Journal of Oleo Science

Copyright ©2015 by Japan Oil Chemists’ Society

doi : 10.5650/jos.ess14258
J. Oleo Sci. 64, (5) 485-496 (2015)

Method for Attaining Rosemary Essential Oil with

Differential Composition from Dried or Fresh
Material
Valtcho D. Zheljazkov1＊ , Tess Astatkie2, Ivan Zhalnov3 and Tonya D. Georgieva3
1
Oregon State University, Columbia Basin Agricultural Research Center, 48037 Tubbs Ranch Road, P.O. Box 370, Pendleton, OR 97801, U.S.A.
2
Dalhousie University, Faculty of Agriculture, 50 Pictou Road, P.O. Box 550, Truro, NS B2N 5E3, Canada
3
Agricultural University, 12 "Mendeleev" Blvd., Plovdiv 4000, Bulgaria

Abstract: Rosemary (Rosemarinus officinalis L.) is a well-known medicinal and essential oil plant, utilized
by humankind since ancient times. The objective was to determine the effect of steam distillation time (DT)
and material (dry or fresh biomass) on essential oil yield, composition, and bioactivity; and to develop
regression models that can predict oil yield and composition at specific DT. The oil yield (content) from dry
biomass was higher (0.43%) than that from fresh biomass (0.35%) and ranged from 0.18% in the 1.25 min
DT to 0.51% in the 40 min DT. There was no yield advantage in extending the DT beyond 40 min, which is
much shorter than the DT used by industry. In this study, the antioxidant capacity of the rosemary oil using
the ORAC oil method was 4,108 mmolVE/L. Rosemary oil did not exhibit significant antileishmanial,
antimalarial, or antimicrobial activity. In general, the low-boiling constituents eluted earlier than the higher
boiling constituents of the essential oil, resulting in a great variation of essential oil composition obtained at
different DT. The most important constituents are α-pinene, eucalyptol, and camphor. The highest α-pinene
concentration in the oil (30.4%) was obtained from dry biomass at 2.5 min DT; eucalyptol (23.3% of the
total oil) from fresh biomass at 2.5 min DT; and camphor (15.9% of the total oil) from fresh biomass at 160
min DT. The DT could be used as an inexpensive tool to alter essential oil composition of the essential oil
from fresh or dried rosemary biomass, and to produce rosemary oils with elevated or lowered concentration
of specific targeted oil constituents to meet specific market demands.

Key words: steam distillation time, Rosemarinus officinalis, α-pinene, eucalyptol, camphor

1 INTRODUCTION surface treatments of various bruises, sprains, and dislo-

Rosemary（Rosemarinus officinalis L.）is a well-known
medicinal and essential oil plant that has been utilized by
humankind for over thousands of years, and is mentioned
in the Greek mythology and in the Bible. This plant has
been used in folk medicines in Asia（India, China）, in the
Mediterranean, and other regions, as well as in medicinal,
culinary, and decorative products since ancient times1, 2）.
For example, the Bulgarian traditional medicine has been
using rosemary as brain-stimulating herb and as a mild
sedative. Rosemary is prescribed to treat physical and
nervous fatigue and is used widely as a biological insecti-
cide against clothes moths2）. Rosemary herbage is used in
the preparation of various dishes. Rosemary extract and
essential oil have been and continues to be used as the
main constituent of various balsamic preparations for
cated joints, as well as for the treatment of some paralyses.
All aboveground plant parts of rosemary contain highly aro-
matic and pleasantly scented essential oil, which has nu-
merous applications in the commercial production of
liquors, scented consumer products, perfumes, cosmetics,
and aromatherapy2, 3）. The biomass residue after the ex-
（distillation waste）could also be
traction of the essential oil
utilized as a source for antioxidant compounds4, 5）, or for
rosmarinic acid, hesperidin, and carnosol in the polypheno-
lic extracts following extraction with petroleum ether and
subsequent methanol extraction in a Soxhlet6）. Rosemary
essential oil composition and the ratio between different oil
constituents plays a role in its bioactivity. Rosemary oil
composition depends on various environmental and genetic
factors, as well as on postharvest processing factors.

