Kristiani et al Tropical Journal of Pharmaceutical Research May 2016; 15 (5): 959-964 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v15i5.9 Original Research Article Characterization of volatile compounds of Albertisia papuana Becc root extracts and cytotoxic activity in breast cancer cell line T47D Elizabeth BE Kristiani1*, Laurentius H Nugroho2, Sukarti Moeljopawiro2 and Sitarina Widyarini3 1 2 3 Faculty of Biology,Universitas Kristen Satya Wacana, Salatiga, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia *For correspondence: Email: betty@staff.uksw.edu; Tel: +62 298 321212; Fax: +62 298 321433 Received: 8 December 2015 Revised accepted: 28 April 2016 Abstract Purpose: To evaluate the cytotoxic activity of chloroform and water root extracts of Albertisia papuana Becc. on T47D cell line and identify the volatile compounds of the extracts. Methods: The plant roots were extracted with chloroform and water using maceration and boiling methods, respectively. The cytotoxicity of the extracts on T47D were determined using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Doxorubicin was used as reference drug in the cytotoxicity test while Probit analysis was used to calculate the Median Growth Inhibitory Concentration IC50 of the extracts. The volatile compounds in the chloroform and water root extracts were analyzed using Gas Chromatography-Mass Spectrophotometry GC-MS. Results: The IC50 of the chloroform and water extracts were 28.0 ± 6.0 and 88.0 ± 5.5 µg/mL, respectively whereas that of doxorubicin was 8.5 ± 0.1 µg/mL. GC-MS results showed that there were 46 compounds in the chloroform extract, out of which the five major components are ethyl linoleate (49.68 %), bicyclo (3.3.1) non-2-ene (29.29 %), ethyl palmitate (5.06 %), palmitic acid (3.67 %) and ethyl heptadecanoate (1.57 %).The water extract consisted of three compounds, butanoic acid (15.58 %); methyl cycloheptane (3.45 %), and methyl 2-O-methylpentofuranoside (80.96 %). Conclusion: The chloroform root extract of A. papuana Becc. had a fairly potent anticancer activity against breast cancer cells and may be further developed as an anticancer agent. Its major components were fatty acids and fatty acid esters. Keywords: Albertisia papuana Becc., Cytotoxicity, Breast cancer, T47D cell lines, Methyl 2-Omethylpentofuranoside Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION Cancer is considered to be the most killer disease in the world and has become a serious problem for the society, both in the developed countries and developing countries. The ineffectiveness of cancer treatment and side effects from the use of current cancer drugs have encouraged the search of alternative cancer drugs from natural products. Many studies have been carried out to obtain new alternative drugs for cancer treatment. Plants which usually used as traditional medicines have been exploited as a source of active compounds with anticancer activity [1,2]. Albertisia papuana Becc., from the family of Menispermaceae, is recognized as a traditional Trop J Pharm Res, May 2016; 15(5): 959 Kristiani et al medicinal plant in Sumatra and Kalimantan, Indonesia [3]. Dayak, a tribe in Kalimantan, usually use the root of A. papuana Becc. for cancer treatment by boiling it. This plant demonstrated cytotoxic activity on HeLa cell [4]. Some plants from the genus of Albertisia also showed many pharmacological activities. Albertisia delagoensis showed anti-plasmodium and cytotoxic activities on breast cancer, blood cancer, and kidney. It contained alkaloid as bioactive compound [5]. Alkaloid from A. vilosa had antibacterial, antifungal, anti-plasmodium, and cytotoxic activities [6]. Alcoholic extract of A. laurifolia had antitumor activity [7]. Based on these reports, A. papuana Becc. could be a potential source in the discovery of bioactive compound for anticancer. Most of the active compounds were alkaloids. In this study, we evaluated the cytotoxic activity of the chloroform and water extracts of A. papuana Becc. root on T47D cell lines using MTT assay and identified the volatile compounds which might be the active compounds for anticancer activity. The volatile chemical contents were identified using GC-MS analysis. EXPERIMENTAL