From Paramignya trimera plants that were collected in the Hon Heo Commune (Khanh Hoa Province, November 2013), n-hexane extracts of its stem bark and leaves was obtained which showed moderate cytotoxic activity towards KB, Hep-G2, Lu and MCF7 cell lines (with IC50 values ranging from 5.7 to 30.89 µg/mL).
JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2014, Vol 59, No 9, pp 11-16 This paper is available online at http://stdb.hnue.edu.vn CYTOTOXIC ACTIVITY OF Paramignya trimera (OLIV.) GUILLAUM PLANT EXTRACTS AND THEIR VOLATILE CHEMICAL CONSTITUENTS Nguyen Thi Thanh Hoa1 , Souknaly Thoumma1,2 Lam Thi Hai Yen1 and Pham Huu Dien1 Faculty of Chemisty, Hanoi National University of Education Faculty of Education, National University of Laos Abstract From Paramignya trimera plants that were collected in the Hon Heo Commune (Khanh Hoa Province, November 2013), n-hexane extracts of its stem bark and leaves was obtained which showed moderate cytotoxic activity towards KB, Hep-G2, Lu and MCF7 cell lines (with IC50 values ranging from 5.7 to 30.89 µg/mL) A GC-MS analysis of the volatile plant components showed that esters, saturated alcohols and sesquiterpenes, including β-farnesene and α-gurjunene, are the major compounds Keywords: Paramignya trimera, cytotoxicity, volatile compounds Introduction The genus Paramignya, family Rutaceae, subfamily Aurantioideae, consists of fifteen species and two subspecies that are scattered across S.E Asia from India to Indonesia and the Philippines [7] Plants of the genus are a woody climber growing at low or moderate altitudes and they are used as medicinal plants in many countries, including Sri Lanka, India and the Philippines In Vietnam three species have been found, P amata, P monophylla and P trimera, all of which are widely distributed from Da Nang to Khanh Hoa Province [1, 2] P trimera (Xao tam phan) was recently reported to show hepatoprotective and cytotoxic activity towards the five cancer cell lines: Hep-G2, HTC116, MDA MB231, DVCAR-8 and Hela [3] Studies on the chemical constituents of the genus Paramignya are very limited Scientists from Sri Lanka found that the fruit of P monophylla contained triterpenes of the tirucallane type, including flindissone, and four pyranocoumarins, including poncitrin and nordentatin, are present in the stem bark [4, 5] Three flavanone, one triterpene and one Received June 27, 2014 Accepted November 27, 2014 Contact Pham Huu Dien, e-mail address: dienph@hnue.edu.vn 11 Nguyen Thi Thanh Hoa, Souknaly Thoumma, Lam Thi Hai Yen and Pham Huu Dien chromene derivatives were isolated from the stems of Paramignya griffithii [8] Recently, in Vietnam, 3,7-dimethyl-2,6-octadienyl)-7-hydroxy-2H-benzopyran-2-one was isolated from the stem bark of P trimera which showed moderate cytotoxic activity in vitro against cancer cell lines [3] In this paper we report on the cytotoxic activity of the extracts of P trimera plants that were collected in Hon Heo Commune (Khanh Hoa Province, November 2013) and their volatile chemical composition Content 2.1 Materials and methods * Plant material Paramignya trimera (Oliv.) Guillaum plants was collected in Hon Heo Commune, Khanh Hoa Province in November 2013 by Mr Pham Huu Quang (Vietxopetro) and identified by Dr Do Huu Thu (Institute of Ecol and Biol Resources, VAST) A voucher specimen (XTP-01) has been deposited in the Herbarium at the Faculty of Chemistry, Hanoi University of Education, Vietnam * Plant volatile composition analysis Leaves and stem bark (1.0 g each, separated) of Paramignya trimera were extracted using ethyl acetate and were subjected to volatile composition analysis A GC-MS of the samples was carried out on 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 µm) with He as the carrier gas (1 mL/min) The temperature programming of the GC-MS analysis was