＊
Correspondence to: Valtcho D. Zheljazkov, Oregon State University, Columbia Basin Agricultural Research Center, 48037 Tubbs
Ranch Road, P.O. Box 370, Pendleton, OR 97801, U.S.A.
E-mail: valtcho.jeliazkov@oregonstate.edu, valtcho.pubs@gmail.com
Accepted January 9, 2015 (received for review November 16, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

485
 V. D. Zheljazkov, T. Astatkie and I. Zhalnov et al.
Traditionally, the essential oil of rosemary has been ex-
tracted via steam distillation as it is the easiest and the
most economical method, although other extraction
methods such as hydrodistillation6, 7）, supercritical CO2 ex-
traction 8, 9）, and innovative hydrodiffusion and gravity
（MHG）10） also have been shown to hold promise. Micro-
wave irradiation was also proposed as a useful pre-treat-
ment to improve the conventional steam distillation of
rosemary11）. There is no agreement in the literature regard-
ing the optimum time for steam distillation of rosemary
biomass, and most of the reports have been utilizing 2-4 h
duration. Comparing literature reports on rosemary oil
yield and composition is difficult due to the various length
of the distillation process utilized. It is not known how the
duration of the steam distillation affects essential oil yield
and essential oil composition, two parameters of impor-
tance for producers, processors and end users. Recent
studies on plants from the same family as rosemary dem-
onstrated that the duration of the DT may alter essential
oil composition substantially, and in some cases alter the
bioactivity of peppermint 12, 13）, Japanese cornmint 14）,
oregano15）, lavender16）, and garden sage17）. The hypothesis
of this study was that the duration of the steam distillation
time（DT） will significantly modify the essential oil yield and
composition, and bioactivity of rosemary. To test the hy-
pothesis, we designed a study with a specific objective of
evaluating the duration of DT on rosemary essential oil
yield, composition, and antioxidant activity and developing
regression models that predict oil yield and composition at
specific steam DT.
2 MATERIALS AND METHODS
2.1 Plant Material
Rosemary（Rosmarinus officinalis L.）seeds were kindly
provided by Dr. Natasha Kovatcheva at the Research Insti-
tute for Roses and Medicinal Plants in Bulgaria. In March
2006, the rosemary seeds were sown in plastic cells filled
with greenhouse growth medium（Metromix 300, The
Scotts Co., Marysville, OH） . The rosemary transplants were
produced in a controlled-environment greenhouse for 50
days, with a day temperature of 22 to 25℃ and a night
temperature of 18℃. The rosemary seedlings were fertil-
ized once a week with a 1.8 g of 20-20-20 N-P2O5-K2O dis-
solved in 300 mL of water and applied to one tray contain-
ing 32 cells, and irrigated daily as needed. The rosemary
plantation was established in June 2006, at the Mississippi
State University, North Mississippi Research and Extension
Center at Verona, MS（34°43′22″N and -88°43′22″W）.
The rosemary plots were in 4 replicates, as part of a ran-
domized complete block design experiment that included
over 30 different aromatic plant species. Only well-devel-
oped transplants around 12-cm height were used for trans-
486
J. Oleo Sci. 64, (5) 485-496 (2015)
planting in previously prepared raised beds covered with
black plastic mulch. The land preparation was as described
earlier for lavender and hyssop18）. Briefly, a bed-shaping
machine was used to form raised beds （15 cm height, 75 cm
wide） , the drip tape was placed in the middle of the bed at
around 5-cm depth, under the plastic. The fertilizers（80
kg/ha of P2O, and 100 kg/ha of K2O, and N at 130 kg/ha）
were based on soil test results, and were applied prior to
bed formation and incorporated by disking. The rosemary
plantlets were transplanted in previously prepared holes in
the plastic mulch, in two rows on each bed, in an offset
manner, at 45-cm in-row and 30-cm between-row spacing.
2.2 Steam Distillation and Distillation Times （DT）
The distillation study experiments were conducted with
fresh or dried rosemary biomass in the fall of 2010, using
the biomass from the 3 replicates of a 5-year old, well-es-
tablished rosemary plantation. Rosemary aboveground
plant parts（ including stems, leaves, and flowers）were
used, the sample replicates correspond to the replicates in
the field experiment. The study with the fresh rosemary
biomass was conducted using freshly harvested biomass,
which was distilled within 1 h, and for the dried material,
the study was conducted with air-dried biomass, which was
air-dried to a constant weight under a shade in a well-aer-
ated barn, to avoid oil losses due to direct exposure to sun
and high temperatures. Fresh （400 g）or dried subsamples
（250 g each）were extracted via steam distillation in 2-L
steam distillation units as described previously19）. Eight
distillation times（DT; 1.25, 2.5, 5, 10, 20, 40, 80, and 160
min） were used. The combinations of material and DT were
randomized and replicated 3 times, giving a total of 48
runs. The beginning of each DT time was recorded at the
moment when the first drop of essential oil dropped into
the separator. At the end of each specific DT, the power
was turned off; the bioflask was separated from the steam
generator and from the separator. The oil and the water
were eluted to a glass vial and put in a freezer. The follow-
ing day, the oil was separated from the water, measured on
analytical scale, and kept in the dark in a freezer. The rose-
mary essential oil yield for each replication was calculated
as grams of oil per 100 g of fresh or dry rosemary biomass.
2.3 Gas Chromatography（GC）Analysis of the Rosemary
Essential Oil
The 48 rosemary essential oil samples were analyzed for
oil profile on a gas chromatograph （Hewlett Packard 6890
