Cytotoxic activity and volatile constituents of fruit and leaves of Pandanus tectorius Parkin ex. J. P. du Roi

JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2015-00072 Chemical and Biological Sci. 2015, Vol. 60, No. 9, pp. 9-13 This paper is available online at Received July 30, 2015. Accepted November 23, 2015. Contact Pham Huu Dien, e-mail address: dienhp@gmail.com 9 CYTOTOXIC ACTIVITY AND VOLATILE CONSTITUENTS OF FRUIT AND LEAVES OF Pandanus tectorius Parkin ex. J. P. du Roi Pham Cong Vu1, Tran The Tung1, Lam Thi Hai Yen1, Nguyen Quyet Tien2, Dang Ngoc Quang1 and Pham Huu Dien1 1 Faculty of Chemistry, Hanoi National University of Education 2 Institute of Chemistry, Vietnam Academy of Sciences and Technology Abstract: Most fractional extracts from fruits and leaves of P. tectorius ex. J.P. du Roi collected in the Bach Ma National Park, Thua Thien - Hue Province, have remarkable cytotoxic activity against KB cell lines. Of these, ethyl acetate extracts have the best activity (with IC50 values of 24 to 48 µg/mL). The main volatile components were identified by gas chromatography/mass spectrometry (GC/MS). These were the secquiterpenes (copaene, isocaryophyllene) found in the fruit and the methyl esters and alcohols found in the leaves. Keywords: Pandanus tectorius Parkins ex. J. P. du Roi, cytotoxic, volatile constituents. 1. Introduction Within the genus Pandanus (Pandanaceae) are approximately 600 species that are widely distributed throughout the tropical and subtropical regions. Several Pa danus species are used as a remedy for toothache and rheumatism, and as a diuretic and cardiotonic [1]. Many volatile compounds, including monoterpe es and sesquiterpenes, were isolated from P. latifolius [2], and several alkaloids were found in P. amaryllifolius [3]. Fatty acid content of the fruit of P. conoideus was analyzed by Southwell [4]. In Vietnam, 4 Pandanus species have been identified. P. amaryllifolius, P. tectorius, P. humilis and P. tonkinensis are growing widespread from Northern to Southern Vietnam [1]. Pandanus tectorius flourishes mainly on islands and along beaches in the Middle of Vietnam. In this paper we report on volatile compon nts of the fruit and leaves of Pandanus tectorius ex. J. P. du Roi plants, collected in the Bach Ma National Park, Thua Thien - Hue Province. Pham Cong Vu, Tran The Tung, Lam Thi Hai Yen, Nguyen Quyet Tien, Dang Ngoc Quang and Pham Huu Dien 10 2. Content 2.1. Subjects and methods * General procedure GC-MS was carried out using a Hewlett Packard mass selective detector 5971 A and a gas chromatograph 5890 Series II using a fused silica gel column coated with DB-17 (30 m x 0.25 mm i.d., film thickness 0.25 m) with He as the carrier gas (1 mL/min). The GC-MS analysis was performed at 50 oC, then at 50 - 250 oC at 4 oC/min and finally isothermal at 250 oC for 10 min. * Subjects Pandanus tectorius ex. J. P. du Roi fruit and leaves were collected in the Bach Ma National Park, Thua Thien - Hue Province in November 2012 and identified by MSc. T.A. Nguyen (Institute of Chemistry, VAST). A voucher specimen (No. PAN201211) was deposited at the Herbarium of Faculty of Chemistry, HNUE, Hanoi, Vietnam. * Cytotoxicity assay Dried powdered leaves and fruit of Pandanus tectorius plants (10 g each) were extracted alternately with n- exane, ethyl acetate, n-buthanol and methanol (100 mL x 3 each) using an ultrasonic bath. T e solvent was removed to give corresponding crude extracts. The crude extracts were tested against KB cell lines from the American Type Culture Collection according to the method described by Scudiero et al. (1988) [5]. Cell lines were cultured in a RPMI 1640 medium supplemented with 10% foetal bovine s rum (FBS) in standard condition, sterile, with 5% CO2, 37 o , 98% humidity and harvested in log phase for assays. In this assay, 200 L volumes of cells at a concentration of 3 x104 cells per mL-1 were inoculated into a 96-well plate in a RPMI 1640 medium. The crude extracts were applied at final concentrations 128, 32, 8, 2 and 0.5 L-1 and the cultures were incubated for 3 days at 37 oC with 5% CO2. Then, 50 L of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, prepared at 1 mg per mL-1 in FBS, was added to the microculture well. After incubation for 4 h, 250 L of the supernatant was removed from each well and 100 L of DMSO was added followed by thorough mixing. Absorbance was measured at 540 nm in a Genios TECAN spectrophotometer. Th IC50 value was calculated based on percent growth inhibition (ODcontrol-ODsample)/ODcontrol. Ellipticin was used as a reference compound. 