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Comparative studies of volatile

oil composition of Rhododendron
anthopogon by hydrodistillation,
supercritical carbon dioxide extraction
and head space analysis
a a a a
Shailja Guleria , Vikas Jaitak , Rikki Saini , Vijay K. Kaul ,
b a a b
Brij Lal , G.D. Kiran Babu , Bikram Singh & R.D. Singh
a
Natural Plant Products Division, Institute of Himalayan
Bioresource Technology (CSIR) , Post Box No. 6, Palampur – 176
061, Himachal Pradesh, India
b
Biodiversity Division, Institute of Himalayan Bioresource
Technology (CSIR) , Post Box No. 6, Palampur – 176061, Himachal
Pradesh, India
Published online: 19 Aug 2011.

To cite this article: Shailja Guleria , Vikas Jaitak , Rikki Saini , Vijay K. Kaul , Brij Lal , G.D.
Kiran Babu , Bikram Singh & R.D. Singh (2011) Comparative studies of volatile oil composition of
Rhododendron anthopogon by hydrodistillation, supercritical carbon dioxide extraction and head
space analysis, Natural Product Research: Formerly Natural Product Letters, 25:13, 1271-1277, DOI:
10.1080/14786419.2011.576395

To link to this article: http://dx.doi.org/10.1080/14786419.2011.576395

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 Natural Product Research
Vol. 25, No. 13, August 2011, 1271–1277

Comparative studies of volatile oil composition of Rhododendron

anthopogon by hydrodistillation, supercritical carbon dioxide
extraction and head space analysisy
Shailja Guleriaa, Vikas Jaitaka, Rikki Sainia, Vijay K. Kaula*, Brij Lalb,
G.D. Kiran Babua, Bikram Singha and R.D. Singhb
Downloaded by [University of California, San Francisco] at 19:43 23 August 2014

a
Natural Plant Products Division, Institute of Himalayan Bioresource Technology (CSIR),
Post Box No. 6, Palampur – 176 061, Himachal Pradesh, India; bBiodiversity Division,
Institute of Himalayan Bioresource Technology (CSIR), Post Box No. 6, Palampur –
176061, Himachal Pradesh, India
(Received 28 July 2010; final version received 27 March 2011)

Volatile oil composition of the leaves of Rhododendron anthopogon

(Ericaceae) growing wild in alpine Western Himalaya was studied using
different extraction techniques including SC-CO2 extraction and hydro-
distillation (HD). Results from different extraction methodologies were
compared with headspace analysis (HS) and evaluated for the effectiveness
of techniques in characterisation of various terpene categories and to assess
their influence on the yield and composition of volatiles. Variability in
constituents and in quantitative yields was observed. The results varied
with different extraction methods. A total of 27 constituents in SC-CO2
extraction, 31 in HD and 17 in HS analysis were identified. Constituents in
SC-CO2 and HD oils were identified by gas chromatography mass
spectrometry analysis. SC-CO2 extraction was carried out at 40 C and
140 bar pressure and the oil represented by major constituents as
-caryophyllene (5.96%), -humulene (4.06%) and p-menthadiene-2,9-
diol (7.28%); in HD, oil limonene (11.26%), -caryophyllene (11.62%),
-humulene (7.22%), and E-nerolidol (5.83%) dominated the oil and in HS
analysis, limonene (24.14%), -terpinene (40.73%), -terpinene (4.92%),
-phellandrene (3.44%) and -ocimene (7.15%) were present as major
constituents.
Keywords: Rhododendron anthopogon; Ericaceae; volatile oil; SC-CO2;
hydrodistillation; GC–MS; headspace analysis; comparison

1. Introduction
Genus Rhododendron (family Ericaceae), a perennial plant, is represented by 1000
species globally (http://en.wikipedia.org/wiki/Rhododendron) having habitat in East
Asia and China (Zhao et al., 2006). In India, 80 species are found confined mainly in
Western Himalayas between 3000 and 4500 m (Aswal & Mehrotra, 1994). Among
these, Rhododendron anthopogon is an aromatic, dwarf, woody shrub with pale

*Corresponding author. Email: vkaul2002@yahoo.co.in

y
IHBT Communication No. 1039.