GC）. The carrier gas was helium, at 40 cm/sec, 11.7 psi
（60℃）, 2.5 ml/min constant flow rate; the injection was
split 60:1, 0.5 μL, the injector temperature was 220℃, and
the oven temperature program was as follows: 60℃ for 1
min, 10℃/min to 250℃. The GC was fitted with a HP-IN-
NOWAX column（cross-linked PEG; 30 m×0.32 mm×0.5
μm）; the flame ionization detector（FID）temperature was
 Method for Attaining Rosemary Oil with Differential Composition
275℃. Individual peaks representing different oil constitu-
ents of rosemary oil were identified using internal stan-
dards（for all the major constituents）, by retention time
and also using mass-spectroscopy.
2.4 Assays for In Vitro Antimicrobial, Antileishmanial, and
Antimalarial Activity, and Assay for Antioxidant Capac-
ity
Selected rosemary essential oils were screened for anti-
microbial and antimalarial activities as described previous-
ly20）. The antileishmanial activity in vitro on a culture of
Leishmania donovani promastigotes was conducted as
described previously21）. The antimicrobial, antileishmanial,
and antimalarial activity of rosemary oils were conducted
at the National Center for Natural Products Research at
the University of Mississippi. The antioxidant capacity of
representative samples of rosemary oils from this study
were tested for antioxidant activity using the ORAC oil
method by Brunswick Laboratories（Southborough, MA,
USA）as described previously22）. As reported by the Bruns-
wick Laboratories,“the ORAC analyses provides a measure
of the scavenging capacity of antioxidants against the
peroxyl radical, which is one of the most common reactive
oxygen species （ROS） found in the body. ORACoil reflects oil
antioxidant capacity. Vitamin E is used as the calibration
standard and the ORAC results are expressed as micromole
VE equivalent （VE）per liter” . requirements25）.
Primary screening for antimicrobial activity of rosemary
essential oils from the two studies from the 160 min DT
were tested against Candida albicans, Candida glabrata,
Candida krusei, Aspergillus fumigatus, Cryptococcus
neoformans, Staphylococus aureus, methicillin-resistant
S. aureus, Escherichia coli, Pseudomonas aerogenosa,
and Mycobacterium intracellulare, at a concentration of
50 μg/ml. The results showing ％ inhibition were calculated
as described previously20）.
2.5 Statistical Analysis
The effect of material（dry vs fresh） and DT（eight steam
distillation times）on oil content（％） , and the concentration
（％）and yield（mg/100 biomass）of α-pinene, camphene,
β-pinene, myrcene, eucalyptol, linalool, camphor, borneol,
verbenone, bornyl acetate, and β-caryophyllene was deter-
mined using a two-factor factorial analysis of variance. For
each response, the validity of model assumptions was veri-
fied by examining the residuals as described previously23）.
For responses with significant material by DT interaction
effect, multiple means comparison of the 16 combinations
of material and DT was completed using the lsmeans state-
ment of Proc GLM of SAS24） that compares all possible
pairs of the least squares means at the 1％ level of signifi-
cance to protect Type I experimentwise error rate from
over inflation. However, when the interaction effect is not
significant, but the main effects are significant, the corre-
J. Oleo Sci. 64, (5) 485-496 (2015)
sponding means were compared at the 5％ level of signifi-
cance. The analysis was completed using the GLM Proce-
dure of SAS24）.
Nonlinear regression modeling to describe the relation-
ships between DT and the concentrations and yields of the
constituents was completed for dry and fresh materials
separately. For dry material, the relationships between DT
and the concentrations of α-pinene and camphene were
adequately modelled by the Power model（Eq. 1）. For dry
material, the relationships between DT and the concentra-
t i o n s o f c a m p h o r, b o r n e o l , b o r n y l a c e t a t e , a n d
β-caryophyllene, as well as the relationships between DT
and the yields of linalool and β-caryophyllene were ade-
quately modelled by the Asymptotic model（Eq. 2）. For
fresh material, the relationships between DT and oil
content was described by the Asymptotic regression model
（Eq. 2） , and the relationship between DT and the concen-
trations of eucalyptol, bornyl acetate, and β-caryophyllene
was adequately described by the Power regression model
（Eq. 1）. For fresh material, the relationships between DT
and the yields of camphor, borneol, bornyl acetate, and
β-caryophyllene were very well described by the Michaelis-
Menten model（Eq. 3）. The parameters of these three non-
linear regression models （Power, Asymptotic and Michaelis-
Menten）were estimated iteratively using the NLIN
Procedure of SAS24）and the fitted models met all adequacy
Y＝θ 1−θ 2eθ X＋ε3
（Eq. 1）
Y＝θ 1X θ ＋ε2
（Eq. 2）
θ 1X
Y＝ ＋ε （Eq. 3）
θ 2＋X
Where Y is the dependent（response）variable, X is the
independent（DT） variable, and the error term ε is assumed
to have normal distribution with constant variance. Validity
of the normality, constant variance and independence as-
sumptions on the error terms were verified by examining
the residuals25）.
3 RESULTS
The 51 constituents listed in Table 1 were identified in
the rosemary oil from this study. However, we did statisti-
cal analysis of the 11 constituents that have the highest
concentration in the total oil.
The Analysis of Variance results that show the signifi-
cance of the main and interaction effects of material and
DT are presented in Table 2 and Table 3. Essential oil
content was significantly affected by both material and DT,
（Table 2）. Dry
but the interaction effect was not significant
material gave higher（ 0.43％）oil content（Table 4）, and
there was no increase in oil yield beyond 10 min DT （Table
487
 V. D. Zheljazkov, T. Astatkie and I. Zhalnov et al.

