2016

NPC Natural Product Communications Vol. 11

No. 12
Chemical Composition, Enantiomeric Distribution, and Biological 1895 - 1898
Activities of Rhododendron anthopogon Leaf Essential Oil from Nepal
Noura S. Dosoky, Prabodh Satyal, Suraj Pokharel and William N. Setzer*

Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA

wsetzer@chemistry.uah.edu

Received: April 23rd, 2016; Accepted: August 5th, 2016

Rhododendron anthopogon D. Don., a small compact Himalayan shrub growing in Nepal, is a known medicinal plant used to treat sore throat, colds, blood
disorders, bone disease, potato allergies, and vomiting, and to relieve liver disorders, headaches and back pain. The present study investigated the chemical
composition and bioactivities of the leaf essential oil from R. anthopogon from Dhankuta, Nepal. The essential oil from leaves was obtained by
hydrodistillation and a detailed chemical analysis was conducted by gas chromatography – mass spectrometry (GC-MS). The enantiomeric distribution of
monoterpenoid components was determined using chiral gas chromatography and represents the first chiral examination of R. anthopogon essential oil. The
essential oil was screened for antimicrobial activity using the microbroth dilution test, and for cytotoxic activity against MCF-7, MDA-MB-231, and 5637
using the MTT assay. A total of 70 volatile components were identified from the essential oil. The major components were α-pinene (21.5%), δ-cadinene
(13.8%), β-pinene (9.5%), limonene (5.9%), δ-amorphene (4.6%), α-muurolene (4.5%), and (E)-caryophyllene (3.2%) with other minor constituents (< 3%).
The essential oil showed marginal antibacterial and cytotoxic activities, but no antifungal effects.

Keywords: Rhododendron anthopogon, Chiral gas chromatography, Essential oil composition, Antimicrobial activity, Cytotoxicity.