ISSN 1478–6419 print/ISSN 1029–2349 online

ß 2011 Taylor & Francis
DOI: 10.1080/14786419.2011.576395
http://www.informaworld.com
 1272 S. Guleria et al.

yellow or creamy white tubular flowers. The plant is locally known as ‘Balu’ or
‘Nachni’. Its leaves are reported as being used for the treatment of bronchitis, cough
and cold by the natives of Himalaya (Hajra & Chakraborty, 1981; Uniyal &
Chauhan, 1973). Its flowers are used by local people in herbal tea and in the
treatment of ailments like gonorrhoea and stomach disorders (Aswal & Mehrotra,
1994). Its flowers have sweet taste and are also used in Tibetan herbalism for the
treatment of inflammations, lung disorders and for general body weakness. Leaves
have diaphoretic and digestive properties and are used in cough and various skin
disorders (Tsarong, 1994). In continuation to our programme on screening of
aromatic flora from Western Himalaya (Jaitak, Singh, & Kaul, 2008; Saini et al.,
2010; Singh et al., 2008), volatile oil constituents of R. anthopogon leaves were
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characterised by gas chromatography mass spectrometry (GC-MS) using different

extraction techniques of SC-CO2 and hydrodistillation (HD). SC-CO2 avoids
degradation of thermally labile compounds and the natural odour of the end product
is retained (Mukhopadhyay, 2000; Taylor, 1996). Results of GC-MS were compared
with headspace (HS) analysis for comparative evaluation of various terpene
categories to understand the variability in volatile oil composition. To the best of
our knowledge, there is no report of comparative extraction of essential oil
composition of R. anthopogon using different extraction techniques and HS analysis.

2. Results and discussion

Results of GC-MS analysis of hydrodistilled (HD), SC-CO2 oil and their comparison
with HS analysis are given in Table 1. The constituents are listed in order of their
elution order on BP-20 capillary column. Thirty-one constituents were identified in
HD oil, representing 91.32% of the total identifications. Twenty-seven constituents
were identified in SC-CO2 oil, representing 79.20% of total identifications. However,
in HS, only 17 constituents were identified representing 98.80% of the identifica-
tions. In HD, oil major constituents are represented by limonene (11.26%), -terpi-
nene (6.05%), -caryophyllene (11.62%), -humulene (7.22%) and E-nerolidol
(5.83%). In SC-CO2, major constituents are represented by -caryophyllene (5.96%),
-humulene (4.06%) and eicosanol (16.66%). In HS analysis, the major constituents
are limonene (24.14%), -terpinene (40.73%) and -humulene (3.49%).
Comparing the yield of major terpene categories, monoterpene hydrocarbon
category represented 89.77% in HS analysis followed by 26.88% in HD oil and a very
low value (3.48%) in SC-CO2. This variability is explained because in comparison to
HD and SC-CO2 extraction, HS analysis represented highly volatile monoterpene
hydrocarbons originally present in the plant material which were easily released, get
fully saturated in the auto-sample vial and are quickly picked up in GC-MS analysis.
In HD, due to heating, highly volatile hydrocarbons are partially lost or get
rearranged, while in SC-CO2, affinity of the extraction of oxygenated monoterpenes is
more than hydrocarbons at selective temperature and pressure. Contrary to this,
oxygenated monoterpenes were very low (1.73%) in HS, 15.86% in SC-CO2 and
15.22% in HD oil. High boiling sesquiterpene hydrocarbons were also low (7.31%) in
HS analysis, but were observed to be maximum in HD oil (37.22%) and (27.58%) in
SC-CO2 oil. Surprisingly, oxygenated sesquiterpenes were absent in HS analysis but
represented 10.63% in HD oil and 8.69% in SC-CO2 oil. Non-terpenes were absent in
 Natural Product Research 1273

Table 1. Comparison of essential oil composition of R. anthopogon on polar BP-20 capillary

column using different extraction techniques.