Table 1 List of the 51 constituents identified in the rosemary oil from this study.
No. Constituent No. Constituent No. Constituent No. Constituent
1. a-pinene 14. thuja-2 27. chrysanthanone 40. trans-cadina-1(6)
2. camphene 15. 4(10)-diene 28. trans-pinocamphone 41. 4-diene
3. b-pinene 16. 1-octen-3-ol 29. pinocarvone 42. g-muurolene
4. myrcene 17. a-phellandrene 30. cis-pinocamphone 43. a-amorphene
5. eucalyptol 18. a-terpinene 31. 4-terpineol 44. b-selinene
6. linalool 19. paracymene 32. para-cymen-8-ol 45. g-amorphene
7. camphor 20. limonene 33. a-terpineol 46. cis-cadina-1
8. borneol 21. cis-ocimene 34. myrtenol 47. d-amorphene
9. verbenone 22. trans-ocimene 35. a-ylangene 48. d-cadinene
10. bornyl acetate 23. g-terpinene 36. a-copaene 49. a-calacorene
11. b-caryophyllene 24. sabinene hydrate 37. sativene 50. caryophyllene oxide
12. cis-3-hexenol 25. terpinolene 38. methyl eugenol 51. humalene epoxide II
13. tricyclene 26. a-pinene oxide 39. a-humalene

Table 2 P-values that show the main and interaction effect of Material (M) and DT on essential oil (EO) content and the
concentrations of α-pinene, camphene, b-pinene, myrcene, eucalyptol, linalool, camphor, borneol, verbenone,
borylacetate, and b-caryophyllene.
camph eucaly camph verbe boryl b-caryo
SV† EO a-pinene b-pinene Myrcene linalool borneol
ene ptol or none acetate phyllene
M 0.001†† 0.001 0.445 0.001 0.001 0.001 0.815 0.001 0.011 0.008 0.001 0.001
DT 0.001 0.001 0.001 0.001 0.331 0.002 0.121 0.001 0.001 0.966 0.001 0.001
M*DT 0.113 0.032 0.683 0.003 0.272 0.503 0.350 0.049 0.115 0.367 0.001 0.001
†
SV = Source of Variation.
††
Significant effects that require multiple means comparison are shown in bold.

Table 3  -values that show the main and interaction effect of Material (M) and DT on the yields of α-pinene, camphene,
P
b-pinene, myrcene, eucalyptol, linalool, camphor, borneol, verbenone, borylacetate, and b-caryophyllene.
camph eucaly verbe Boryl b-caryo
SV† a-pinene b-pinene myrcene linalool camphor borneol
ene ptol none acetate phyllene
M 0.001†† 0.073 0.021 0.001 0.255 0.001 0.606 0.024 0.311 0.001 0.001
DT 0.010 0.020 0.001 0.001 0.001 0.001 0.001 0.001 0.280 0.001 0.001
M*DT 0.151 0.537 0.165 0.354 0.106 0.790 0.710 0.016 0.318 0.002 0.001
†
SV = Source of Variation.
††
Significant effects that require multiple means comparison are shown in bold.

