Chemical composition and biological activities of essential Oil from leaf and bark of Nyctanthesarbor-tristis L. from Nepal

Abstract

The essential oil from the leaves and barks of Nyctanthes arbor-tristis, collected from Biratnagar, Nepal, was hydrodistilled and analyzed by GC-MS. A total of 26 compounds were identified in the leaf oil, accounting for 100% of the oil while a total of 20 compounds were identified in the bark oil accounting for only 89.4% of the oil. Both the leaf and bark oil had similar quantities of hexadecanoic acid (26.4% and 34.3%, respectively) and octadecanoic acid (3.9 and 6.2%, respectively). However, the leaf oil also consisted of linalool (11.0%), (E)-phytol (13.6%) and (3Z)-hexenyl benzoate (11.0%), which were absent in the bark oil. Besides fatty acids, the bark oil exhibited significantly different composition with mostly β-eudesmol and other eudesmol isomers (27.5%). The oil was screened for antimicrobial activity and showed marginal activity against Bacillus cereus and Aspergillus niger (MIC = 625 μg mL). N. arbor-tristis leaf oil was inactive in the brine shrimp lethality test (LC50 > 100μg/mL).

Full text

Introduction
Nyctanthes arbor-tristis L. (Oleaceae) is widely
distributed along subtropical, tropical to sub-
Himalayan regions in the South East Asia (Das et al.,
2010; Khatune et al., 2003). It has been extensively
used as a therapeutic agent in the Ayurvedic healing
traditions of South Asia (Rathee et al., 2007; Saxena
et al., 1986; Tuntiwachwuttikul et al., 2003).
Traditionally used to treat sciatica, arthritis and
malaria, however, reports from the literature have
also indicated that the leaf oil from N. arbor-tristis
include hepatoprotective, anti-leishmanial, antiviral
and antifungal activities (Rathee et al., 2007). Local
people of Andhra Pradesh, India use the whole
tree for cancer, root for fever, sciatica, anorexia
and bark as expectorant (Rathod et al., 2010). Other
research into the leaf extract of the N. arbor-tristis
have shown considerable immunological activity
and water soluble ethanol extracts from the leaves
are reported to possess anti-inammatory activity
which, however, accompany development of ulcers
in test rats (Rathee et al., 2007; Saxena et al., 1986).
In addition, anti-oxidant studies on the acetone-
soluble ethyl acetate leaf extracts have shown
signicant activity against hydroxy and superoxide
radicals, as wells as peroxide scavenging activity
(Rathee et al., 2007). Likewise, activity-guided
isolation of compounds in N. arbor-tristis owers
yielded iridoid glucosides that have exhibited
antiplasmodial activity against Plasmodium
falciparum (Tuntiwachwuttikul et al., 2003).
Besides these compounds, 4-hydroxyhexahydro-
benzofuran-7-one (Khatune et al., 2003), nyctoside
A, arborside C, arborside D, 6-hydroxyloganin,
arbortristoside A, arbortristoside B (Sasmal et al.,
2007) and nyctanthoside (Jensen et al., 2002) have
been reported.
Besides from these sparse reports into the study
of biological activities of leaf and ower extracts
of N. arbor-tristis, to our knowledge this is the rst
examination of the leaf and bark essential oils of this
medicinal plant from Nepal. The objective of this
study is to analyze the chemical compositions, and
examine the microbial and brine shrimp lethality
activities of leaf and bark oils of N. arbor-tristis.
Materials and methods
Plant material
Nyctanthes arbor-tristis was collected from city of
Biratnagar (26°28’N, 87°16’E, 72 m above sea level)
in Morang district in Koshi Zone in Nepal on 15 May
2011. The plant was identied by Tilak Gautam,
and a voucher specimen (1024) has been deposited
in the herbarium of the Tribhuvan University, Post-
Graduate Campus, Botany Department, Biratnagar.
The fresh leaf sample (102 g) and the fresh bark
Chemical composition and biological activities of
essential Oil from leaf and bark of Nyctanthes
arbor-tristis L. from Nepal
Prabodh Satyal1, Prajwal Paudel1, Ambika Poudel2 and William N. Setzer1*
1Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA
2Department of Chemistry, Tribhuvan University, MMAMC campus, Biratnagar, Nepal
*Corresponding author. E-mail: wsetzer@chemistry.uah.edu
1
Open Access Journal of Medicinal and Aromatic Plants Vol. 3(1): 1-4
Abstract: The essential oil from the leaves and barks of Nyctanthes arbor-tristis, collected from Biratnagar, Nepal, was hydrodistilled
and analyzed by GC-MS. A total of 26 compounds were identied in the leaf oil, accounting for 100% of the oil while a total of 20
compounds were identied in the bark oil accounting for only 89.4% of the oil. Both the leaf and bark oil had similar quantities
of hexadecanoic acid (26.4% and 34.3%, respectively) and octadecanoic acid (3.9 and 6.2%, respectively). However, the leaf oil also
consisted of linalool (11.0%), (E)-phytol (13.6%) and (3Z)-hexenyl benzoate (11.0%), which were absent in the bark oil. Besides fatty
acids, the bark oil exhibited signicantly different composition with mostly β-eudesmol and other eudesmol isomers (27.5%). The oil
was screened for antimicrobial activity and showed marginal activity against Bacillus cereus and Aspergillus niger (MIC = 625 μg mL).
N. arbor-tristis leaf oil was inactive in the brine shrimp lethality test (LC50 > 100μg/mL).
Key words: Nyctanthes arbor-tristis, essential oil composition, eudesmol, Nepal, antimicrobial, brine shrimp
lethality
Manuscript received: 24 March, 2012 Manuscript accepted: 13 June, 2012

