat,
Comparison of two isolation methods for essential oil from rosemary leaves:
hydrodistillation and microwave hydrodiffusion and gravity, Food Chem.
114 (2009) 355e362.
[38] A. Filly, X. Fernandez, M. Minuti, F. Visinoni, G. Cravotto, F. Chemat, Solvent-
free microwave extraction of essential oil from aromatic herbs: from labo-
ratory to pilot and industrial scale, Food Chem. 150 (2014) 193e198.
[39] I. Calinescu, I. Asofiei, A.I. Gavrila, A. Trifan, D. Ighigeanu, D. Martin, C. Matei,
M. Buleandra, Integrating microwave-assisted extraction of essential oils and
polyphenols from rosemary and thyme leaves, Chem. Eng. Commun. 204
(2017) 965e973.

rino-Issartier, C. Ginies, G. Cravotto, F. Chemat, A comparison of essential
[40] S. Pe
oils obtained from lavandin via different extraction processes: ultrasound,
microwave, turbohydrodistillation, steam and hydrodistillation,
J. Chromatogr., A 1305 (2013) 41e47.
[41] G. Khalili, A. Mazloomifar, K. Larijani, M.S. Tehrani, P.A. Azar, Solvent-free
microwave extraction of essential oils from Thymus vulgaris L. and Melissa
officinalis L. Ind. Crops Prod. 119 (2018) 214e217.
[42] P. Tatke, Y. Jaiswal, An overview of microwave assisted extraction and its
applications in herbal drug research, Res. J. Med. Plant 5 (2011) 21e31.
[43] M. Jacotet-Navarro, N. Rombaut, A.-S. Fabiano-Tixier, M. Danguien, A. Bily,
F. Chemat, Ultrasound versus microwave as green processes for extraction of
350 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351
rosmarinic, carnosic and ursolic acids from rosemary, Ultrason. Sonochem.
27 (2015) 102e109.
[44] Gerlon de AR Oliveira, Anselmo E. de Oliveira, Edemílson C. da Conceiça ~o,
Maria I.G. Leles, Multiresponse optimization of an extraction procedure of
carnosol and rosmarinic and carnosic acids from rosemary, Food Chem. 211
(2016) 465e473.
[45] I.G. Moorthy, J.P. Maran, S. Ilakya, S.L. Anitha, S.P. Sabarima, B. Priya, Ultra-
sound assisted extraction of pectin from waste Artocarpus heterophyllus
fruit peel, Ultrason. Sonochem. 34 (2017) 525e530.
[46] B. Trusheva, D. Trunkova, V. Bankova, Different extraction methods of bio-
logically active components from propolis: a preliminary study, Chem. Cent.
J. 1 (2007) 1e13.
[47] Y. Pico, Ultrasound-assisted extraction for food and environmental samples,
TrAC Trends Anal. Chem. 43 (2013) 84e99.
[48] F. Chemat, N. Rombaut, A.-G. Sicaire, A. Meullemiestre, A.-S. Fabiano-Tixier,
M. Abert-Vian, Ultrasound assisted extraction of food and natural products.
Mechanisms, techniques, combinations, protocols and applications. A re-
view, Ultrason. Sonochem. 34 (2017) 540e560.
[49] M. Toma, M. Vinatoru, L. Paniwnyk, T.J. Mason, Investigation of the effects of
ultrasound on vegetal tissues during solvent extraction, Ultrason. Sonochem.
8 (2001) 137e142.
[50] K. Vilkhu, R. Mawson, L. Simons, D. Bates, Applications and opportunities for
ultrasound assisted extraction in the food industry d a review, Innov. Food
Sci. Emerg. Technol. 9 (2008) 161e169.
[51] S. Albu, E. Joyce, L. Paniwnyk, J.P. Lorimer, T.J. Mason, Potential for the use of
ultrasound in the extraction of antioxidants from Rosmarinus officinalis for
the food and pharmaceutical industry, Ultrason, Sonochem 11 (2004)
261e265.
[52] J. Bernatoniene, U. Cizauskaite, L. Ivanauskas, V. Jakstas, Z. Kalveniene,
D.M. Kopustinskiene, Novel approaches to optimize extraction processes of
ursolic, oleanolic and rosmarinic acids from Rosmarinus officinalis leaves, Ind.
Crops Prod. 84 (2016) 72e79.
[53] L. Paniwnyk, H. Cai, S. Albu, T.J. Mason, R. Cole, The enhancement and scale
up of the extraction of anti-oxidants from Rosmarinus officinalis using ul-
trasound, Ultrason. Sonochem. 16 (2009) 287e292.