performed at 50 ◦ C, then at 50 250 ◦ C at ◦ C /min and finally isothermal at 250 ◦ C for 10 * Cytotoxicity assay An extract was made from dried Paramignya trimera stem bark powder (10 g) alternating n-hexane, ethyl acetate and methanol (100 mL × each) An extract was made from dried leaf powder using methanol in an ultrasonic bath The solvent was removed to give corresponding crude extracts The crude extracts were tested against KB, Hep-G2, Lu and MCF7 cell lines from the American Type Culture Collection according to the method described in [6] Cell lines were cultured in an RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) under standard conditions, sterile with 5% CO2 , 37 ◦ C, 98% humidity and harvested at log phase for assays In this assay, 200 µL volumes of cells at the concentration of × 104 cells mL−1 were inoculated into a 96-well plate in an RPMI 1640 medium The crude extracts were applied at final concentrations of 128, 32, 8, and 0.5 µL−1 and cultures were incubated for days at 37 ◦ C with 5% CO2 Then, 50 µL of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, prepared at mg mL−1 in FBS, was added to the microculture well After h of incubation, 250 µL of the supernatant was removed from each well and 100 µL 12 Cytotoxic activity of Paramignya trimera (Oliv.) Guillaum plant extracts of DMSO was added and thoroughly mixed Absorbance was measured at 540 nm in a Genios TECAN spectrophotometer The IC50 value was calculated based on percentage of growth inhibition (ODcontrol -ODsample )/ODcontrol Ellipticin was used as the reference compound 2.2 Results and discussion 2.2.1 Cytotoxic activity of plant extracts Table Cytotoxic activity of Paramignya trimera plant extracts against KB, Lu and MCF7 cell lines (IC50 value, µg/mL) No Sample KB HepG2 Lu MCF7 SB-N 5.7 9.9 18.66 30.89 SB-E > 128 8.96 >128 71.31 SB-M > 128 > 128 > 128 > 128 L-E 57.4 > 128 > 128 > 128 Different extracts of the Paramignya trimera plants exhibited different cytotoxic activity against the four cell lines (ellipticin was used as the control sample, see Table 1) IC50 value of Ellipticin: 0.31 µg/mL; SB means stem bark, L mean leaves, N: -n-hexane, E:- ethyl acetate, M: - methanol extracts, respectively Table shows that: - The n-hexane extract from stem bark obtained a moderate cytotoxicity towards four tested cancer cell lines (with IC50 values ranging from 5.7 to 30.89 µg/mL) - The ethyl acetate extracts from stem bark and leaves have a moderate cytotoxicity towards Hep-G2, MCF7 and KB cancer cell lines (with IC50 values of 8.96, 71.31 and 57.4 µg/mL, respectively) The methanol extracts from stem bark showed no activity towards the four tested cancer cell lines 2.2.2 Chemical constituents of the volatile composition Figure The GC-MS chromatogram of ethyl acetate extract of P trimera stem bark 13 Nguyen Thi Thanh Hoa, Souknaly Thoumma, Lam Thi Hai Yen and Pham Huu Dien Table The volatile composition of P trimera stem bark Number Name Retention time (min) % Hydro propyl 5.05 1.83 propanedioate Butyl acetate 5.97 1.27 2-Ethylhexanol 10.52 8.55 p-Allylanisole 19.21 5.69 Tetradecane 21.18 1.11 α-Guaiene 24.30 1.79 Aristolen 24.72 1.68 Dodecanol 25.44 1.87 α-Gurjunene 26.73 21.11 10 Dimethyl o-phtalate 28.85 3.95 11 Ethyl p-ethoxybenzoate 29.27 1.19 12 Falcarinol 43.40 3.59 To obtain the volatile compounds from Paramignya trimera, the fresh stem bark and leaves was extracted with ethyl acetate, separately The ethyl acetate extracts were filtered through a small glass column packed with silica gel to give a green oil, which was directly analyzed by thin layer chromatography (TLC) and GC-MS The GC-MS chromatograms were shown in Figures and Each component that appeared on GC-MS was identified by comparing its retention time and mass spectrum with our library databases Table describes the chemical