2
OAJMAP (2012)
sample (104 g) were crushed and hydrodistilled
using a Clevenger apparatus for 4 h to give clear,
colorless essential oils of 0.002 g and 0.005 g,
respectively, which were stored at 4°C until analysis.
Gas chromatographic – mass spectral
analysis
The leaf and bark essential oils of N. arbor-tristis
were 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 = 45-400 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, lm 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 ow rate of 1.0 mL/min. Injector
temperature was 200°C and detector 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 CH
2
Cl
2
was prepared and 1 μL was injected using
a splitless injection technique.
Identication 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 (Adams 2007) 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.
The essential oil compositions of N. arbor-tristis are
summarized in Table 1.
Antimicrobial screening
The essential oils were screened for antimicrobial
activity against Gram-positive bacteria
Bacillus cereus (ATCC No. 14579) and Staphylococcus
aureus (ATCC No. 29213), and Gram-negative
bacteria Pseudomonas aeruginosa (ATCC No. 27853)
and Escherichia coli (ATCC No. 10798). Minimum
inhibitory concentrations (MICs) were determined
using the microbroth dilution technique (Setzer et al.,
2003). Dilutions of the essential oils were prepared
in cation-adjusted Mueller Hinton broth (CAMHB)
beginning with 50 μL of 1% w/w solutions of oils in
DMSO plus 50 μL CAMHB. The oil solutions were
serially diluted (1:1) in CAMHB in 96-well plates.
Organisms at a concentration of approximately 1.5 ´
108 colony-forming units (CFU)/ mL were added to
each well. Plates were incubated at 37 ± 1 °C for 24
hours; the nal 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. Antifungal activity against
Aspergillus niger (ATCC No. 16888) was determined
as above using YM broth inoculated with A. niger
hyphal culture diluted to a McFarland turbidity of
1.0. Amphotericin B was the positive control.
Brine shrimp lethality assay
The brine shrimp (Artemia salina) lethality test was
carried out using a modication (Satyal et al., 2012)
of the procedure by McLaughlin (1991). Artemia
salina eggs were hatched in a sea salt solution
(Instant Ocean®, 38 g/L) with an incandescent light
bulb as the heat source. After 48 hours, the newly
hatched nauplii were counted using a micropipette
and transferred to 20-mL vials. Nine vials each
containing 10 A. salina nauplii in 10 mL of sea
salt solution (same as the hatching solution) were
prepared. Three vials were labeled as controls with
rst one containing no DMSO, another with 10 μL,
and the last one with 100 μL DMSO. Three replicate
vials contained 10 μL of 1% essential oil solution
in DMSO, and the other three were prepared by
adding 100 μL of 1% essential oil solution in DMSO.
Surviving A. salina was counted after 24 hours.
Results and Discussion
The essential oils of N. arbor-tristis were obtained
in 0.002% and 0.005% yield for the leaf and bark
samples, respectively. A total of 26 compounds were
identied in N. arbor-tristis leaf oil while only 20
compounds were identied for the bark essential oil.
The composition of the leaf essential oil of N. arbor-
tristis was notably different from the bark oil. While
the leaf and bark oils were mostly composed of
similar quantities of fatty acids: palmitic acid (26.4
and 34.3%, respectively) and stearic acid (3.9 and
6.2%, respectively), minor components in leaf oil
included (E)-phytol (13.6%), (3Z)-hexenyl benzoate
(11.0%), and linalool (11.0%). The result from this
work is in agreement with a previous analysis of