Constituents KI KI0 SC-CO2 HD HS

-Terpinene 1190 1226 – 0.62  0.02 4.92  0.03

Limonene 1210 1237 0.83  0.02 11.26  0.03 24.14  0.02
-Phellandrene 1220 1241 – 0.83  0.02 3.44  0.03
(Z)- -Ocimene 1240 1257 0.67  0.02 3.72  0.01 7.15  0.02
-Terpinene 1251 1263 0.85  0.03 6.05  0.03 40.73  0.03
(E)- -Ocimene 1253 1267 – 1.11  0.01 3.23  0.02
p-Cymene 1275 1281 – 1.25  0.01 1.19  0.02
-Terpinolene 1288 1288 – 0.71  0.02 3.03  0.03
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cis-3-Hexen-1-ol 1378 1378 – – 0.12  0.01

Linalool 1550 1550 – 1.52  0.01 –
Calarene 1573 1568 1.13  0.02 1.34  0.02 1.93  0.02
Bornyl acetate 1598 1599 – 1.62  0.02 –
Terpinen-4-ol 1599 1601 5.54  0.02 2.15  0.12 0.42  0.03
-Caryophyllene 1615 1617 5.96  0.03 11.62  0.03 1.73  0.03
Citronellyl formate 1636 1638 3.04  0.02 5.51  0.03 1.13  0.01
Aromadendrene 1648 1650 5.55  0.03 6.30  0.02 1.64  0.02
-Gurjunene 1652 1656 – 2.03  0.06 –
-Humulene 1668 1670 4.06  0.03 7.22  0.02 3.49  0.02
(E)- -Farnesene 1669 1671 2.34  0.02 0.93  0.02 –
-Terpineol 1682 1685 – 3.06  0.03 0.17  0.02
Ledene – – 2.64  0.04 2.74  0.04 0.44  0.03
-Muurolene 1728 1727 1.66  0.03 2.42  0.02 –
(Z,E)- -Farnesene 1730 1728 1.51  0.02 – –
E,E- -Farnesene 1730 1728 2.25  0.02 1.05  0.02 –
-Cadinene 1764 1761 1.76  0.02 2.89  0.01 –
Citronellol 1767 1765 – 1.36  0.02 –
Benzyl alcohol 1875 – 0.76  0.02 – –
Phenylethyl alcohol 1908 – 1.04  0.02 – –
(E)-Nerolidol 2044 2044 3.26  0.02 5.83  0.02 –
Globulol 2108 2104 0.84  0.02 1.13  0.02 –
Spathulenol 2155 2153 1.13  0.02 1.35  0.02 –
-Eudesmol 2235 2237 1.13  0.03 0.70  0.02 –
-Eudesmol 2250 2248 2.33  0.02 1.63  0.03 –
Tetracosane 2400 – – 0.74  0.03 –
Eicosanol 2590 – 16.66  0.03 – –
2-Furan carboxylic acid 2609 – 1.28  0.02 0.63  0.03 –
p-Menth-8(10)-ene-2,9-diol 2717 – 7.28  0.02 – –
Isobutylpthalate 2791 – 2.54  0.03 – –
Phenylethylbenzoate 2801 – 1.31  0.01 – –
Monoterpene hydrocarbons (%) 3.48  0.04 26.88  0.07 89.77  0.14
Oxygenated monoterpenes (%) 15.86  0.02 15.22  0.08 1.73  0.01
Sesquiterpenes hydrocarbons (%) 27.58  0.11 37.22  0.17 7.31  0.02
Oxygenated sesquiterpenes (%) 8.69  0.11 10.64  0.04 –
Non-terpenes (%) 23.58  0.03 1.36  0.04 –
Total (%) 79.20  0.17 91.32  0.23 98.80  0.15

Notes: SC-CO2, supercritical carbon dioxide extraction; HD, hydrodistillation; HS, headspace
analysis. Constituents present in SC-CO2 and HD are listed in the order of their elution from
BP-20 Column; KI, Kovats index on BP-20 column experimentally determined using
homologous series of C8–C30 alkanes; KI0 , Kovats index on BP-20 column from the literature.
 1274 S. Guleria et al.