Rhododendron (Ericaceae) is a large genus of medicinal plants
made up of about 1024 species. Rhododendron anthopogon D. Don.
is a small compact evergreen shrub, inhabiting the alpine Himalayas
from Himachal Pradesh to Bhutan at altitudes from 2,000 to 5,000
m asl. R. anthopogon is simply known as rhododendron or rose tree.
It is commonly known as taalisri in the Punjabi area while in Nepal,
it is known as balu karpo, dhali karpo and palu [1]. The plant is
about 30-60 cm high with rough, densely scaly branches [2]. Leaves
are aromatic, ovate, 2.5-4 cm long, cinnamon brown beneath as the
lower surface is densely covered with red-brown scales. R.
anthopogon blooms in June-July. Flowers are small bell-shaped,
white to pale yellow or pink in color, and appear in numerous short
terminal cymes [1,2]. Because its wood is easy to cut and the
branching trunks are easy to transport, rhododendrons are often
used for fuel wood in the Himalayas.
R. anthopogon has a reputed use in traditional medicine. For many
years, the leaves, stem and flowers of R. anthopogon have been
used for fever, pain and inflammation of limbs due to their
antibacterial properties. Tender leaves are applied on the forehead
to relieve headaches [1]. The leaves are sedative, stimulant and
expectorant [1,3]. A tea of fresh R. anthopogon leaves and flowers
is used to promote digestive heat, stimulate appetite and relieve
liver disorders, headaches and back pain. This tea is also used to
treat sore throat, colds, blood disorders, bone disease, potato
allergies, and vomiting, and to counteract water-earth illness [4].
The vapor of boiled leaves is inhaled to treat cold, cough and
insomnia. Fresh leaves mixed with mustard oil and paste are used in
treating wounds and cuts. A mixture of R. anthopogon leaves and
Abies webbiana leaves has been used as a treatment of respiratory
diseases [3]. Moreover, the dried leaves, young shoots and flowers
are widely used as incense (dhoop) during Buddhist rituals and
celebrations [5].
In Nepal, the essential oil of R. anthopogon is known as Sunpati oil
or Anthopogon oil and is obtained from the aerial parts by steam
distillation [4b]. Due to its sweet herbal and slightly balsamic scent,
R. anthopogon oil is used to soothe inflamed skin, sore muscles, and
joints. The oil is also used to add shine and to naturally perfume the
hair [4b]. R. anthopogon extracts were reported to have antiviral,
antibiotic, antimycobacterial, anti-inflammatory, analgesic,
antipyretic, antiproliferative and cytotoxic properties [4d,6]. R.
anthopogon has been reported to have a strong antioxidant capacity
[7]. The leaves of R. anthopogon were reported to contain
quercetin, myricetin, taxifolin, kaempferol derivatives, ursolic acid,
epi-friedinol, β-sitosterol, betulinic acid and rutin [3]. Qin and co-
workers [8] isolated five known monoterpenes from the leaves of R.
anthopogon, namely ranhuadujuanine A, cannabiorcicyclolic acid,
ranhuadujuanine B, ranhuadujuanine C and ranhuadujuanine D. The
components of leaf essential oil of R. anthopogon from Rolwaling
area of Dolakha district, Nepal [6c] and Himachal Pradesh, India [9]
have been previously reported. This study was conducted to
investigate the composition of the leaf essential oil of R.
anthopogon from Dhankuta, Nepal as well as its biological
activities and enantiomeric distribution of monoterpenoids.
As shown in Table 1, a total of 70 compounds were identified from
R. anthopogon leaf essential oil. The oil is mainly composed of α-
pinene (21.5%), δ-cadinene (13.8%), β-pinene (9.5%), limonene
(5.9%), δ-amorphene (4.6%), α-muurolene (4.5%), and (E)-
caryophyllene (3.2%) with other minor constituents (< 3%). The
essential oil composition and percentages from the current study
revealed that there are important differences from the previously
published reports from Rolwaling area of Dolakha district, Nepal
[6c] or Himachal Pradesh, India [9]. Innocenti et al. [6c] identified
17 compounds from the oil of R. anthopogon aerial parts (leaves
and flowers) from Nepal and the major constituents in their study
were α-pinene (37.4%), β-pinene (16.1%), limonene (13.3%), and
δ-cadinene (9.1%). Limonene (11.3%), (E)-caryophyllene (11.6%),
α-humulene (7.2%), and (E)-nerolidol (5.8%) dominated the oil
from India [9]. Also, the essential oil composition of R. anthopogon
is different from the leaf essential oil of R. anthopogonoides, which
was mainly composed of benzylacetone (40-50%), selina-3,7(11)-
diene (20-25%), and neofuranodiene (10-12%) [10]. The essential
1896 Natural Product Communications Vol. 11 (12) 2016 Dosoky et al.