Table 4  ean essential oil (EO) content (% oil in fresh or dry biomass), and
M
mean concentration (% of the total oil) of myrcene, eucalyptol,
borneol, and verbenone from dry and fresh materials.
EO content† Myrcene eucalyptol borneol verbenone
Material
in biomass ------------------- % of total oil ---------------------------
Dry 0.430 a 4.79 a 19.3 b 2.57 b 0.98 b
Fresh 0.346 b 3.96 b 22.6 a 2.90 a 1.46 a
†
Means sharing the same letter are not significantly different at the 5% level of
significance.

488
J. Oleo Sci. 64, (5) 485-496 (2015)
 Method for Attaining Rosemary Oil with Differential Composition

Table 5 Mean essential oil (EO) content (% oil in biomass), and mean
concentration (% in the oil) of camphene, eucalyptol, and borneol
from the eight steam distillation times (DT).
DT (min) EO content camphene eucalyptol borneol
†
1.25 0.182 d 9.54 a 22.0 abc 1.93 d
2.5 0.353 c 8.94 ab 23.3 a 1.90 d
5 0.342 c 9.64 ab 18.9 d 2.69 bc
10 0.443 abc 8.00 abcd 22.2 ab 2.53 c
20 0.494 ab 7.70 bcd 21.0 bcd 2.99 b
40 0.509 a 8.07 abc 19.7 d 3.06 b
80 0.403 abc 7.19 cd 20.1 cd 3.22 ab
160 0.381 bc 6.54 d 20.6 bcd 3.57 a
†
Means sharing the same letter are not significantly different at the 5% level of
significance.

Fig. 1 I nteraction plot of the concentration of essential oil constituents (in % of total oil) of α-pinene, β-pinene,
camphor, and bornyl acetate obtained from the combinations of the two materials and the eight distillation
times. Within each plot, means sharing the same letter are not significantly different at the 1% level of
significance.

489
J. Oleo Sci. 64, (5) 485-496 (2015)
 V. D. Zheljazkov, T. Astatkie and I. Zhalnov et al.

Fig. 2 I nteraction plot of the concentration of β-caryophyllene (in % of total oil), and the yields (mg/100 biomass) of
the essential oil constituents borneol, bornyl acetate, and β-caryophyllene obtained from the combinations of
the two materials and the eight distillation times. Within each plot, means sharing the same letter are not
significantly different at the 1% level of significance.

5）. However, there was significant interaction effect on
α-pinene, β-pinene, camphor, boryl acetate, and
β-caryophyllene（Table 2）concentrations suggesting that
the differences in the means obtained from the different
DTs are not uniform for fresh and dry materials.
The highest concentration of α-pinene（30.4％; Fig. 1）
was obtained from dry material at 2.5 min DT, of β-pinene
（4.13％; Fig. 1）from fresh material at 2.5 min DT, of
camphor（15.9％; Fig. 1）from fresh material at 160 min DT,
of boryl acetate（5.79％; Fig. 1）from fresh material at 160
min DT, and of β-caryophyllene（3.51％; Fig. 2）from dry
material at 80 min DT.
Generally, the low boiling essential oil constituents
α-pinene（Fig. 1）, camphene（Table 5）, β-pinene（Fig. 1）,
and eucalyptol（1,8-cineole; Table 5）were eluted early in
the distillation process, resulting in their higher concentra-
tions in the oils obtained at 1.25-2.5 min DT compared to
the oils obtained later. Although camphene was not affect-
490
J. Oleo Sci. 64, (5) 485-496 (2015)
ed by material（Table 2）, eucalyptol was significantly af-
fected by material, and higher concentration （22.6％ of the
total oil）was obtained from fresh biomass（Table 4） .
Conversely, the concentration of higher boiling point oil
constituents, camphor（Fig. 1）, and borneol（Table 5）were
higher in the oils obtained at longer DT（80 or 160 min） and
lower in the oils obtained at the shorter DT（1.25 or 5 min）.
The concentration of the highest boiling constituent, ver-
benone was not significantly affected by DT（Table 2） , but
by material, with fresh material giving higher concentration
（1.46％ of the total oil; Table 4）. The concentrations of
myrcene（ Table 2）and linalool（with an overall mean of
2.78％; Table 2） were not affected by the DT, but myrcene
was affected by material, with dried material giving higher
（4.79％ of the total oil; Table 4） concentration.
The yield of various constituents was calculated from the
oil yield at any specific DT and the concentration of a given
constituent in the oil, and it was expressed as mg of con-
 Method for Attaining Rosemary Oil with Differential Composition