HS analysis but were observed to be maximum (23.58%) in SC-CO2 and very low
(1.36%) in HD oil. Comparing the monoterpene hydrocarbon category between SC-
CO2 oil with HD oil, it was very low (3.48%) in SC-CO2 than in HD (26.88%). This
may be explained that during HD, temperature and pH are responsible for the
rearrangement of volatile constituents leading to artefact formation, which adds on to
the formation of these monoterpene hydrocarbons in the extracted oil and are not
actually true constituents. Further operating SC-CO2 extraction at selective temper-
ature of 40 C and 140 bar afforded very low percentage of undesired monoterpene
hydrocarbons. SC-CO2 oil was rich in oxygenated sesquiterpenes, which explains that
the selected temperature and pressure were highly suitable for the extraction of
oxygenated terpene category. The main constituents responsible for the typical green
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and sweet notes of R. anthopogon oil are linalool, citronellyl formate, citronellol and
nerolidol. Earlier volatile oil composition of R. anthopogon HD oil was reported from
Nepal (Innocenti et al., 2010), reporting 17 constituents mostly mono and sesquiter-
pene hydrocarbons with major ones as -pinene (37.4%), -pinene (15.98%), cis-
ocimene (5.32%) and -cadinene (9.10%). The oil is devoid of any oxygenated
terpenes. Recently, in another species Rhododendron lepidotum from Kashmir
Himalaya, -pinene (56%) is reported as the major constituent along with other
constituents -pinene (13.49%), limonene (6.58%), -terpinene (2.22%), bornyl
acetate (2.87%) and -humulene (4.26%) (Rathar, 2010). Volatile oil compositions of
five Turkish Rhododendron species, R. ponticum, R. luteum, R. sochadzae, R. ungernii
and R. smirnovii are reported (Tasdemir et al., 2003) representing mostly non-terpene
constituents, styrene (10%), undecane (4%), 2-ethyl hexanol (13.3%), 5,15-rosadiene
(42.8%), 1-butanol (3.3%), (Z)-3-hexanol (4.5%), 6-methyl-5-hepten-2-one (3.2%),
-butyrolactone (13.5%), benzyl alcohol (11.7%) and phenyl ethyl alcohol (4%).
However, in our studies, none of the above non-terpenes were found in SC-CO2 oil.
However, in HD and HS analysis, this category was represented by low percentage of
different constituents. In some species of Rhododendron, -humulene, caryophyllene,
limonene and , -pinenes have been reported as the major constituents (Belousov,
Dembitsky, Berezovskaya, & Tikhonov, 1995; Doss, Hatheway, & Hrutfiord,1986;
Ma, Sun, & Wu, 1983; Shi, 1981). The volatile compositions of other R. anthopogon
species are different from those found in our results. These species are devoid of
citronellol, citronellyl formate and E-nerolidol, which are the key fragrance
constituents of R. anthopogon from Western Himalayan region of India. It is likely
that altitude and environment play an important role in the biosynthesis of volatile
constituents. It is, therefore, further desired to investigate R. anthopogon volatile oil
from more locations varying in altitudes for their categorisation so that these could be
placed in separate groups based on terpene categories, representing different
geographical zones.

3. Experimental
3.1. Plant material
Leaves of R. anthopogon were collected in August 2008 during flowering stage from
alpine Western Himalaya at an altitude of 3000–4500 m above sea level m.s.l N 32
180 1400 and E 78 010 1000 and a voucher specimen was deposited in IHBT herbarium
section (Voucher No. P2P-13027). The leaves were shade-dried and used for
hydrodistillation and SC-CO2 extractions. Fresh leaves were used for HS analysis.
 Natural Product Research 1275

3.2. Reagents
HPLC grade dichloromethane and n-hexane were purchased from M/S SD fine
chemicals (Mumbai, India). Anhydrous Na2SO4 of analytical grade was procured
from M/S Merck Chemicals (Mumbai, India). Liquid carbon dioxide (99.99%)
purity was procured from M/s Jalan Sales Corporation, Rajpura (Punjab), India.
Standard authentic terpene samples, i.e. linalool, neryl acetate, nerolidol and
citronellol were procured from Haarman and Reimier, Germany.

3.3. Isolation of volatile oils

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3.3.1. Hydrodistillation
In this analysis, 500 g of shade dried leaves were charged along with 2 L water in a
round bottom flask fitted to a Clevenger-type apparatus. The plant material was
hydrodistilled for 3 h. Light yellow-coloured volatile oil in 0.4% yield (w/v) was
collected in 2 mL hexane as collector solvent. The oil was dried on anhydrous sodium
sulphate, filtered and the solvent was evaporated under nitrogen flow. It was stored
in a refrigerator at 4 C before GC and GC-MS analysis.

