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7/21/2019 Steam Distillation Extraction Kinetics Regression Models to Predict Essential Oil Yield, Composition, And Bioactivity …
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Industrial Crops and Products 58 (2014) 61–67
Contents lists available at ScienceDirect
Industrial Crops and Products
j ournal homepage : www.elsevier .com/ locate / indcrop
Steam distillation extraction kinetics regression models to predict
essential oil yield, composition, and bioactivity of chamomile oil
Archana Gawdea,b, Charles L. Cantrella, Valtcho D. Zheljazkov b,∗,Tess Astatkiec, Vicki Schlegeld
a Natural Products UtilizationResearch Unit, Agricultural Research Service, UnitedStates Department of Agriculture, P.O. Box 8048,University, MS 38677,
USAb University of Wyoming, Sheridan Research andExtension Center, 663Wyarno Road, Sheridan,WY 82801, USAc DalhousieUniversity, Faculty of Agriculture, 50 PictouRoad, P O Box550, Truro, NS B2N5E3, Canadad University of Nebraska–Lincoln, Department of Food Science andTechnology, 327 Food Technology Complex, Lincoln, NE 68583, USA
a r t i c l e i n f o
Article history:
Received 17 December 2013
Received in revised form 23 March 2014
Accepted 1 April 2014
Keywords:
Matricaria chamomilla
Matricaria recutita
Essential oil profile
Chamomile antioxidant capacity
Chamomile antimicrobial activity
a b s t r a c t
Chamomile (Matricaria chamomilla L.) is one of the most widely spread and used medicinal and essential
oil crops in the world. Chamomile essential oil is extracted via steam distillation of the inflorescences
(flowers). In this study, distillation time (DT) was found to be a crucial determinant of yield and composi-
tion of chamomile essential oil, but not of the antioxidant capacity. Essential oil obtained at 30, 60, 90, 120,
180, 240, 360, 480, 600, and 720min showed significant increase in oil yield with increasing DT, reaching
a maximum of 3.1 g oil per 1000 g of flowers at 720min. The major compounds that were identified and
quantified were anethole,-farnesene, spathulenol,-bisabolol oxide B,-bisabolone oxide A, chamazu-
lene,-bisabolol oxide A, and spiroether.-farnesene showed a decrease in content with increasing DT,
whereas-bisabolol oxide A, spiroether, and chamazulene rapidly increased up to 240 min, after which it
started to plateau showing negligible change. Anethole content showed a steady decrease over time from
approximately 2.4% at 30min to 0.54% at 720min. Yields of spathulenol,-bisabolol oxide B,-bisabolol
oxide A,-bisabolone oxide A, chamazulene,and spiroether essential oil constituents expressed as g/100 g
of dried chamomile inflorescences showed a steady increase that was described well by the Michaelis-Menton model. If higher concentrations of -bisabolol oxide A and chamazulene, and higher oil yields are
desired, chamomile flowers must be steam distilled for 480 min. However, if oil with high -farnesene
concentration is desirable, then chamomile flowers should be distilled for 30min. Distillation time can
be used as a modifier of chamomile essential oil yield and composition. The kinetics regression models
developed in this study can be utilized to predict essential oil yield, and composition of chamomile oil.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Chamomile (Matricaria chamomillaL. synonym:Matricariarecu-
tita) is a member of the Asteraceae family. Chamomile flowers
are most commonly used for making chamomile tea known for
its calming effect and the essential oil is used in the pharma-
ceutical and cosmetic industries (Salamon, 2007; Valussi, 2012;
Wheatley, 2005). Chamomileflowers and extracts havebeen exten-
sively used in traditional medicine in many countries, especially in
the Mediterranean region. For example, Bulgarian folk medicine
has utilized chamomile flower extract against insomnia, hysteria,
∗ Corresponding author. Tel.: +1 307 737 2415.