Table 6 Mean yield (mg/100 rosemary biomass) of α-pinene, camphene, β-pinene, myrcene, eucalyptol,
linalool, and camphor from the eight steam distillation times (DT).
DT (min) α-pinene camphene β-pinene myrcene eucalyptol linalool camphor
†
1.25 52.4 c 18.3 c 6.7 c 8.9 b 41.3 d 4.9 c 19.9 d
2.5 93.4 ab 29.9 abc 10.6 b 15.8 ab 76.7 c 8.1 c 33.7 cd
5 85.9 abc 33.1 abc 8.8 bc 15.8 ab 62.7 cd 4.1 c 43.5 bc
10 102.3 a 35.6 ab 14.3 a 19.2 a 97.2 ab 13.9 ab 55.4 ab
20 103.5 a 38.3 ab 14.8 a 20.3 a 102.3 a 15.5 a 67.5 a
40 102.5 a 41.2 a 14.2 a 21.5 a 97.1 ab 12.7 ab 62.5 ab
80 74.8 abc 29.4 abc 11.5 ab 17.7 a 80.6 bc 12.1 b 52.6 b
160 64.2 bc 25.2 bc 10.5 b 16.1 a 78.1 c 13.2 ab 54.0 ab
†
Means sharing the same letter are not significantly different at the 5% level of significance.

Table 7 Mean yield (mg/100 rosemary biomass) of

α-pinene, β-pinene, myrcene, and linalool
from dry and fresh materials.
Material α-pinene β-pinene myrcene linalool
†
Dry 101 a 10.5 b 20.1 a 12.2 a
Fresh 68 b 12.3 a 13.7 b 8.9 b
†
Means sharing the same letter are not significantly different
at the 5% level of significance.

stituent per 100 g of rosemary biomass. As shown in Table
3 either material or DT affected the yields of all constitu-
ents except that of verbenone. Only the yields of borneol,
boryl acetate, and β-caryophyllene were significantly af-
fected by the interaction of material and DT（Table 3） . The
highest yield of borneol（17.97 mg; Fig. 2）, and of
β-caryophyllene（13.57 mg; Fig. 2）were obtained from dry
material at 40 min DT, and of bornyl acetate （18.85 mg; Fig.
2） was obtained from fresh material at 160 min DT.
Generally, the yield of most constituents was low in the
shorter DT （1.25 and 2.5 min） and reached maximum at 10
min DT（for α-pinene, camphene, β-pinene, myrcene, euca-
lyptol, camphor, and linalool） （for borneol）
, at 20 min , or at
40 min（for bornyl acetate, and β-caryophyllene） （Table 6,
Fig. 2）. Regarding material, higher yield of α-pinene（101
mg）, myrcene（20.1 mg）, and linalool（12.2）was obtained
from dry material and higher yield of β-pinene（12.3 mg）
was obtained from fresh material （Table 7） .
The fitted nonlinear regression models shown in the
plots of Fig. 3, Fig. 4 and Fig. 5 can be used to predict the
concentrations and yields at any given DT. The Asymptotic
regression model described the relationship between DT
and essential oil content from fresh material（Fig. 3）, the
concentrations of camphor, borneol, bornyl acetate,
β-caryophyllene from dry material（Fig. 4）, and the yields
of linalool and β-caryophyllene from dry material（Fig. 5）,
with the potential maximum values of 0.41％, 12.94％,
3.26％, 2.228％, 15.08 mg, and 14.58 mg respectively. The
J. Oleo Sci. 64, (5) 485-496 (2015)
Michaelis-Menten nonlinear regression model was the most
appropriate model for the yields of camphor, borneol,
bornyl acetate, and β-caryophyllene from fresh material.
The two parameters of this model are physically meaning-
ful representing the long term maximum（θ1）and the DT
required to reach half of the long term maximum（θ2）. Ac-
cordingly, the long term maximum yield of camphor,
borneol, bornyl acetate, and β-caryophyllene from fresh
material was 63.5, 14.42, 19.4, and 9.53 mg respectively.
The DT needed to reach half of these maximum yields
were 2.11, 3.71, 7.16, and 5.5 min respectively.
The Power model was the best nonlinear regression
model to describe the relationship between DT and the
concentration of eucalyptol, bornyl acetate, and
β-caryophyllene from fresh material （Fig. 3）, and the con-
centration of α-pinene, and camphene from dry material
（Fig. 4）. The relationships between DT and the other re-
sponse variables were either not strong enough, or there
was no pattern to develop an appropriate regression model.
In this study, the antioxidant capacity of the rosemary oil
using the ORACoil method was 4,108 μmolVE/L. Rosemary
oil antimicrobial activity from this study was screened
against ten microorganisms and exhibited low inhibition
against Candida krusei（14％ inhibition）, and negligible
activity against Aspergillus fumigatus（2％）, Cryptococ-
cus neoformans（4％）and Methicillin-resistant Staphylo-
coccus aureus ATCC 33591（MRS） （2％） .
The antimicrobial activity of rosemary oil against the
491
 V. D. Zheljazkov, T. Astatkie and I. Zhalnov et al.