composition of the ethyl acetate extract of P trimera stem bark Accordingly, three esters (butyl acetate, dimethyl o-phtalate and ethyl p-ethoxybenzoate) together with two saturated alcohols (2-ethylhexanol and dodecanol) were detected However, they are only minor compounds The remaining compounds are sesquiterpenes, of which α-gurjunene (21.11 %) is a major compound Figure The GC-MS chromatogram of ethyl acetate extract of P trimera leaves 14 Cytotoxic activity of Paramignya trimera (Oliv.) Guillaum plant extracts Table describes the chemical composition of the ethyl acetate extract of P trimera leaves Beside esters, which were found in stem bark (see Table 2), some sesquiterpenes were detected, β-farnesene (25.03%), germacrene-D (10.55%) and α-gurjunene (5.11%) being the major compounds Selected structures of volatile components in P trimera are shown in Figure Table The volatile composition of P trimera leaves Number Name Retention time (min) % Butyl acetate 5.95 4.47 2-Acetoxypropanol 7.42 4.66 Ethylidene diacetate 10.52 2.37 Geyrene 15.69 2.73 Ethoxy(methoxy)methylsilan 16.02 3.69 Pregeijerene 21.39 2.44 trans-Caryophyllene 24.02 4.46 β-Farnesene 24.72 25.03 α-Humulene 25.14 2.24 Germacrene-D 25.98 10.55 10 α-Gurjunene 26.73 5.11 11 Nerolidol 28.15 1.36 12 Dimethyl o-phthalate 28.87 2.73 Because no work on the volatile composition of P trimera has been published, this study presents the first published evidence that β-farnesene, α-gurjunene and other sesquiterpenes are chemotaxonimic markers for P trimera α-Gurjunene β-Farnesene Germacrene-D Figure Selected structures of volatile components of P trimera 15 Nguyen Thi Thanh Hoa, Souknaly Thoumma, Lam Thi Hai Yen and Pham Huu Dien Conclusion From Paramignya trimera plants that were collected in Hon Heo Commune (Khanh Hoa Province) it was found that sesquiterpenes, including β-farnesene and α-gurjunene, are major compounds of its volatile composition Furthermore, n-hexane extracts of its stem bark and leaves showed moderate cytotoxic activity towards KB, Hep-G2, Lu and MCF7 cell lines (with IC50 values ranging from 5.7 to 30.89 µg/mL) REFERENCES [1] Vo Van Chi, 1999 Dictionary of Vietnamese Medicinal Plants Med Press House, p 348 [2] Pham Hoang Ho, 2006 Herbal plants in Vietnam Young Press House, pp 363-364 [3] Nguyen Minh Khoi, Pham Thi Nguyet Hang, Do Thi Phuong, 2013 Study on acute toxicity, hepatoprotective activity and cytotoxic activity of P trimera (Oliv.) Guillaum J of Med plants, Vol.18, No.1, pp.14-20 (in Vietnamese) [4] Vijaya Kumar, N M Mohamed Niyaz, D B Mahinda Wickramatne, 1991 Tirucallane derivatives from Paramignya monophylla fruits Phytochem., Vol 30, No 4, pp 1231-1233 [5] Vijaya Kumar, N M Mohamed Niyaz, D B Mahinda Wickramatne, 1995 Coumarins from stem bark of Paramignya monophylla Phytochem., Vol 38, No 3, pp 805-806 [6] D A Scudiero, R H Shoemaker, K D Paull, K D., A Monks, S Tierney, 1988 Cancer Res., Vol 48, pp 4827-4833 [7] Swingle W T et al., 1967 The Citrus including, Univ of California Press, Berkeley and Los Angeles, Vol 1, pp.190-430 [8] Watanapiromsakul C., Waterman P G., 2000 Flavanone, triterpene and chromene derivatives from the stems of Paramignya griffithii Phytochem., Vol 55, No 3, pp 269-273 16 ... After h of incubation, 250 µL of the supernatant was removed from each well and 100 µL 12 Cytotoxic activity of Paramignya trimera (Oliv.) Guillaum plant extracts of DMSO was added and thoroughly... GC-MS chromatogram of ethyl acetate extract of P trimera leaves 14 Cytotoxic activity of Paramignya trimera (Oliv.) Guillaum plant extracts Table describes the chemical composition of the ethyl acetate... stem bark of P trimera which showed moderate cytotoxic activity in vitro against cancer cell lines [3] In this paper we report on the cytotoxic activity of the extracts of P trimera plants that