E-mail addresses:[email protected], [email protected]
(V.D. Zheljazkov).
gastritis, various cystic formations, headache, stomach pain, as
wound epithelialization, gas relieve, antispasmodic, and sweat-
ing agent (Stojanov, 1973). The bioactivity of essential oils and
their effective use in aromatherapy is largely dependent on the
composition of major volatile compounds. Cultivation methods,
management practices (Zheljazkov and Astatkie, 2011), abi-
otic factors (Razmjoo et al., 2008), and post-harvest processing
(Shahhoseini et al., 2013) are known to largely affect the yields and
composition of essential oils. Planting date and seedling age influ-
enced the content and composition of chamomile (Mohammad
et al., 2010) whereas irrigation affected the compositions in
chamomile (Pirzad et al., 2006). Post-harvest processing parame-
ters like drying methods (Shahhoseini et al., 2013; Pirbalouti et al.,
2013) areknown to affectessentialoil composition andbioactivity.
Four different drying methods showed significant effects on com-
position in Roman chamomile essential oil (Omidbaigi et al., 2004).
http://dx.doi.org/10.1016/j.indcrop.2014.04.001
0926-6690/© 2014 Elsevier B.V. All rights reserved.
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62 A. Gawde et al. / Industrial Crops and Products 58 (2014) 61–67
Distillation parameters like processing of plant material and dis-
tillation apparatus, along with the specific method and time may
additionally affect the essential oil quality (Hosni et al., 2010). Pre-
viously, it was found that the duration of the distillation time (DT)
significantly affected essential oil yield and composition in pep-
permint (Mentha× piperita L.), lemongrass (Cymbopogon flexuosus
Steud.), and palmarosa (CymbopogonmartiniiRoxb.) (Cannon et al.,
2013), fennel (Foeniculum vulgare Mill.) (Zheljazkov et al., 2013a),
Rocky Mountain juniper ( Juniperus scopuloum L.) (Zheljazkov et al.,
2013b), sweet sagewort ( Artemisia annua L.) (Zheljazkov et al.,
2013c), lavender (Lavandula angustifolia Mill.) (Zheljazkov et al.,
2013d), and oregano (Origanum vulgare L.) (Zheljazkov et al.,
2012a).
Currently, there is no information about how DT would affect
the yield and composition of chamomile. The optimal DT for dried
chamomile flowers is also unknown. We hypothesized that by
changing the duration of the DT of chamomile flowers, we could
obtain essential oil with diverse composition and bioactivity and
optimize the DT for maximum oil yield. Such standardizations
can be commercially utilized by identifying the best suitable DT
for desired compositions of essential oils. Therefore, the objective
of this study was to evaluate the effect of DT (steam distillation
extractionkinetics) on yield, composition, and antioxidant capacity
of chamomile essential oil. Furthermore, this study evaluated the
essential oil’s antimicrobial activities tested against ten bacterial
and fungal species.
2. Material and methods
2.1. Plant material
Bulk certified dried chamomile flowers were obtained from
Starwest Botanicals (Rancho Cordova, CA). The country of origin
was Egypt.
2.2. Steam distillation and distillation time (DT)
The steam distillation study/extraction kinetics experiment
was carried out in 2013 at the University of Wyoming, Sheridan
Research and Extension Center using a sample of 200 or 250g of
dried flower. The steam DT investigated in this study were 30, 60,
90, 120, 180, 240, 360, 480, 600, and720 min. These DTswere based
on our preliminary studies and literature reports. Also, this spac-
ing of the DT allowed us to develop extraction kinetics regression
models that can be utilized to predict chemical composition andoil
yield at any specific DT. All DTs were performed in triplicate in a
2 l steam distillation unit as described previously for peppermint,
lemongrass, and palmarosa (Cannon et al., 2013), and for laven-
der inflorescences (Zheljazkov et al., 2013c). Briefly the apparatus
includes a 2l pear shaped flask filled with water on a hotplate and
a 2 l bioflask positioned above. The bioflask contains the flowers.The still head is attached to the top of the bioflask and directs the
steam to the condenser that allows the co-distilled steam and oil
to simultaneously collect and separate in a collector. This system is
analogous to large commercial installations; the collector is similar
to the Florentine vessel used in commercial installations.
