GC-MS of Anthopogon Oil and Its Biological Properties

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Original paper

GC-MS of Essential Oil ofRhododendron anthopogon D. Don and Its

Biological Properties

Khilendra Gurung*, Gabbriella Innocenti**, Stefano Dall Acqua**, Maria Carrara***, Aurelia

Tubaro**** and Mariagnese Barbera***

* Himalayan Bio Trade P. Ltd., Kathmandu, Nepal; E-mail: [email protected]

** Dipartimento di Scienze Farmaceutiche, University of Padova, Via Marzolo 5, 35121 Padova,Italy; E-mail: [email protected]

*** 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 preliminary

data on antibacterial and antifungal activities were obtained.

GC-MS analysis of the anthopogon oil led to the identification of the majority of the components

as -Pinene (37.39%), -Pinene (15.98 %), Limonene (13.26%), and -Cadinene (9.91%).

For the three of the cell lines; ovarian (2008), cervix (A-431) and colon (LoVo), a verysignificant cell 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 IC50

experimentally 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 and

217.6 for LoVo cells. These preliminary data showed thatanthopogon oil is characterised by a

cytotoxic activity which is independent from cell line and treatment protocol used.The evaluation of antifungal and antibacterial activities showed that anthopogon oil was active

towardsBacillus subtilis andEnterococcus faecalis.

Key words index: Anthopogon oil, GC-MS, Antiprolipherative, Antibacterial, Antifungal

IntroductionRhododendron anthopogon D. Don(Ericaceae) is an evergreen shrub growing at an altitude of3300-5100m from east to western regions of Nepal. The leaves of this shrub are aromatic and

used locally as incense. Traditionally, the leaves and flowers ofRhododendron anthopogon are

used 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 by

steam distillation of the aerial parts ofRhododendron anthopogon. Also known as Sunpati oil

locally, is good natural source of sweet herbal, faint balsamic essence. Anthopogon oil is

essentially used in perfumery. Herbs Production and Processing Company Limited (HPPCL) hadinvestigated the organoleptic properties and physico-chemical properties of anthopogon oil as its

specification in Nepal.


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Little information has been published aboutRhododendron 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 preliminary

data on antibacterial and antifungal activities were obtained.

Materials and Methods

Chemical Analysis

Rhododendron anthopogon was collected at an altitude ranging between 4000-4500m from

Rolwaling 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: 0.25m; temperatureprogramme: 60C (3min) to 280C at rate of 3/min; inj. temp. 200C.

Antiprolipherative ActivitySamples

Anthopogonoil 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

maintained in 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 37C for

further 3 or 24 hours with different concentrations (from 100 to 600 mg/ml) ofanthopogonoil.

Then, cells treated for 3 hours were washed and incubated in culture medium for 21 hours. Cell

growth was evaluated by MTT reduction assay.

MTT Test

20 l of MTT solution (5 mg/ml in PBS) were added to each well, plates incubated at 37C, 4

hours later culture media discarded and dark blue crystals dissolved in DMSO (150 l/well).Absorbance was measured on a micro-culture plate reader (Titertek Multiscan) using a test

wavelength of 570 nm and a reference wavelength of 630 nm.

Statistical Analysis

For each assay five different experiments were performed in triplicate. The results were

statistically evaluated by Student's t-test. The IC50, 95% confidence limits, and the potency ratioswere estimated using the Litchfield and Wilcoxon method.


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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 -Pinene (37.39%), -Pinene (15.98 %),Limonene (13.26%), and -Cadinene (9.91%). The anthopogon oilconsisted mainly

monoterpenes.Whilethe major constituents of Anthopogon oil as reported by Yonzon etal.,(2005) were o-cadinene (11.4%), -pinene (8.3%), -caryophyllene (6.5%) and -pinene

(6.2%).