2.2. Results and discussion * Cytotoxic activity Different extracts of Pandanus tectorius plants exhibited different degrees of cytotoxic activity against KB cell lines (ellipticin was used as the control sample, see the Table 1). Table 1. Cytotoxic activity of Pandanus tectorius fruit and leaf extracts against KB cell lines (IC50 value, µg/mL) No. Sample IC50 No. Sample IC50 1 F-1 94.11 5 L-1 48 2 F-2 24 6 L-2 37.05 3 F-3 > 128 7 L-3 102.4 4 F-4 > 128 8 L-4 > 128 Here, IC50 value of Ellipticin: 0.31 g/mL; F indicates fruit, L indicates leaves; 1-n-h xane, 2- ethyl acetate, 3- n-buthanol and 4- methanol extracts, respectively. From Table 1, we see that: Cytotoxic activity and volatile constituents of fruit and leaves of Pandanus tectorius 11 - Most fractional extracts from the fruit and leaves of P. tectorius plants show remarkable cytotoxic activity against KB cell lines, the strongest of them being the ethyl acetate extracts (IC50 values from 24 to 48 µg/mL). The ethyl acetates usually contain volatile components in addition to other constituents such as flavonoids and alkaloids. In our previous papers [6, 7] we reported on the chemical constituents of P. tectorius fruit and leaves. In this paper, we concentrate on the volatile components of the fruit and leaves of the plant. - The methanol extracts made from the fruit and leaves had on cytotoxic activity towards the KB cell lines. * Volatile composition To obtain volatile compounds from P. tectorius fruit and leaves, fresh samples were extracted with diethyl ether. The diethyl ether extracts were filtered through a small glass column packed with silica gel using diethyl ether to give oils, which were analyzed using thin layer chromatography (TLC) and GC-MS. Each component that appeared on the GC-MS was identified by comparing the retention time and mass spectrum with known samples [8] and our library database. Table 2 describes the chemical composition of the diethyl ether xtract of P. tectorius fruit. Table 2. The volatile composition of P. tectorius fruit No. Name Formula Retention time (min) % 1 Copaene C15H24 11.36 15.0 2 1,5-dimethyl-8-(1-metyletyl) cyclodeca -1,5-diene C15H24 11.50 3.4 3 1,7-dimethyl-7-(4-methyl-3-pentenyl)- tricyclo[2.2.0(2,6)]heptane C15H24 11.87 5.2 4 Isocaryophyllene C15H24 12.23 8.0 5 α-caryophyllene C15H24 12.42 7.8 6 2,4a,5-9,9a-octahydro-3,5,5-trimethyl - 9-methylene-1H-benzocycloheptene C15H24 12.81 0.4 7 1-4,4a,5,6,8a-octahydro-7-methyl-4- metylene-1-(1-methyletyl)naphtalene C15H24 13.19 0.2 8 Caryophyllene oxide C15H24O 14.23 1.0 9 Methyl hexadecanoate C17H34O2 19.27 2.6 10 Methyl (Z,Z)-octadec-9,12-adienoate C19H34O2 21.85 2.3 11 Methyl (E)-octadec-9-enoate C16H32O2 21.95 15.2 12 Methyl 16-methylheptadecanoate C19H38O2 22.34 8.6 13 Isopropyl palmitate C19H38O2 24.11 1.8 14 Octadecanoic acid C18H36O2 24.78 1.8 15 Oleic acid C18H34O2 27.21 3.9 16 Octadecanoic acid C18H36O2 27.56 1.2 17 bis-(2-Ethylhexyl) phtalate C24H38O4 27.94 1.2 18 7-methyl-Z-tetradec-7-enyl acetate C17H32O2 30.09 1,2 19 Squalene C30H50 31.44 0.6 20 2,6,10-trimethyltetradecane C17H36 32.64 1.0 21 (E)-tetradec-2-enol C28H48O 34.93 0.4 22 24-methylcholest-5-en-3-ol C28H48O 38.64 2.5 23 4,4-dimethylcholesta-22,24-dien-3-ol C29H48O 39.31 3.6 24 (E)-10,13,13-trimethyltetradec-11-enyl acetate C19H48O 39.58 0.6 Pham Cong Vu, Tran The Tung, Lam Thi Hai Yen, Nguyen Quyet Tien, Dang Ngoc Quang and Pham Huu Dien 12 From Table 