Gc-ms Of Anthopogon Oil And Its Biological Properties

Original paper GC-MS of Essential Oil of Rhododendron 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 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 significant 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 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 showed that anthopogon 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 towards Bacillus subtilis and Enterococcus faecalis. Key words index: Anthopogon oil, GC-MS, Antiprolipherative, Antibacterial, Antifungal Introduction 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 and used locally as incense. Traditionally, the leaves and flowers of Rhododendron 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 of Rhododendron 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) had investigated the organoleptic properties and physico-chemical properties of anthopogon oil as its specification 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 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: 0.25µm; temperature programme: 60°C (3min) to 280°C at rate of 3/min; inj. temp. 200°C. Antiprolipherative Activity Samples 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 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 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. Cell growth was evaluated by MTT reduction assay. MTT Test 20 µl of MTT solution (5 mg/ml in PBS) were added to each well, plates incubated at 37°C, 4 hours later culture media discarded and dark blue crystals dissolved in DMSO (150 µl/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 ratios were 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 on comparison 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 mainly monoterpenes. 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 oil S.N. Compound Percentage 1 α -Thujene 0.21 2 α - Pinene 37.39 3 Camphene 0.23 4 β - Pinene 15.98 5 β - Myrcene 1.07 6 ρ - cymene 2.59 7 Limonene 13.26 8 Cis- ocimene 5.33 9 Trans- terpinene 1.48 10 α - copaene 0.75 11 trans - β - caryophyllene 2.53 12 α - humulene 0.23 13 allo - aromandrene 0.20 14 Germacrene 1.77 15 α - Muurolene 2.74 16 α – Amorphene 3.15 17 δ - Cadinene 9.91

KI 931 939 953 980 1118 1026 1031 1050 1062 1376 1418 1454 1461 1480 1499 1524

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 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

showed that anthopogon oil is characterised by a cytotoxic activity which is independent from cell line and treatment protocol used.

200 ug/ml 400 ug/ml 600 ug/ml

100 80 60

IC50 246,1µg/ml

% Survival

(223,4-271,2)

IC50 213,5µ g/ml (184,6-247,0)

40

IC50 236,6µg/ml (187,9-252,0)

20 0 2008

Figure: 1

A-431

LoVo S

100

80

100 ug/ml

IC50 224,0µg/ml (196,9-254,9)

200 ug/ml

IC50 218,6µg/ml

400 ug/ml

IC50 217,6µg/ml

(186,9-255,6)

% Survival

60

(202,2-276,8)

40

20

0

2008

A-431

LoVo S

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 1-20µl/well. The anthopogon oil was active towards Bacillus subtilis and Enterococcus 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 of anthopogon oil. We are especially thankful to Mr. Marco Valussi, without whose support and dedication, we could not have achieved the expected results.

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, Council of Scientific and Industrial Research, New Delhi. ™ Pohle, P.1990. Useful Plants of Manang District: A Contribution to the Ethnobotany of Nepal-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.