Table 1: Chemical composition of Rhododendron anthopogon leaf essential oil from Nepal.
RIa Compound % RIa Compound %
925 α-Thujene 0.1 1447 trans-Muurola-3,5-diene 0.4
932 α-Pinene 21.5 1453 (E)-β-Farnesene 1.5
947 α-Fenchene 0.1 1457 Alloaromadendrene 0.8
948 Camphene 0.3 1460 cis-Cadina-1(6),4-diene 0.2
977 β-Pinene 9.5 1470 Dauca-5,8-diene 1.5
989 Myrcene 1.1 1473 trans-Cadina-1(6),4-diene 2.8
1009 δ-3-Carene 0.1 1477 γ-Curcumene 0.2
1017 α-Terpinene 0.2 1479 Germacrene D 0.2
1024 p-Cymene 0.7 1480 ar-Curcumene 0.1
1029 Limonene 5.9 1483 trans-Muurola-4(14),5-diene 0.1
1035 (Z)-β-Ocimene 2.5 1486 β-Selinene 1.2
1046 (E)-β-Ocimene 0.5 1489 Viridiflorene 0.5
1057 γ-Terpinene 2.4 1490 γ-Amorphene 0.5
1085 Terpinolene 0.5 1493 α-Selinene 1.7
1098 6-Methyl-2-heptyl acetate 0.1 1497 α-Muurolene 4.5
1100 Linalool 0.7 1501 Epizonarene 0.1
1119 exo-Fenchol 0.1 1504 (E,E)-α-Farnesene 0.3
1128 allo-Ocimene 0.1 1509 β-Curcumene 0.5
1171 Borneol 0.1 1511 δ-Amorphene 4.6
1180 Terpinen-4-ol 0.4 1517 δ-Cadinene 13.8
1195 α-Terpineol 1.2 1521 Zonarene 1.3
1245 (3Z)-Decenol 0.1 1530 trans-Cadina-1,4-diene 0.6
1283 Bornyl acetate 0.3 1535 α-Cadinene 0.8
1346 α-Cubebene 0.5 1539 α-Calacorene 0.1
1350 Citronellyl acetate 0.4 1561 (E)-Nerolidol 0.1
1367 α-Ylangene 0.1 1601 Viridiflorol 0.1
1374 α-Copaene 1.1 1613 Juneol 0.1
1382 α-Bourbonene 0.1 1625 1-epi-Cubenol 0.3
1388 β-Elemene 0.1 1629 γ-Eudesmol 0.1
1390 4-Phenylbut-2-yl acetate 0.1 1640 α-Muurolol (= Torreyol) 1.3
1401 Sesquithujene 0.1 1642 epi-α-Muurolol 1.6
1405 α-Gurjunene 0.3 1644 δ-Cadinol 0.5
1417 (E)-Caryophyllene 4.1 1653 α-Cadinol 2.2
1428 β-Copaene 0.3 1686 α-Bisabolol 0.1
1436 Aromadendrene 0.5
Compounds Identified 70
1444 cis-Muurola-3,5-diene 0.1
a
RI determined with respect to a homologous series of n-alkanes on an HP-5ms column.