Steam DT of30, 60, 90, 120,180, 240,360, 480,600, and 720 min
were recorded as the times required from the beginning of distil-
lation (the moment the first drop condenses) to the time when
distillation was turned off. The distilled oil was separated from
waterand collectedin glassvialsequippedwitha Teflonlinedscrew
cap. The oils were weighed on an analytical scale and were stored
at −5 ◦C for gas chromatography (GC) identification. Essential oil
yield was calculated as grams of oil per weight (g) of 100g dried
flowers.
Fig. 1. Gas chromatography-FID chromatogram of chamomile flowers essential oil.
2.3. Gas chromatography–flame ionization detector (FID)
quantification of essential oil components
A total of eight constituents were identified and quantified in
chamomile flower essential oil (Fig. 1). Oil samples were analyzed
by GC-FID on a Agilent CP-3800 GC equipped with a DB-5 fused
silica capillary column (30 m×0.25mm, with a film thickness of
0.25m) operated usingthe following conditions: injector temper-
ature, 240◦C; column temperature, 60–120 at 3 ◦C/min, then held
at 240 ◦C at 20 ◦C/min for 5 min; carrier gas, He; injection volume,
1l (split on FID, split ratio 50:1); FID temperature was 300 ◦C.
Compounds anethole,-farnesene, spathulenol, -bisabolol oxide
B, -bisabolone oxide A, chamazulene, -bisabolol oxide A, and
spiroether were identified in oilsamples by Kovat analysis (Adams,2007), and comparison of mass spectra with those reported in
the NIST mass spectra database. Compounds were quantified by
performing area percentage calculations based on the total com-
bined FID area. For example, the area for each reported peak was
divided by total integrated area from the FID chromatogram from
all reported peaks and multiplied by 100 to arrive at a percent-
age. The percentage is a peak area percentage relative to all other
constituents integrated in the FID chromatogram.
2.4. Antioxidant capacity
The antioxidant capacity of the oil extracts from all DT in three
replicateswas determinedby the oxygen radical absorbance capac-
ity (ORAC) method as described by Huang et al. (2002a,b). Samplesof extracted oil were prepared for antioxidant capacity tests by
mixing 10±1 mg oil with 1ml of water and acetone (1:1) with 7%
methyl--cyclodextrins (w:v). The test was prepared in a 96-well
plate by first transferring 25l of 74 mM phosphate buffer saline
(pH 7.4) to each well. The test sample (25l) or Trolox (25l) that
served as the standard was added to different wells at concentra-
tion of 0.2, 0.4, 3.3, 6.5, 10, 13, 25, 50g/ml) followed by 150l
of fluorescein (8.16×10−5 mM). The samples were incubated at
37 ◦C for 10 min, with 3 m in of intermittent shaking. After incu-
bation, the reaction was activated by adding 153mM 2,2-azobis
(2-amidinopropane)hydrochloride (25l) toeachwell.All samples
and standards were prepared in 96 well plates and the fluores-
cence was measured every 1.5 min with a microplate reader set
at an excitation and emission wavelength of 485nm and 520nm,
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A. Gawde et al. / Industrial Crops and Products 58 (2014) 61–67 63
respectively, until the decreasing fluorescence values plateaued.
The area under the decay curve was calculated, and the results
were expressed in mole Trolox equivalents/g of extract. Trolox
equivalent is a unit of antioxidant capacity of a given substance,
as compared to the standard, Trolox, i.e., a water soluble analog of
vitamin E.
2.5. Antimicrobial activity testing
Antimicrobial testing was performed at the National Center
for Natural Product Research (NCNPR), University, MS. Primary
screening for antimicrobial activity of chamomile essential oil
distilled from 720 min DT was tested against Candida albicans, Can-
dida glabrata, Candida krusei, Aspergillus fumigatus, Cryptococcus
neoformans, Staphylococus aureus, methicillin-resistant S. aureus,
Escherichia coli, Pseudomonas aerogenosa, and Mycobacterium intra-
cellulare, at a concentration of 50g/ml, and % inhibition was
calculated as described previously (Bharate et al., 2007).