Table 1: Chemical composition of anthopogon oil

S.N. Compound Percentage KI

1 -Thujene 0.21 9312 - Pinene 37.39 939

3 Camphene 0.23 953

4 - Pinene 15.98 980

5 - Myrcene 1.07 1118

6 - cymene 2.59 1026

7 Limonene 13.26 1031

8 Cis- ocimene 5.33 1050

9 Trans- terpinene 1.48 1062

10 - copaene 0.75 1376

11 trans - - caryophyllene 2.53 1418

12 - humulene 0.23 145413 allo - aromandrene 0.20 1461

14 Germacrene 1.77 1480

15 - Muurolene 2.74 1499

16 Amorphene 3.15

17 - Cadinene 9.91 1524

Antiprolipherative Activity

Generally, when cells were exposed for 3 hours at different concentrations (200, 400, 600 mg/ml)

of anthopogonoil and incubated with culture medium for 21 hours, results revealed a dose

dependent cytotoxic effect. In particular, for the three of them cell lines, a very significant cell

growth 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 data


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showed thatanthopogon oil is characterised by a cytotoxic activity which is independent from

cell line and treatment protocol used.

Figure: 1

0

20

40

60

80

100

2008 A-431 LoVo S

%Su

rvival

200 ug/ml

400 ug/ml

600 ug/ml

IC50

246,1g/ml(223,4-271,2)

IC50

213,5g/ml(184,6-247,0)

IC50

236,6g/ml(187,9-252,0)


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Figure: 2

Microbiological Activity

The evaluations of antifungal and antibacterial activities were performed with aromatogram on

some reference species. Anthopogon oil was assayed at doses 1-20l/well. The anthopogon oilwas active towardsBacillus subtilis andEnterococcus faecalis. Preliminary data are reported in

Table 2.

Table: 2 Evaluation of Antifungal and Antibacterial Activity

Micro organisms Activity

Bacillus subtilis +++

Enterococcus faecalis +++

Staphylococcus aureus +

Candida spp. +

Escherichia coli -

Pseudomonas aeruginosa -

Acknowledgements

We are grateful to the local people who collected the Sunpati leaves from Rolwaling valley and

the staffs of Pathibhara Essential Oil Industry, Changkhu Village Development Committee,

Dolakha district for providing the sample ofanthopogon oil. We are especially thankful to Mr.Marco Valussi, without whose support and dedication, we could not have achieved the expected

results.

0

20

40

60

80

100

2008 A-431 LoVo S

%S

urvival

100 ug/ml

200 ug/ml

400 ug/ml

IC50

224,0g/ml(196,9-254,9)

IC50

218,6g/ml(186,9-255,6) IC50

217,6g/ml(202,2-276,8)


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References

HMG. 1970.Medicinal Plants of Nepal. Bulletin of the Department of Medicinal Plants No.3, His Majesty's Government of Nepal, Ministry of Forest and Soil Conservation,

Kathmandu. Joshi, YC, MP Dobhal, BC Joshi and FSK Barar. 1981. Pharmazie, pp 36, 381. CSIR. 1986. The Useful Plants of India. Publications and Information Directorate, Councilof Scientific and Industrial Research, New Delhi.

Pohle, P.1990. Useful Plants of Manang District: A Contribution to the Ethnobotany ofNepal-Himalaya. Franz Steiner Verlag Wiesbaden GMBH, Stuttgart, pp 32.

Rajbhandari, DM and T Schopke. 1999. Pharmazie, pp 54, 232-234. Shakya, PR. 1999. Phytogeograhy and Ecology of Nepalese Rhododendron. In:Nepal

Nature's Paradise (Majupuria TC and RK Majupuria eds). M. Devi, Gwalior, India. Shrestha, TB. 1999. Rhododendrons. In:Nepal Nature's Paradise (Majupuria TC and RK

Majupuria eds). M. Devi, Gwalior, India. IUCN. 2000.National Register of Medicinal Plants. IUCN Nepal, Kathmandu. Lama, YC, SK Ghimire and Y Aumeeruddy-Thomas. 2001.Medicinal Plants of Dolpo:

Amchis Knowledge and Conservation. WWF Nepal Program, Kathmandu. Yonzon, M, DJ Lee, T Yokochi, Y Kawano and T Nakahara. 2005. Antimicrobial Activities

of Essential Oils of Nepal. In:Journal of Essential Oil Research, Jan/Feb.

GC-MS of Anthopogon Oil and Its Biological Properties

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