oil of R. mucronulatum was reported to have β-pinene (16.1%), R. anthopogon leaf oil was richer in l-α-pinene and d-β-pinene than
camphene (11.9%), δ-3-carene (11.4%), limonene (9.5%), and γ- the other enantiomers, respectively. This enantiomeric distribution
terpinene (9.5%) as the main constituents [11] and the major is somewhat unusual, but it has been observed in peppermint oil
components of R. nivale oil were δ-cadinene (15.5%), α-cadinol from Morocco [21]. Camphene in R. anthopogon leaf oil was
(10.2%), (E)-farnesol (8.0%), methyl carvacrol (8.1%), and an α- exclusively the l-enantiomer. Spearmint leaf oil, on the other hand,
cadinol isomer (7.8%) [12]. According to Olennikov et al. [13], the has been found to be composed of exclusively the d-enantiomer
leaf essential oil of R. dauricum was dominated by (E)- [21]. Similarly, l-α-terpineol was the exclusive enantiomer in R.
caryophyllene (10-19%), γ-cadinene (14.0%), β-gurjunene (10.5%), anthopogon essential oil, which was also found in Tarchonanthus
and humulene (10.2%), while R. aureum was dominated by calarene camphoratus essential oil [22]. Bornyl acetate was also exclusively
(16-48%). The major components of R. tomentosum leaf oil were the l-enantiomer in R. anthopogon. However, both linalool and
palustrol (15.9-53.5%), ledol (11.8-18.3%), γ-terpineol (0-31.2%), terpinen-4-ol were nearly racemic. This study represents first chiral
p-cymene (0.1-13.9%), lepalone (0.7-6.5%), lepalol (1.0-6.5%) and examination of R. anthopogon essential oil.
cyclocolorenone (1.0-6.4%) [14].
The essential oil of R. anthopogon was screened for potential
The enantiomeric distribution of monoterpenoids in the leaf antimicrobial activity. The results showed that there was only
essential oil was determined using chiral gas chromatography marginal antibacterial activity against Bacillus cereus (MIC = 313
(Table 2). The d-enantiomer of limonene is the more common, μg/mL) and Staphylococcus aureus (MIC = 156 μg/mL) and no
particularly in Citrus oils such as orange [15], lemon [16], mandarin antifungal activity against Aspergillus niger (MIC = 625 μg/mL) or
[17], bergamot [18], and Australian finger lime [19]. The Candida albicans (MIC = 1250 μg/mL). The essential oil of
predominant enantiomer of limonene in R. anthopogon leaf R. anthopogon showed no in-vitro cytotoxic activity against MCF-7
essential oil as revealed in this study was the l-enantiomer. cells (36.27 ± 12.42% kill at 100 µg/mL), MDA-MB 231 (25.82 ±
l-Limonene is also the predominant enantiomer in pine, spruce [20], 8.72% kill at 100 µg/mL), and only a marginal effect against 5637
peppermint, and spearmint [21] essential oils. (77.61 ± 2.02% kill at 100 µg/mL). Our results are in contrast with
previous reports on the biological activity of R. anthopogon.
Table 2: Enantiomeric excess (ee) and distribution (ed) of monoterpenes found in Innocenti et al. [6c] found a significant antibacterial activity of
Rhododendron anthopogon leaf essential oil from Nepal. against Staphylococcus aureus, Enterococcus fecalis, and Bacillus
Compounds Relative % ee (%) ed [d to l] (%) subtilis as well as an antifungal activity against Mycobacterium
Bornyl acetate 0.25 100 0 to 100 tuberculosis and Candida pseudotropicalis. Gyawali et al. [23]
α-Terpineol 1.15 100 0 to 100
Camphene 0.3 100 0 to 100 reported a high antibacterial activity of the commercially available
α-Thujene 0.12 42.4 28.8 to 71.2 essential oil of R. anthopogon against S. aureus. These activities
α-Pinene 21.52 47.2 26.4 to 73.6
β-Pinene 9.49 34.6 67.3 to 32.7
were attributed to the presence of different monoterpenes (α-pinene,
Limonene 5.86 95.4 2.3 to 97.7 β-pinene, limonene), a sesquiterpene (δ-cadinene) and β-eudesmol
Linalool 0.7 10 55.0 to 45.0 [4a,6c,14]. Neither α-pinene nor limonene are particularly
Terpinen-4-ol 0.36 9.2 54.6 to 45.4
antibacterial [24], but δ-cadinene has shown antibacterial effects
[25]. According to Baral et al. [26], the aerial parts extract showed
Rhododendron anthopogon essential oil Natural Product Communications Vol. 11 (12) 2016 1897