[54] B.E. Richter, B.A. Jones, J.L. Ezzell, N.L. Porter, Accelerated solvent extraction:
a technique for sample preparation, Anal. Chem. 68 (1996) 1033e1039.
[55] A. Mustafa, C. Turner, Pressurized liquid extraction as a green approach in
food and herbal plants extraction: a review, Anal. Chim. Acta 703 (2011)
8e18.
[56] D.T.V. Pereira, A.G. Tarone, C.B.B. Cazarin, G.F. Barbero, J. Martínez, Pressur-
ized liquid extraction of bioactive compounds from grape marc, J. Food Eng.
240 (2019) 105e113.
[57] A. Basile, M.M. Jime
nez-Carmona, A.A. Clifford, Extraction of rosemary by
superheated water, J. Agric. Food Chem. 46 (1998) 5205e5209.
[58] E. Iban~ ez, A. Kub
atov
~ ora
a, F.J. Sen
ns, S. Cavero, G. Reglero, S.B. Hawthorne,
Subcritical water extraction of antioxidant compounds from rosemary
plants, J. Agric. Food Chem. 51 (2003) 375e382.
[59] M.B. Hossain, C. Barry-Ryan, A.B. Martin-Diana, N.P. Brunton, Optimisation of
accelerated solvent extraction of antioxidant compounds from rosemary
(Rosmarinus officinalis L.), marjoram (Origanum majorana L.) and oregano
(Origanum vulgare L.) using response surface methodology, Food Chem. 126
(2011) 339e346.
[60] E. V
azquez, M.R. García-Risco, L. Jaime, G. Reglero, T. Fornari, Simultaneous
extraction of rosemary and spinach leaves and its effect on the antioxidant
activity of products, J. Supercrit. Fluids 82 (2013) 138e145.
[61] M. Sihvonen, E. Ja €rvenp€ aa€, V. Hietaniemi, R. Huopalahti, Advances in su-
percritical carbon dioxide technologies, Trends Food Sci. Technol. 10 (1999)
217e222.
[62] C. Yi, J. Shi, S.J. Xue, Y. Jiang, D. Li, Effects of supercritical fluid extraction
parameters on lycopene yield and antioxidant activity, Food Chem. 113
(2009) 1088e1094.
[63] H. Bendif, K. Adouni, M. Djamel, Miara, Renata Baranauskiene, _ [87]
Paulius Kraujalis, P.R. Venskutonis, S.M. Nabavi, F. Maggi, Essential oils (EOs),
pressurized liquid extracts (PLE) and carbon dioxide supercritical fluid ex-
tracts (SFE-CO2) from Algerian Thymus munbyanus as valuable sources of
antioxidants to be used on an industrial level, Food Chem. 260 (2018)
289e298.
[64] Raul N. Carvalho Jr., Lucinewton S. Moura, Paulo T.V. Rosa, M. Angela
A. Meireles, Supercritical fluid extraction from rosemary (Rosmarinus offici-
nalis): kinetic data, extract's global yield, composition, and antioxidant ac-
tivity, J. Supercrit. Fluids 35 (2005) 197e204. https://doi.org/10.1016/
j.supflu.2005.01.006.
[65] M. Herrero, M. Plaza, A. Cifuentes, E. Ib
an~ ez, Green processes for the
extraction of bioactives from Rosemary: chemical and functional character-
ization via ultra-performance liquid chromatography-tandem mass spec-
trometry and in-vitro assays, J. Chromatogr., A 1217 (2010) 2512e2520.
[66] M. Younes, P.J. Aggett, F. Aguilar, R. Crebelli, B. Dusemund, M. Filipi c,
M. Frutos, P. Galtier, D. Gott, U. Gundert-Remy, G.G. Kuhnle, C. Lambre
,
I.T. Lillegaard, P. Moldeus, A. Mortensen, A. Oskarsson, I. Stankovic,
I. Waalkens-Berendsen, R.A. Woutersen, M.C. Wright, P.E. Boon, O. Lindtner,
C. Tlustos, A. Tard, J. Leblanc, Refined exposure assessment of extracts of
rosemary (E 392) from its use as food additive, EFSA J. 16 (2018) 5373.
[67] I. Rodríguez-Meizoso, M. Castro-Puyana, P. Bo € rjesson, J.A. Mendiola,
C. Turner, E. Ib
~ ez, Life cycle assessment of green pilot-scale extraction
an
processes to obtain potent antioxidants from rosemary leaves, J. Supercrit.
Fluids 72 (2012) 205e212.