Fig. 3 P
 lot of Distillation time vs. essential oil concentration (%) and the concentrations (%) of eucalyptol, bornyl
acetate, and β-caryophyllene from fresh material along with the fitted (solid line) Asymptotic and Power
nonlinear regression models.

other organisms: Candida albicans, Candida glabrata, S.
aureus, methicillin-resistant S. aureus, Escherichia coli,
Pseudomonas aerogenosa, and Mycobacterium intracel-
lulare was zero. Rosemary essential oil tested at a concen-
tration of 15867 ng/mL did not show any antimalarial activ-
ity. Also, there was no significant antileishmanial activity
for the rosemary oil tested at 80 ug/mL.
4 DISCUSSION
In this study, most of the oil from fresh or dried rosemary
biomass was extracted in the first 20 min suggesting that
there would not be yield advantage in extending the steam
distillation time beyond 20 min. These findings are impor-
tant, since the shorter DT may save significant amount of
time and energy resources for the growers（who have their
own or co-op distillation facility） , and for the essential oil
industry. Previous reports used much longer steam distilla-
tion time, between 2 and 4 hours. For example, in one
study, 2 h was reported as the optimal distillation time26）.
Another study used 2 h hydrodistillation time to extract
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J. Oleo Sci. 64, (5) 485-496 (2015)
the essential oil from air-dried leaves of rosemary7）, and
third study used 3 h for hydrodistillation of rosemary 6）.
Furthermore, hydrodistillation method was compared with
the innovative hydrodiffusion and gravity（MHG）methods
for oil extraction from fresh rosemary leaves by using 3 h
extraction time for the hydrodistillation method and 15
min for the MHG method10）. In another study, an ultra-
sound-assisted dynamic extraction（using ethanol）method
was compared with 3h steam distillation to extract oil from
rosemary27）. The authors reported that the ultrasound-as-
sisted dynamic extraction method was 165-176 min shorter
（fewer minutes）than the steam distillation used in the
same study27）. In another study, essential oil was extracted
using 4 h of hydrodistillation5）. Some authors did not report
the time for the hydrodistillation of rosemary28）.
This study demonstrated that the length of the DT could
alter essential oil yield and composition of either fresh or
dried rosemary. This result is logical because different oil
constituents elute at different time points during the steam
distillation process. The regression models developed for
the steam distillation of either fresh or dried rosemary
biomass may predict specific oil composition. The DT could
 Method for Attaining Rosemary Oil with Differential Composition

Fig. 4 P
 lot of Distillation time vs concentration (%) of α-pinene, camphene, camphor, borneol, bornyl acetate, and
β-caryophyllene from dry material along with the fitted (solid line) Power and Asymptotic nonlinear regression
models.

be used to obtain oil with a definite profile to meet the folia Mill.） , peppermint（Mentha×piperita L.）
16） 12, 13）
, and
demands of specific markets. Other recent studies with Japanese cornmint（Mentha canadensis L.） . 14）

crops from the same family, Lamiaceae, also have shown The oil yield in this study from the dried materials was
that the length of the DT can alter both yield and composi- higher than that from the fresh material, due to difference
tion of garden sage（ Salvia officinalis L.）17）, oregano in water content. In general, the rosemary oil yield in this
（Origanum vulgare L.） 15）
（Lavandula angusti-
, lavender study was similar to the oil content reported previously
493
J. Oleo Sci. 64, (5) 485-496 (2015)
 V. D. Zheljazkov, T. Astatkie and I. Zhalnov et al.

Fig. 5 P
 lot of Distillation time vs. yields (mg/100 biomass) of linalool, and β-caryophyllene from dry material; and
yields (mg/100 biomass) of camphor, borneol, bornyl acetate, and β-caryophyllene along with the fitted (solid
line) from fresh material Asymptotic and Michaelis-Menten nonlinear regression models.

