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GC-MS of Essential Oil of
Rhododendron anthopogon
D. Don and ItsBiological Properties
Khilendra Gurung
*
, Gabbriella Innocenti
**
, Stefano Dall’ Acqua
**
, Maria Carrara
***
, AureliaTubaro
****
and Mariagnese Barbera
***
*
Himalayan Bio Trade P. Ltd., Kathmandu, Nepal; khilendragurung@yahoo.com
**
Dipartimento di Scienze Farmaceutiche, University of Padova, Via Marzolo 5, 35121 Padova,Italy; gabbriella.innocenti@unipd.it
***
Dipartimento di Farmacologia ed Anestesiologia, University of Padova, Largo E.Meneghetti 2, 35121 Padova, Italy.
****
Dipartimento di Economia e Merceologia, University of Trieste, Via A. Valerio 6, Trieste,Italy
.
Abstract
The chemical composition of a sample of anthopogon oil was investigated by means of GC-MS.The antiproliferative activities on human cancer cells were also evaluated. In addition preliminarydata on antibacterial and antifungal activities were obtained.GC-MS analysis of the anthopogon oil led to the identification of the majority of the componentsas
α
-Pinene (37.39%),
β
-Pinene (15.98 %), Limonene (13.26%), and δ-Cadinene (9.91%).For the three of the cell lines; ovarian (2008), cervix (A-431) and colon (LoVo), a very significantcell growth inhibition was pointed out with the two highest doses used. Decrease of cell proliferation occurred in the same way on 2008, A-431 and LoVo: the values of IC50experimentally obtained were 246.1, 213.5 and 236.6 mg/ml, respectively.A remarkable inhibition of cell growth resulted after exposure at the highest dose, and the valuesof IC50 were similar to that previously obtained: 224.0 for 2008 cells, 218.6 for A-431 cells and217.6 for LoVo cells. These preliminary data showed that
anthopogon oil is characterised by acytotoxic activity which is independent from cell line and treatment protocol used.The evaluation of antifungal and antibacterial activities showed that anthopogon oil was activetowards
Bacillus subtilis
and
Enterococcus faecalis
.
Key words index: Anthopogon oil, GC-MS, Antiprolipherative, Antibacterial, AntifungalIntroduction
Rhododendron anthopogon
D. Don
(Ericaceae) is an evergreen shrub growing at an altitude of 3300-5100m from east to western regions of Nepal. The leaves of this shrub are aromatic andused locally as incense. Traditionally, the leaves and flowers of
Rhododendron anthopogon
areused in stomach, liver and lungs disorders, indigestion, sore throat and phlegm diseases. Also,used as an appetizer, diuretic and in vomiting (Lama
et al.,
2001). Anthopogon oil is obtained bysteam distillation of the aerial parts of
Rhododendron anthopogon.
Also known as Sunpati oillocally, is good natural source of sweet herbal, faint balsamic essence. Anthopogon oil isessentially used in perfumery. Herbs Production and Processing Company Limited (HPPCL) hadinvestigated the organoleptic properties and physico-chemical properties of anthopogon oil as itsspecification in Nepal.
Little information has been published about
Rhododendron anthopogon
and Anthopogon oil(HMG, 1970; Joshi
et al
., 1981; CSIR, 1986; Pohle, 1990; Rajbhandari and Schopke, 1999;Shakya, 1999; Shrestha, 1999; IUCN, 2000, Lama
et al.,
2001 and Yonzon
et al
., 2005).
The chemical composition of a sample of anthopogon oil was investigated by means of GC-MS.The antiproliferative activities on human cancer cells were also evaluated. In addition preliminarydata on antibacterial and antifungal activities were obtained.
Materials and MethodsChemical Analysis
Rhododendron anthopogon
was collected at an altitude ranging between 4000-4500m fromRolwaling area of Dolakha district, Nepal, during August-September 2003. Fresh aerial parts(leaves and flowers) were used for the extraction of anthopogon oil by steam distillation method.GC-MS was obtained from a Hewlett-Packard 6890-5973 system operating on EI mode,equipped with a capillary column HP-5MS 30m x 0.25 mm; film thickness: 0.25
µ
m; temperature programme: 60°C (3min) to 280°C at rate of 3/min; inj. temp. 200°C.