3
OAJMAP (2012)
RI
a
Compound Leaf (%) Bark (%)
856 (3Z)-Hexenol 2.5 ---
941 α-Pinene --- tr
b
1008 (3Z)-Hexenyl acetate 0.5 ---
1024 p-Cymene --- tr
1028 Limonene --- 0.2
1031 1,8-Cineole --- 1.3
1043 Phenylacetaldehyde 0.3 ---
1065 Acetophenone 0.8 ---
1100 Linalool 11.3 tr
1104 Nonanal 0.4 ---
1112 2-Phenylethyl alcohol 0.3 ---
1143 Camphor --- 0.8
1176 Terpinen-4-ol 0.2 ---
1181 m-Cymen-8-ol 0.6 ---
1184 p-Cymen-8-ol 0.5 ---
1187 (3Z)-Hexenyl butanoate 0.3 ---
1189 α-Terpineol 4.7 ---
1192 Methyl salicylate 5.6 ---
1226 Nerol 0.7 ---
1251 Geraniol 3.7 ---
1311 p-Vinylquaiacol 0.8 ---
1324 (3Z)-Hexenyl tiglate 0.7 ---
1337 Methyl anthranilate 0.7 ---
1356 Eugenol 1.2 ---
1414 (E)-β-Damascone 0.4 ---
1475 n-Dodecanol 5.5 6.8
Table 1 : Leaf and bark essential oil composition of Nyctanthes arbor-tristis from Nepal.
fatty acids of N. arbor-tristis bark oil from
Bangladesh, which was reported to contain
a similar quantity of palmitic acid (16.4%),
but additional fatty acids such as oleic acid
(13.3%), behenic acid (24.8%), and nervonic
acid (12.4%) (Rahman & Shahajan, 2011).
However, the bark oil had a considerably
different composition with eudesmol isomers
(27.5%) along with smaller quantities of
n-dodecanol (6.8%), elemol (5.8%) and
cryptomeridiol (4.8%) also present. Overall,
100% of the leaf essential oil was identied as
oppose to only 89.4% for the bark essential oil.
The essential oils of N. arbor-tristis were
screened for potential antimicrobial activity
against Staphylococcus aureus, Escherichia
coli, Bacillus cereus, Pseudomonas aeruginosa,
and Aspergillus niger, but showed no activity
against any of the microorganisms (MIC>
1250 μg/mL) except for B. cereus and A. niger (MIC
= 625 μg/mL by leaf oil and MIC = 313 μg/mL by
bark oil). Additionally, neither N. arbor-tristis leaf
nor bark oil showed activity in the brine shrimp
(Artemia salina) lethality test (LC50 > 100 μg/mL).
As an established traditional medicine and from
literature reports suggesting important biological
activities, the essential oils from N. arbor-tristis
may be potential candidates for therapeutic use,
and therefore, further research into the biological
activities of the oil is currently being pursued.
Acknowledgment: PP and PS are grateful
to Tribhuvan University-MMAMC campus for
helping with the plant collection and access to
laboratory equipments. WNS is grateful to an
anonymous private donor for the gift of the GC-MS
instrumentation. We thank Dr. Bernhard Vogler for
technical assistance with GC-MS data collection.