2.6. Antileishmanial and antimalarial activity testing
Antileishmanial activity of the oils was tested in vitro on a cul-
ture of LeishmaniadonoVanipromastigotes.In a 96-well microplateassay, the oil at concentration of 80g/ml was added to the
Leishmania promastigotes culture (2×106 cells/ml). The plates
were incubated at 26◦C for 72 h , and growth of Leishmania pro-
mastigotes was determined by Alamar Blue assay (Mikus and
Steverding, 2000). Pentamidine and Amphotericin B were used as
the standard antileishmanialagents. IC50(the concentrations caus-
ing 50% inhibition in growth) and IC90 (the concentrations causing
90% inhibition in growth) were computed from the growth inhi-
bition curve. Antimalarial activity was tested using a previously
described procedure (Bharate et al., 2007).
2.7. Statistical analysis
The effect of distillation time (DT) on oil content (%), and theconcentration (%) and component yield (g/100g dry flowers) of
anethole, farnesene, spathulenol,-bisabololoxide B,-bisabolone
oxide A, camazulene,-bisabolol oxideA, andspiroether was deter-
mined using a one-way analysis of variance. For each response,
the validity of model assumptions was verified by examining the
residuals as described in Montgomery (2013). Since the effect of
DT was significant (P < 0.05) on all responses, except antioxidant
capacity, multiple means comparison was completed using Dun-
can’s multiple range test at the 5% level of significance, and letter
groupings were generated. The analysis was completed using the
GLM Procedure of SAS (SAS Institute Inc, 2010).
The relationships between DT and oil content (%), and between
DT and the area percentage (%) of anethole and farnsene (steam
distillation extraction kinetics) were adequately described by thePower model (Eq. (1)). The relationships between DT and concen-
trations (%) of chamazulene,-bisabolol oxide A, and spiroether, as
well as the yields (g/100g dry flowers) of spathulenol,-bisabolol
oxide B, -bisabolone oxide A, chamazulene, -bisabolol oxide
A, and spiroether were adequately described by the Michaelis-
Menten model(Eq. (2)). The relationships between DT andthe other
response variableswere notstrong enoughto be describable byany
regression model. Since both the Power and the Michaelis-Menten
models are nonlinear, their parameters were estimated iteratively
using the NLIN Procedure of SAS (SAS Institute Inc, 2010), and the
fitted models met all model adequacy requirements described in
Bates and Watts (2007).
Y = 1 X 2 + ε (1)
Y = 1 X
2 + X + ε (2)
where Y is the dependent (response) variable, X is the independent
(DT)variable, andthe error term isε assumed to have normal distri-
bution with constant variance. Validity of the normality, constant
variance, and independence assumptions on the error terms were
verified by examining the residuals (Bates and Watts, 2007).
3. Results
3.1. Effect of DT on essential oil yield
Essential oils obtained by treating chamomile flowers to dif-
ferent DT showed significant increases in oil yield (content) with
increasing DT, and reached a maximum at 720 m in DT (Fig. 2,
Table 1).
3.2. Effects of DT on essential oil composition
The major compounds identified and quantified in chamomile
essential oil were anethole, -farnesene, spathulenol, -bisabolol
oxide B, -bisabolone oxide A, chamazulene, -bisabolol oxide A,and spiroether. -farnesene and -bisabolol oxide A were major
components accounting for almost 60–70% of the total oil com-
position. -farnesene showed a decrease in concentration with
increasing DT (explained by a Power model, Fig. 2), whereas %
-bisabolol oxide A, rapidly increased with an increase in DT up
to 240min, after which it reached a plateau showing negligible
change (explained by the Michaelis-Menton model, Fig. 2). Sim-
ilarly, spiroether and chamazulene, the next major constituents
in order, showed a rapid increase up to 240min DT after which
they remained fairly unchanged. Both kinetics were very well
modeled by the Michealis-Menton model(Fig.2). Anethole showed
a steady decrease over time from approximately 2.42% at 30min
DT to 0.54 at 720min DT (Table 1, Fig. 2). Plots of yields of essen-
tial oil constituents expressed as mg/100g of dried flowers vs. DTshoweda steady increase in yields of spathulenol,-bisabolol oxide
B, -bisabolol oxide A, -bisabolone oxide A, chamazulene, and
spiroether that was adequately modeled by the Michaelis-Menton
model (Table 2, Fig. 3).