remarkable antimicrobial effects against Klebsiella pneumonia,
Salmonella typhimurium and Fusarium eridiforme.
Experimental
Plant Material: Leaves of Rhododendron anthopogon, collected
from city of Dhankuta, (2200 m asl) in Nepal on 04/05/2014, were
used in this study. The plant material was identified by Mr. Kiran
Kumar Pokharel. The essential oil was obtained from fresh leaves
(100 g) that were crushed and hydrodistilled using a Clevenger type
apparatus for 4 h. The pale yellow essential oil (0.5 g) was stored at
4°C until analyzed.
Gas Chromatographic – Mass Spectral Analysis: The essential oil
of Rhododendron anthopogon was analyzed by GC-MS using an
Agilent 6890 GC with Agilent 5973 mass selective detector [MSD,
operated in the EI mode (electron energy = 70 eV), scan range = 40-
400 amu, and scan rate = 3.99 scans/sec], and an Agilent
ChemStation data system. The GC column was an HP-5ms fused
silica capillary with a (5% phenyl)-polymethylsiloxane stationary
phase, film thickness of 0.25 μm, a length of 30 m, and an internal
diameter of 0.25 mm. The carrier gas was helium with a column
head pressure of 48.7 kPa and a flow rate of 1.0 mL/min. Inlet
temperature was 200°C and interface temperature was 280°C. The
GC oven temperature program was used as follows: 40°C initial
temperature, hold for 10 min; increased at 3°C/min to 200°C;
increased 2°/min to 220°C. A 1 % w/v solution of the sample in
dichloromethane was prepared and 1 μL was injected using a 10:1
split ratio.
Identification of the oil components was based on their retention
indices determined by reference to a homologous series of
n-alkanes, and by comparison of their mass spectral fragmentation
patterns with those reported in the literature [27] and stored
on the MS library [NIST database (G1036A, revision
D.01.00)/ChemStation data system (G1701CA, version
C.00.01.080]. The percentages of each component are reported as
raw percentages based on total ion current without standardization.
Chiral Gas Chromatographic – Mass Spectral Analysis: Chiral
analysis of the essential oils was performed on a Shimadzu GCMS-
QP2010S operated in the EI mode [(electron energy = 70eV), scan
range = 3.0 scans/sec]. GC equipped with a Restek B-Dex 325
capillary column (30 m × 0.25 mm ID × 0.25 μm film). Oven
temperature was started at 50°C, and then gradually raised to 120°C
at 1.5°C/min. The oven was then raised to 200°C at 2°C/min and
held for 5 min. Helium was the carrier gas and was flow rate was
maintained at 1.8 mL/min. Samples were diluted 3% w/v with
CH2Cl2 and then a 0.1 µL sample was injected in a split mode with
a split ratio of 1:45.
Antimicrobial Screening: The essential oil of R. anthopogon was
screened for antimicrobial activity against Bacillus cereus (ATCC
No. 14579), Staphylococcus aureus (ATCC No. 29213), Aspergillus
niger (ATCC No. 16888) and Candida albicans (ATCC No.10231).
The minimum inhibitory concentration (MIC) was determined using
the microbroth dilution technique. For the bacteria, dilutions of the
samples were prepared in cation-adjusted Mueller Hinton broth
(CAMHB) beginning with 50 μL of 1% w/w solutions of samples in
DMSO plus 50 μL CAMHB. The sample solutions were serially
diluted (1:1) in CAMHB in 96-well plates to give concentrations of
2500, 1250, 625, 313, 156, 78, 39, and 19.5 μg/mL. Organisms at a
concentration of approximately 1.5  108 colony-forming units
(CFU)/mL were added to each well. Plates were incubated at 37°C
for 24 hours; the minimum inhibitory concentration (MIC) was
determined as the lowest concentration without turbidity.
Gentamicin was used as a positive antibiotic control; DMSO was
used as a negative control (50 μL plus 50 μL CAMBH, serially
diluted as above).
Antifungal activity was determined, as described above for bacteria,
using C. albicans in a yeast-nitrogen base growth medium with
approximately 7.5 × 107 CFU/mL. Antifungal activity against A.
niger was determined as above using yeast mold (YM) broth
inoculated with A. niger hyphal culture diluted to a McFarland
turbidity of 1.0. Amphotericin B was the positive control.
Cytotoxicity screening: R. anthopogon oil was tested for
cytotoxicity against MCF-7 (human breast cancer cells, ATCC No.
HTB-22), MDA-MB-231 (human breast cancer cells, ATCC No.
HTB-26), and 5637 (human bladder cancer cells, ATCC No. HTB-
9) cells. MCF-7 and MDA-MB-231 cells were grown in RPMI
1640 supplemented with 10% Fetal bovine serum (FBS), 30mM
HEPES, sodium bicarbonate, and Penicillin-Streptomycin. 5637
cells were grown in RPMI 1640 supplemented with 10% FBS, 1.0
mM sodium pyruvate, 2.5 g/L glucose, 30mM HEPES, sodium
bicarbonate, and Penicillin-Streptomycin. In-vitro cytotoxic activity
was performed using the 96-well MTT assay as previously reported
[28].
Acknowledgments – The authors are grateful to Mr. Kiran Kumar
Pokharel for assisting in collection of plants.

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