3.3.2. SC-CO2 extraction

SC-CO2 extraction was performed on a computer-controlled SFE system from Thar
Technologies Inc., Pittsburgh, USA. In this method, 950 g of shade-dried leaves were
charged in an extraction vessel. Initially, the vessel was pressurised with CO2 up to
140 bar and kept at 40 C for 1 h. Then, extraction was carried out in a dynamic mode
for 3 h, at 140 bar, 40 C with 60 g min1 flow rate of CO2. The first separator was set
at a pressure of 80–90 bar, temperature ()9 C to ()10 C. The second separator
was set at 20–30 bar and temperature ()5 C to 0 C. The oil was collected in second
separator. The waxes in the oil were removed and the remaining oil analysed by GC
and GC-MS.

3.3.3. HS analysis
In this analysis, 1 g sample of fresh leaves was placed in 20 mL auto-sample vial with
crimp top, sealed immediately and used for HS analysis. The analysis was carried out
using Shimadzu headspace auto-injector AOC-5000 connected to Shimadzu GC-MS
equipment GC2010 and QP2010, Kyoto, Japan, and loaded with fused silica BP-20
column (30 m  0.25 mm ID, 0.25 mm film thickness) stationary phase 100%
polyethylene glycol, oven temperature 40 C held for 5 min, temperature programmed
at 5 min1 to 220 C, carrier gas helium 1 mL min1. Hamilton bonadnz AG
Switzerland syringe MSH 02-00B, 2.5 mL capacity, 60 mm scale and 0.1 mm id was
used. HS syringe temperature was set at 100 C, syringe flash time 45 min, sample
heater temperature 80 C, incubation time 45 min, agitator on 5 s and off 2 s, plunger
fill speed 100 mL s1, pulling delay 1.0 s, plunger injection speed 500 mL s1, post-
injection delay 500 mL s1.
 1276 S. Guleria et al.

3.4. Essential oil analysis

3.4.1. GC analysis
GC analysis was performed using Shimadzu GC 2010 with an auto-sampler system
AOC-20i equipped with flame ionisation detector (FID). Constituents were
separated on a fused silica BP-20 capillary column (30 m  0.25 mm id, 0.25 mm
film thickness) 100% polyethyleneglycol stationary phase J&W column, Scientific
Folsom, CA, USA. The operating conditions were: injector and detector temper-
atures 250 , carrier gas nitrogen, flow rate 1 mL min1, split ratio 1:20, oven
temperature 40 C hold for 5 min and programmed at 5 C min1 to 220 C and finally
hold at 220 C for 5 min. The sample injection size was 1 mL in HPLC grade
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dichloromethane.

3.4.2. GC-MS analysis

GC-MS analysis of HD and SC-CO2 extracted oils was carried out on Shimadzu
GC-2010 series gas chromatograph interfaced with mass spectrometer (QP 2010),
Kyoto, Japan, equipped with FID, AOC-5000 auto-sampler coupled to fused silica
BP-20 capillary column (30 m  0.25 mm id, 0.25 mm thickness), stationary phase
100% polyethyleneglycol, J&W column, Scientific Folsom, CA, USA, equipped with
split–splitless injector. Initial column temperature was 40 C fixed for 5 min,
programmed to 220 C at 5 C min1 and held for 5 min at 220 C, injector
temperature 220 C; sample injection volume of 2 mL was prepared in HPLC grade
dichloromethane and injected in split mode ratio 1:20. The spectrum was scanned
with acquisition mass range 50–600 m/z, scan rate 1.6 scans sec1. All mass spectra
were acquired in electron impact mode with ionisation voltage 70 eV. Ion source and
quadrupole temperatures were 200 C, transfer line temperature 220 C and helium
flow 1 mL min1 constant. Kovats indices of each compound were calculated relative
to C8–C30 aliphatic hydrocarbons (Sigma Milan, Italy) by directly injecting these into
the column under the above temperature programme and compared with the
literature data (Davies, 1990). Further, confirmation of the constituents was
achieved by matching mass spectra with the data given in the literature (Adams,
1995; Jennings & Shibamoto, 1980; NIST/05 Mass Spectral Library, 2005).

3.5. Statistical analysis

GC-MS analysis was carried out in triplicate and mean  SD of the constituents was
worked out for three sample replicates using Microsoft Excel program.

Acknowledgements
The authors are grateful to M/s Proctor & Gamble for sponsoring the project (project #
SSP0049) and to Dr P.S. Ahuja, Director, IHBT, for providing necessary facilities. They also
thank Ms Vijaylata Pathania for carrying out GC and GC-MS analysis of the samples.

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