Antiprolipherative ActivitySamples
Anthopogon
oil was dissolved in DMSO and diluted in culture medium in order to obtain a stock solution constituted by: 1% DMSO,
9% anthopogon oil, 90% culture medium.
Cell Lines
Three human tumour cell lines: ovarian (2008), cervix (A-431) and colon (LoVo) were used.2008 and A-431 cells were maintained in RPMI 1640 medium, while LoVo cells were maintainedin MEM Eagle. Both culture media were supplemented with 10% heat-inactivated FCS, 1%antibiotics (all products of Biochrom KG Seromed) and 1% 200 mM glutamine (Merck).
Cytotoxicity
Cells were seeded in 96-well tissue plates (Falcon) and after 24 hours, incubated at 37°C for further 3 or 24 hours with different concentrations (from 100 to 600 mg/ml)
of
anthopogon
oil.Then, cells treated for 3 hours were washed and incubated in culture medium for 21 hours. Cellgrowth was evaluated by MTT reduction assay.
MTT Test
20
µ
l of MTT solution (5 mg/ml in PBS) were added to each well, plates incubated at 37°C, 4hours later culture media discarded and dark blue crystals dissolved in DMSO (150
µ
l/well).Absorbance was measured on a micro-culture plate reader (Titertek Multiscan) using a testwavelength of 570 nm and a reference wavelength of 630 nm.
Statistical Analysis
For each assay five different experiments were performed in triplicate. The results werestatistically evaluated by Student's
t
-test. The IC50, 95% confidence limits, and the potency ratioswere estimated using the Litchfield and Wilcoxon method.
Results and Discussion
Chemical Analysis
GC-MS analysis of the anthopogon oil led to the identification of the majority of components(98.8 % of the total components detected) which are listed in Table: 1, with their relative percentage and their retention indices. The identification of components was based oncomparison of their mass spectra with those of Wiley Library, as well as on comparison of their retention indices with literature values.The major constituents of the anthopogon oil were
α
-Pinene (37.39%),
β
-Pinene (15.98 %),Limonene (13.26%), and δ-Cadinene (9.91%). The anthopogon oil
consisted mainlymonoterpenes.
While
the major constituents of Anthopogon oil as reported by Yonzon
et al.,
(2005) were o-cadinene (11.4%), α-pinene (8.3%), β-caryophyllene (6.5%) and β-pinene(6.2%).
Table 1: Chemical composition of anthopogon oilS.N.CompoundPercentageRI
1α -Thujene0.219312α - Pinene37.399393Camphene0.239534β - Pinene15.989805β - Myrcene1.0711186ρ - cymene2.5910267Limonene13.2610318Cis- ocimene5.3310509Trans- terpinene1.48106210α - copaene0.75137611
trans
- β - caryophyllene2.53141812α - humulene0.23145413
allo
- aromandrene0.20146114Germacrene1.77148015α - Muurolene2.74149916α – Amorphene3.1517δ - Cadinene9.911524
Antiprolipherative Activity
Generally, when cells were exposed for 3 hours at different concentrations (200, 400, 600 mg/ml)of anthopogon
oil and incubated with culture medium for 21 hours, results revealed a dosedependent cytotoxic effect. In particular, for the three of them cell lines, a very significant cellgrowth inhibition was pointed out with the two highest doses used. As shown in Figure 1,decrease of cell proliferation occurred in the same way on 2008, A-431 and LoVo: the values of IC50 experimentally obtained were 246.1, 213.5 and 236.6 mg/ml, respectively.When the exposure was extended to 24 hours using 100, 200 and 400 mg/ml of anthopogon oil,like results were pointed out (Figure 2). A remarkable inhibition of cell growth resulted after exposure at the highest dose, and the values of IC50 were similar to that previously obtained:224.0 for 2008 cells, 218.6 for A-431 cells and 217.6 for LoVo cells. These preliminary datashowed that
anthopogon oil is characterised by a cytotoxic activity which is independent fromcell line and treatment protocol used.
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