（ranging from 0.6 to 1.5％ in fresh biomass29）, and from
0.88 and 1.2％ in fresh herbage）28）. The oil content in the
latter reports was only in the leaves, a determination that
explains the relatively high concentration. On the other
hand, 4.1％ oil content was reported in dried rosemary
leaves26）. It was also reported that oil content can vary
between 1.3 and 3.0％ in dry biomass, depending on the
genotype and the harvesting stage6）. In another study, oil
content of 0.9-1.2％ in fresh biomass was reported;
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J. Oleo Sci. 64, (5) 485-496 (2015)
however, the authors did not state the length of the hydro-
distillation time, the plant parts used for distillation, or the
phenological stage28）.
The main oil constituents of rosemary in this study were
α-pinene, eucalyptol, and camphor. Similar oil composition
（13-15.5％ α-pinene, 19-21％ eucalyptol, 17-19％ camphor
6）
of the total oil） （6-9.4％ α-pinene, 35-56％ eucalyptol
, and
＝1.8 cineole, 9.3-12.6％ camphor of the total oil）7）, was
also reported.
 Method for Attaining Rosemary Oil with Differential Composition
In a study with the effect of harvesting stages on oil
content and composition, 9.7-12.5％ α-pinene, 26％ 1,8
cineole（eucalyptol）and 25.4-26.3％ camphor was report-
ed28）. In this study, DT produced greater concentration
range of chemical constituents in rosemary essential oil
than previously reported concentration ranges for the
same chemotypes or harvesting stage6）, or in pre-treatment
with enzymes in an enzyme-assisted extraction7）, or har-
vesting stage or fertilizer rates28）.
In this study, rosemary essential oil did not show signifi-
cant antimicrobial activity against Candida albicans,
Candida glabrata, Candida krusei, Aspergillus fumiga-
tus, Cryptococcus neoformans, Staphylococus aureus,
methicillin-resistant S. aureus, Escherichia coli, Pseudo-
monas aerogenosa, and Mycobacterium intracellulare,
using a previously developed method20）. A recent study
showed significant antmicrobial effect of rosemary oil
against E. coli, L. monocytogenes and Salmonella ty-
phimurium6）. Higher antimicrobial effects were associated
with higher concentrations of γ-terpinene, terpinolene and
caryophillene oxide. Also, antimicrobial activity of rose-
mary essential oil against Staphylococcus aureus, Entero-
coccus feacium, Staphylococcus agalactiae, Escherichia
coli and Salmonella typhimurium, and the yeast
Candida albicans was reported7）. However, the authors
used different method and much higher concentration of
rosemary oil for testing the antimicrobial activity7）. In addi-
tion, the chemical profile of the rosemary oil in this study,
although similar, was not the same as in previous studies.
The specific ratio between different oil constituents are
also thought to affect antimicrobial activity and antioxidant
capacity of rosemary essential oil6, 7, 28）.
Higher antioxidant activity of rosemary extract obtained
at fruit maturation stage of the biomass was also reported6）.
However, the results from the latter report are not compa-
rable with the results in this study since the authors used
different method, additionally; we report the antioxidant
capacity of rosemary essential oil while the other authors5, 6）,
reported the antioxidant activity of distillation waste.
5 CONCLUSIONS
This study found optimal distillation time for high essen-
tial oil yield from fresh or dried rosemary biomass. The
results demonstrated no oil yield advantage beyond 10-20
min DT.
The results from this study with fresh or dried rosemary
biomass attested that the distillation time must be speci-
fied when reporting essential oil content and composition
of rosemary; otherwise, the results would not be compara-
ble to those of other reports. The DT could be used as in-
expensive tool to alter essential oil composition of the es-
sential oil from fresh or dried rosemary biomass, and to
J. Oleo Sci. 64, (5) 485-496 (2015)
produce rosemary oils with elevated or lowered concentra-
tion of specific targeted oil constituents to meet specific
market demands. The regression models developed in this
study can be used to predict rosemary essential oil profile
at different DT, and possibly, to compare literature reports
in which different duration of the steam distillation was
used.
ACKNOWLEDGEMENTS
This research was funded in part by ARS Specific Crop
Agreement 58-6402-026 with Mississippi State University.
Specific project:“Field establishment of medicinal herbs
and potential for commercial production”awarded to Dr.
V.D. Jeliazkov（Zheljazkov）. Authors thank Thomas Horgan,
Ganisher Abbasov, and S. Marie Rogers, for excellent help
with the field experiments and oil extraction.
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