4
OAJMAP (2012)
References
Adams, R. 2007. Identication of essential oil
components by gas chromatography/
mass spectrometry. Carol Stream, IL, USA:
Allured Publishing.
Das, S., Sasmal, D. and Basu, S. P. 2010.
Antispasmodic and antihelmintic activity
of Nyctanthes arbor tristis Linn. International
Journal of Pharmaceutical Science and
Research 1: 51-55.
Jensen, S.R., Franzyk, H. and Wallander, E. 2002.
Chemotaxonomy of the Oleaceae: iridoids
as taxonomic markers. Phytochemistry 60:
213-231.
Khatune, N. A, Islam, M. E, Rahman, M. A. A.,
Mosaddik, M. A and Haque, M. E. 2003.
In-vivo cytotoxic evaluation of new
benzofuran derivative isolated from
Nyctanthes arbor-tristis L. on Ehrlich Ascite
Carcinoma cells (EAC) in mice. Journal of
Medical Sciences 3: 169-173.
McLaughlin, J. L. 1991. Bench-top bioassays for
the discovery of bioactive compounds in
higher plants. Brenesia 34: 1-14.
Rahman, M. M. and Shahjahan, M. 2011. Analysis of
fatty acid composition of Nyctanthes arbor-
tristis Linn. bark by gas chromatography.
International Journal of Pharmaceutical
Research and Development 3: 1-5.
Rathee, J. S., Hassarajani, S. A. and Chattopadhyay,
S. 2007. Antioxidant activity of Nyctanthes
arbor-tristis leaf extract. Food Chemistry 103:
1350-1357.
Rathod, N., Raghuveer, I., Chitme, H. R. and
Chandra, R. 2010. Free radical scavenging
activity of Nyctanthes arbortristis in
streptozotocin-induced diabetic rats.
Indian Journal of Pharmaceutical Education
and Research 4: 288-294.
Sasmal, D., Das, S. and Basu, S.P. 2007.
Phytoconstituents and therapeutic
potential of Nyctanthes arbor-tristis Linn.
Pharmacognosy Reviews 1: 344-349.
Satyal, P., Paudel, P., Lamichhane, B., and Setzer
W. N. 2012. Volatile constituents and
biological activities of the leaf essential
oil of Jasminum mesnyi growing in Nepal.
Journal of Chemical and Pharmaceutical
Research 4: 437-439.
Saxena, R. S., Gupta, B., Saxena, K. K., Srivastava,
V. K. and Prasad, D. N. 1987. Analgesic,
antipyretic and ulcerogenic activity of
Nyctanthes arbor-tristis leaf extract. Journal
of Ethnopharmacology 19: 193-200.
Setzer, M. C., Moriarity, D. M., Lawton, R. O. ,
Setzer, W. N.,Gentry, G.A. and Haber,W.A
2003. Phytomedicinal potential of tropical
cloudforest plants from Monteverde, Costa
Rica. Revista de Biologia Tropical 51: 647-674.
Tuntiwachwuttikul, P., Rayanil, K. and Taylor,
W. C. 2003. Chemical constituents from
the owers of Nyctanthes arbor-tristis.
ScienceAsia 29: 21-30.
1487 (E)-β-Ionone 1.2 ---
1550 Elemol --- 5.8
1564 Dodecanoic acid --- tr
1569 (3Z)-Hexenyl benzoate 11.0 ---
1631 γ-Eudesmol --- 1.7
1651 β-Eudesmol --- 17.1
1654 α-Eudesmol --- 8.7
1677 n-Tetradecanol --- tr
1722 (2Z,6E)-Farnesol --- 0.9
1809 Cryptomeridiol --- 4.8
1920 Methyl palmitate --- 1.8
1957 Hexadecanoic acid 26.4 34.3
2112 (E)-Phytol 13.6 ---
2123 Methyl stearate --- 1.4
2162 Octadecanoic Acid 6.2 3.9
Total Identied 100.0 89.4