2, three monoterpenes (copaene, isocaryophyllen and caryophyllene oxide) together with methyl esters of hexadecanoate, (Z,Z)- c adec-9,11-adienoate and (E)-octadec- 9-enoate are identified as the main volatile components (up to 15.2%) in the fruit. Table 3 describes the chemical composition of the diethyl ether xtract of P. tectorius leaves. Accordingly, the main volatile constituents from the leaves are esters, alcohols, hydroperoxides (up to 7.2%), quite different from the fruit. Here are some structures of the main constituents of P. tectorius fruit (Figure 2). Copane Isocaryophyllene O Caryophyllene oxide Methyl hexadecanoat O O Methyl (Z,Z)-octadec-9,12-dienoat Methyl (9E)-octadecenoat O O O O 1 16 18 1 1 18 129 9 e e e Figure 2. Selected structures of volatile components of P. tectorius fruit Table 3. The volatile components of P. tectorius leaves No Name Formula Retention time (min) % 1 Pentyl 4-pentylphenyl naphtalen- 2,6-dicarboxylate C28H32O4 4.33 2.54 2 Hexan-3-ol C6H14O 4.80 12.2 3 2-hexyl hydropeoxide C6H14O2 4.98 7.2 4 Hexan-2-one C6H12O 5.23 4.7 5 Butyl acetate C6H12O2 5.58 7.1 6 Ethylbenzene C8H10 6.97 1.3 7 3-methyl-2-(1-benzoyl-1- methylethyl)azirane C19H21NO 9.28 2.1 8 Hex-2-yl hydropeoxide C6H14O2 9.67 1.8 9 Decanal C10H20O 15.58 2.1 10 p-allylanisole C10H12O 17.13 2.0 11 Tetradecane C14H30 20.03 2,8 12 Dodecanol C12H26O 24.84 2.8 13 Botryococcane C33H68 25.95 2.6 14 Dimethyl o-phtalate C10H10O4 28.40 8.9 15 2-ethylhexyl benzoate C15H22O2 32.16 2.5 Cytotoxic activity and volatile constituents of fruit and leaves of Pandanus tectorius 13 3. Conclusion Most of the fractional extracts from the fruit and leaves of P. tectorus ex. J. P. du Roi collected in the Bach Ma National Park, Thua Thien–Hue Province, have remarkable cytotoxic activity against the KB cell lines. Among the extracts, the ethyl acetate extracts have the strongest activity (with IC50 values of 24 to 48 µg/mL). A GC-MS analysis of the ether extracts identified the main volatile components. Copaene(15.0 %) and isocaryophyllene (8.0%) are two major sesquiterpenes in fruit and 2-hecyl hydroperoxide, hexan-3-ol (12.2%) in leaves. This is the first study to identify cytotoxic activity and the volatile components of Vietnamese P. tectorius plants. Acknowledgement: Financial support provided by a grant from Ministry of Education and Training of Vietnam (No. B2013-17-37) is gratefully acknowledged. The authors thank MSc. Nguyen The Anh, Institute of Chemistry, VAST, Vietnam, for identifying the plant materials. REFERENCES [1] Vo Van Chi, 1999. A dictionary of Traditional Vietnamese Medicinal plants. Bio- Pharmaceutical Publishing House, p. 428. [2] Pieris N. M. and Macleod A.J., 1981. Antioxidative Activities of constituents Isolated from Pandanus odoratissimu. Phytochemistry, Vol. 20, No. 9, pp. 2213-2216. [3] Nonato M. G., M. J. Garson, R. J. W. Truscott and J. A Carver, 1993. Structural characterization of piperidine alkaloids from Pandanus amaryllifolius by inverse- detected 2D NMR techniques. Phytochemistry, Vol. 34, pp. 1159-1163. [4] Harris R. and Southevell K., 1992. Chemical characteristics of Pandanus conoideus fruit lipid. J. Sci. Food Agric., Vol. 58, pp. 593-594. [5] Scudiero D. A., Shoemaker R.H., Paull K. D., Monks A. and Tierney S., 1988. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res., Vol. 48, pp. 4827-4833. [6] Luong Thi Cuc Hoa, Lam Thi Hai Yen, Dang Ngoc Quang, Pham Huu Dien, 2014. Study on chemical constituent from the leaves of Pandanus tectorius Parkin. Ex J.P. du Roi collected in Thua Thien - Hue Province. J. of Science, HNUE, Vol. 59, No.1A, pp. 186-193. [7] Nguyen Quyet Tien, Nguyen Quang An, Nguyen Ngoc Tuan, Truong Thi Thanh Nga, Lanh Thi Ngoc, Pham Cong Vu, Pham Huu Dien, 2014. The fatty acids and glycerides from the fruits of Pandanus tectorius Parkins ex. J. P. du Roi, Vietnam J. of Chem., Vol. 52, No. 6A, pp.98-102. [8] Joulin P., Koenig W.A., 1998. The atlas of spectral data of sesquiterpene hydrocarbons. E. B. Verlag, Hamburg.