[68] A. del P. Sa
nchez-Camargo, A. Valde
s, G. Sullinia, V. García-Can ~ as,
A. Cifuentes, E. Iba
n
~ ez, M. Herrero, Two-step sequential supercritical fluid
extracts from rosemary with enhanced anti-proliferative activity, J. Funct.
Foods. 11 (2014) 293e303.
[69]
Martín, S. Varona, A. Navarrete, M.J. Cocero, Encapsulation and co-
A.
precipitation processes with supercritical fluids: applications with essential
oils, Open Chem. Eng. J. 4 (2010) 31e41.
[70] A. Visentin, S. Rodríguez-Rojo, A. Navarrete, D. Maestri, M.J. Cocero, Precip-
itation and encapsulation of rosemary antioxidants by supercritical anti-
solvent process, J. Food Eng. 109 (2012) 9e15.
[71] A. Cha
fer, T. Fornari, A. Berna, E. Iban ~ ez, G. Reglero, Solubility of solid car-
nosic acid in supercritical CO2 with ethanol as a co-solvent, J. Supercrit.
Fluids 34 (2005) 323e329.
[72] A. del P. S
anchez-Camargo, V. García-Can ~ as, M. Herrero, A. Cifuentes,
E. Iba
n~ ez, Comparative study of green sub- and supercritical processes to
obtain carnosic acid and carnosol-enriched rosemary extracts with in vitro
anti-proliferative activity on colon cancer cells, Int. J. Mol. Sci. 17 (2016)
2046.
[73] Q.-W. Zhang, L.-G. Lin, W.-C. Ye, Techniques for extraction and isolation of
natural products: a comprehensive review, Chin. Med. 13 (2018) 20.
[74] J. Lutisan, J. Cvengros, Mean free path of molecules on molecular distillation,
Chem. Eng. J. Biochem. Eng. J. 56 (1995) 39e50.
[75] G.N. Mezza, A.V. Borgarello, J.D. Daguero, M.C. Pramparo, Obtention of
rosemary essential oil concentrates by molecular distillation and free radical
scavenging capacity analysis, Int. J. Food Eng. 9 (2013) 147e153.
[76] G.N. Mezza, A.V. Borgarello, N.R. Grosso, H. Fernandez, M.C. Pramparo,
M.F. Gayol, Antioxidant activity of rosemary essential oil fractions obtained
by molecular distillation and their effect on oxidative stability of sunflower
oil, Food Chem. 242 (2018) 9e15.
[77] C. Perez-Locas, V.A. Yaylayan, The Maillard reaction and food quality dete-
rioration, in: Chem. Deterior. Phys. Instab. Food Beverages, 2010, pp. 70e94.
https://doi.org/10.1533/9781845699260.1.70.
[78] A. Visentín, M. Cismondi, D. Maestri, Supercritical CO2 fractionation of
rosemary ethanolic oleoresins as a method to improve carnosic acid recov-
ery, Innov. Food Sci. Emerg. Technol. 12 (2011) 142e145.
[79] Y. Zhang, J.P. Smuts, E. Dodbiba, R. Rangarajan, J.C. Lang, D.W. Armstrong,
Degradation study of carnosic acid, carnosol, rosmarinic acid, and rosemary
extract (Rosmarinus officinalis L.) assessed using HPLC, J. Agric. Food Chem. 60
(2012) 9305e9314.
[80] C. Wellwood, R. Cole, Relevance of carnosic acid concentrations to the se-
lection of rosemary, Rosmarinus officinalis (L.), accessions for optimization of
antioxidant yield, J. Agric. Food Chem. 52 (2004) 6101e6107.
[81] E. Heidaryan, T. Hatami, M. Rahimi, J. Moghadasi, Viscosity of pure carbon
dioxide at supercritical region: measurement and correlation approach,
J. Supercrit. Fluids 56 (2011) 144e151.
[82] T. Fornari, A. Ruiz-Rodriguez, G. Vicente, E. Va
zquez, M.R. García-Risco,
G. Reglero, Kinetic study of the supercritical CO2 extraction of different
plants from Lamiaceae family, J. Supercrit. Fluids 64 (2012) 1e8.
[83] Z. Ahmed, M. Abdeslam-Hassan, L. Ouassila, B. Danielle, Extraction and
modeling of algerian rosemary essential oil using supercritical CO2: effect of
pressure and temperature, Energy Procedia 18 (2012) 1038e1046.
[84] P. Ramírez, F.J. Sen ~ or
ans, E. Iban~ ez, G. Reglero, Separation of rosemary
antioxidant compounds by supercritical fluid chromatography on coated
packed capillary columns, J. Chromatogr., A 1057 (2004) 241e245.