3.3. Antioxidant capacity
The antioxidant capacity of the chamomile oil in this study
was not significantly affected by the DT. An average activity
of 905mol Trolox equivalents/g of extract was observed in
chamomile essential oil. These results suggest thatsimilar amounts
and/orcombinations of antioxidantswere extractedfor allthe time
points.
3.4. Antimicrobial activity
Antimicrobial activity of the chamomile essential oil from this
study tested against ten microorganisms exhibited low inhibition
against Candida krusei, Cryptococcus neoformans, and Mycobac-
terium intracellulare with percent inhibitions of 38, 39, and 35,
respectively (data not shown). The chamomile oil showed some
activity against Candida globrata, and Pseudomonas aeruginosa
with a 14% and 10% inhibition, respectively. The chamomile oil
had negligible activity against E. coli and Candida albicans, with
3% and 5% inhibition, respectively. The antimicrobial activity of
chamomile oil against Aspergillus fumigatus, Staphylococcus aureus,
and methicillin-resistant S. aureuswas zero.
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64 A. Gawde et al. / Industrial Crops and Products 58 (2014) 61–67
Fig. 2. Plot of distillationtime(DT) vs. essentialoil yield andthe concentration of anthole,-farnsene, chamazulene,-bisabolol oxide A,and spiroether alongwith thefitted
(solid line) Power and Michealis-Menton regression models. The fitted models aregivenwithin each plot.
3.5. Antileishmanial and antimalarial activity
The activity of essential oil tested at a concentration of
15867ng/ml, did not show any antimalarial activity. There was no
significant antileishmanial activity for oils tested at 80g/ml.
4. Discussion
A significant increase in oil yield was observed with increasing
DT up to 480min, after which the oil yield increase was not sig-
nificant. While an increase in DT may be favorable for oil yields, it
Table 1
Mean essential oil (EO) yield and concentration of anethole, farnesene, spathulenol, -bisabolol oxide B, -bisabolone oxide A, chamazulene, -bisabolol oxide A, and
spiroether obtained from the10 distillationtimes (DT).
DT(min) EOyield(g/100) Anethole(%) Farnesene Spathulenol -Bisabolol oxide B -Bisabolone oxide A Chamazulene -Bisabolol oxide A Spiroether
30 0.027 c 2.4 b 41.6 a 2.0 de 9.0 bc 8.2 a 0.43 c 30.1 e 6.2 f
60 0.075 c 6.2 a 34.6 b 1.9 e 8.8 c 7.4 ab 0.89 bc 32.5 de 8.6 ef
90 0.070 c 1.9 bc 32.2 bc 2.2 bc 9.8 a 8.1 a 0.97 b 34.9 cd 10.1 de
120 0.098 c 1.5 cd 28.9 cd 2.3 ab 9.5 ab 8.1 a 1.53 a 36.5 c 11.7 d
180 0.182 b 1.0 de 26.3 de 2.4 a 8.7 c 8.1 a 1.62 a 37.9 bc 14.1 c
240 0.197 b 0.9 de 23.2 ef 2.3 ab 7.8 d 7.3 b 1.81 a 41.7 a 15.0 bc
360 0.204 b 0.7 e 21.5 f 2.1 cd 7.0 e 7.03 b 1.85 a 41.6 a 18.3 a
480 0.283 a 1.7 c 24.4 ef 2.2 b 7.5 de 6.9 b 1.85 a 40.1 ab 15.5 bc
600 0.289 a 0.9 de 22.5 ef 2.1 bc 7.2 de 7.0 b 1.81 a 41.1 ab 16.9 ab
720 0.307 a 0.5 e 22.5 ef 2.2 bc 7.4 de 7.2 b 1.80 a 40.4 ab 17.9 a
Withineach column, means followed by thesame letterare not significantly differentat 5% level of significance.
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A. Gawde et al. / Industrial Crops and Products 58 (2014) 61–67 65
Table 2
Meanyield of anethole,farnesene, spathulenol,-bisabololoxide B,-bisabolone oxideA, chamazulene,-bisabololoxide A, and spiroether obtained fromthe 10 distillation
times (DT).