[85] C.M. Galanakis, V. Goulas, S. Tsakona, G.A. Manganaris, V. Gekas,
A knowledge base for the recovery of natural phenols with different solvents,
Int. J. Food Prop. 16 (2013) 382e396.
[86] M.F.B.A. Razak, P.K. Yong, Z.M. Shah, L.C. Abdullah, S.S. Yee, I.T.C.S. Yaw, The
effects of varying solvent polarity on extraction yield of orthosiphon stam-
ineus leaves, J. Appl. Sci. 12 (2012) 1207e1210.
T.N. Sorrell, Organic Chemistry, Second ed., University Science Books, Cali-
fornia, 2006.
[88] M. Bellumori, M. Innocenti, A. Binello, L. Boffa, N. Mulinacci, G. Cravotto,
Selective recovery of rosmarinic and carnosic acids from rosemary leaves
under ultrasound- and microwave-assisted extraction procedures, Compt.
Rendus Chem. 19 (2016) 699e706.
[89] Z. Jiang, C. Kempinski, J. Chappell, Extraction and analysis of terpenes/ter-
penoids, Curr. Protoc. Plant Biol. 1 (2016) 345e358.
[90] T. Liu, X. Sui, R. Zhang, L. Yang, Y. Zu, L. Zhang, Y. Zhang, Z. Zhang, Application
of ionic liquids based microwave-assisted simultaneous extraction of car-
nosic acid, rosmarinic acid and essential oil from Rosmarinus officinalis,
J. Chromatogr., A 1218 (2011) 8480e8489.
[91] R. Hatti-Kaul (Editor), Aqueous Two-phase Systems Methods and Protocols,
Springer Science & Business Media, Berlin, 2000.
[92] E.E.-S. Patricia Va
zquez-Villegas, M.A. Torres-Acosta, F. Ruiz-Ruiz, M. Rito-
Palomares, O. Aguilar, Factorial and economic evaluation of an aqueous two-
phase partitioning pilot plant for invertase recovery from spent brewery
yeast, Front. Chem. 6 (2018) 454.
[93] M.A. Torres-Acosta, K. Mayolo-Deloisa, J. Gonza
lez-Valdez, M. Rito-Pal-
omares, Aqueous two-phase systems at large scale: challenges and oppor-
tunities, Biotechnol. J. 14 (2018). https://doi.org/10.1002/biot.201800117.
[94] M. Iqbal, Y. Tao, S. Xie, Y. Zhu, D. Chen, X. Wang, L. Huang, D. Peng, A. Sattar,
M.A.B. Shabbir, H.I. Hussain, S. Ahmed, Z. Yuan, Aqueous two-phase system
 A. Ali et al. / Trends in Analytical Chemistry 118 (2019) 338e351
(ATPS): an overview and advances in its applications, Biol. Proced. Online 18
(2016) 18.
[95] D.-Y. Zhang, Y.-G. Zu, Y.-J. Fu, W. Wang, L. Zhang, M. Luo, F.-S. Mu, X.-H. Yao,
M.-H. Duan, Aqueous two-phase extraction and enrichment of two main
flavonoids from pigeon pea roots and the antioxidant activity, Separ. Purif.
Technol. 102 (2013) 26e33.
[96] X. Ji, Q. Peng, Y. Yuan, F. Liu, M. Wang, Extraction and physicochemical
properties of polysaccharides from Ziziphus Jujuba cv. Muzao by ultrasound-
assisted aqueous two-phase extraction, Int. J. Biol. Macromol. 108 (2018)
541e549.
[97] M.H. Zainal-Abidin, M. Hayyana, A. Hayyan, N.S. Jayakumara, New horizons
in the extraction of bioactive compounds using deep eutectic solvents: a
review, Anal. Chim. Acta 979 (2017) 1e23.
351
[98] C. yong Cai, S. Wu, C. Wang, Y. Yang, D. Sun, F. Li, Z. Tan, Deep eutectic
solvents used as adjuvants for improving the salting-out extraction of ursolic
acid from Cynomorium songaricum Rupr. in aqueous two-phase system,
Separ. Purif. Technol. 209 (2019) 112e118.
[99] J. Chen, M. Liu, Q. Wang, H. Du, L. Zhang, Deep eutectic solvent-based
microwave-assisted method for extraction of hydrophilic and hydropho-
bic components from Radix Salviae miltiorrhizae, Molecules 21 (2016)
1383.
[100] B.R.R. Rao, S.K. Kothari, D.K. Rajput, R.P. Patel, K.P. Sastry, Composition of
rosemary (Rosmarinus officinalis L.) oil produced from the semi-arid
tropical climate of south India, J. Med. Aromat. Plant Sci. 32 (2010)
20e23.