DT(min) Anethole(g/100g dry flowers) Farnesene Spathulenol -Bisabolol oxide B -Bisabolone oxide A Chamazulene -Bisabolol oxide A Spiroether
30 0.7 d 11.4 d 0.5 d 2.5 d 2.2 d 0.1 e 8.0 c 1.6 d
60 3.9 ab 26.1 cd 1.4 cd 6.6 cd 5.6 cd 0.7 e 24.2 c 6.5 d
90 1.3 cd 22.6 cd 1.5 cd 6.9 cd 5.8 cd 0.7 e 24.7 c 7.1 d
120 1.5 bcd 28.2 c 2.2 c 9.3 c 7.9 c 1.5 de 35.6 c 11.4 d
180 1.8 abcd 47.6 b 4.3 b 15.8 b 14.7 b 2.9 cd 68.8 b 25.6 c
240 1.8 abcd 45.4 b 4.5 b 15.4 b 14.3 b 3.6 c 82.0 b 29.6 c360 1.3 cd 43.6 b 4.2 b 14.1 b 14.2 b 3.8 bc 84.6 b 37.6 bc
480 4.7 a 68.8 a 6.2 a 21.0 a 19.4 a 5.3 ab 113.7 a 44.0 ab
600 3.6 abc 65.4 a 6.2 a 20.8 a 20.1 a 5.2 ab 118.7 a 48.7 ab
720 1.6 bcd 68.8 a 6.7 a 22.8 a 22.1 a 5.5 a 124.3 a 55.3 a
Withineach column, means followed by thesame letterare not significantly differentat 5% level of significance.
resulted in dissimilar chemical composition over time, confirming
our hypothesis. A specific desired compositional profile may be
a prime factor in making a choice of DT. As mentioned in the
results, -bisabolol oxide A rapidly increased with increasing DT
up to 240 m in after which it remains unaltered. This also means
that -bisabolol oxide A concentrations will not be jeopardized if
longer DTs are utilized to exploit higher oil yields. However, if oil
with high -farnesene concentration is desirable, then chamomile
flowers can be distilled for 30min only. If higher concentrations
of -bisabolol oxide A and chamazulene in the oil, and higher oil
yields are desirable, chamomile flowers need to be steam distilled
for 480 min. Previously, DT was demonstrated to change oil yields,
Fig. 3. Plot of distillationtime (DT) vs.the yieldsof spathulenol,-bisabolol oxideB, -bisabolone oxide A, chamazulene,-bisabolol oxide A, andspiroether along with the
fitted (solid line) Michealis-Menton regression models. The fitted models aregivenwithin each plot.
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A. Gawde et al. / Industrial Crops and Products 58 (2014) 61–67 67
Zheljazkov, V.D., Astatkie, T., Jeliazkova, E.A., Schlegel, V., 2012c. Distillation timealters essentialoil yield, composition, and antioxidantactivityof maleJuniperusscopulorum trees. J. Oleo Sci. 61, 537–546.
Zheljazkov, V.D., Horgan, T., Astatkie, T., Schlegel, V., 2013a. Distillation timemodifies essential oil yield, composition, and antioxidant capacity of fennel(Foeniculum vulgare Mill). J. Oleo Sci. 62, 665–672.
Zheljazkov, V.D., Astatkie, T., Jeliazkova, E.A., Tatman, A.O., Schlegel, V., 2013b.Distillation time alters essential oil yield, composition and antioxidant
activity of female Juniperus scopulorum trees. J. Essent. Oil Res. 25,62–69.
Z he ljazkov , V .D., As tatkie , T., H or gan, T., Schlege l, V. , S imonn et , X. , 2013c.Dist il lation t ime e ffe ct on es se nt ial o il y ield, composition, and ant ioxi-dant capacity of sweet sagewort (Artemisia annua L.) oil. HortScience 48,1288–1292.
Zheljazkov, V.D., Cantrell, C.L., Astatkie, T., Jeliazkova, E., 2013d. Distillation timeeffecton lavender essential oil yield and composition.J. Oleo Sci. 62, 195–199.