Materials Albertisia papuana Becc was collected from Dayak, East Kalimantan, Indonesia in April 2014. The plant was identified by Dr. Joeni Setijo Rahajoe, a taxonomist of Herbarium Bogoriense, Biology Research Center, Bogor, Indonesia. A voucher specimen (No. 001/2014/FBUKSW/KHT) was deposited in the herbarium of Laboratory of Primary Biology, Faculty of Biology, Universitas Kristen Satya Wacana, Salatiga, Indonesia. T47D cell line was obtained from Parasitology Laboratory, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. Preparation of root extract of A. papuana Becc. The roots of about 50 cm in height of A. papuana plants were cleaned using tap water and cut into small pieces. The sample was air-dried for at least a week then dried in an oven at 40 ᵒC for 5 h. The dried root was ground using a blender (Philip HR1538). The chloroform root extract (CE) was macerated using chloroform (1:5 w/v) and then soaked for 24 h. This procedure was repeated four times until the resultant supernatant became clear. The water root extract (WE) was prepared by the boiling method. The powdered sample was boiled with water (1:5 w/v) for five minutes. Each extract was filtered and then dried in a rotary evaporator (Rotavapor R114 Buchi) under vacuum (Eyela A-1000S) at 40 ᵒC. The crude extracts were stored at 4 ᵒC before use. Cytotoxicity assay The in vitro cytotoxicity of chloroform and water extracts and doxorubicin on T47D were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide(MTT) assay with a slight modification. We used SDS instead of DMSO to stop the formation of formazan crystal. After that, the plate was incubated overnight without rotation on shaker. Briefly, an aliquot of 100 µl cell suspension (±1 x 104 T47D cells) was loaded into each well of 96-well plate and incubated for 24 h at 37°C in a 5 % CO2 incubator (Heraeus).The various concentrations of treatments (extracts 0-500 μg/mL and doxorubicin 0-100 μg/mL) were added to each well and then incubated for 24 h at the same condition. Each concentration tested was in triplicates. At the end of treatment, the medium was removed and 10 µLMTT solution 5 mg/mL (Sigma) was added. The plate was incubated in the dark for 3 to 4 h. The reaction was stopped by the addition of 100 µL 10 % SDS solution in 0.01 N HCl (Sigma) and then incubated overnight at room temperature. The absorbance of each well was measured using ELISA reader (SLT 240 ATC) at 595 nm. IC50 values (mean ± SD) were calculated using Probit analysis (SPSS 16.0 for Windows). Identification of chemical compounds The chemical compounds of extracts were analyzed by GC-MS(Agilent GC 6890N 5975B MSD).The capillary column was Agilent 19091S433 model, HP-5MS 5 % Phenyl Methyl Siloxane. The oven temperature was programmed as follows: initial temperature at 100 ᵒC, initial time for 1.00 min, final temperature at 300 ᵒC for 10.0 min. The conditions of front inlet mode splitless were as follows: initial temperature at 300 ᵒC, pressure was 10.45 psi for CE and 9.32 psi for WE, purge flow was 50.0 ml/min, purge time for 0.0 min, total flow 53.8 ml/min, saver flow for 20.0 ml/min, saver time was 2.0 min, and carrier gas was Helium. The sample was dissolved in pure ethanol and injected using a split technique. Identification of components in sample used Wiley7Nist05.L database. Trop J Pharm Res, May 2016; 15(5): 960 Kristiani et al RESULTS The cytotoxic activity of both extracts on T47D cell lines are shown in Table 1. The cytotoxicity of CE (IC50, 28.0 µg/mL) was three-fold higher than that of WE (IC50, 88.0 µg/mL). Both extracts were less cytotoxic than doxorubicin hydrochloride (IC50, 8.5 µg/mL). Based on the GC-MS chromatogram, there were forty-six compounds in the chloroform root extract of A. papuana Becc. (Table 2). The five major components of this extract were ethyl linoleate (49.68 %), bicyclo (3.3.1) non-2-ene (29.29 %), ethyl palmitate (5.06 %), palmitic acid (3.67 %), ethyl heptadecanoate (1.57 %). The other components had a relative concentration less than 1 %. The GC-MS chromatograms of water root extract of A. papuana Becc. consisted of three compounds (Table 3). These were methyl 2-O-methylpentofuranoside (80.98 %), butanoic acid (15.58 %), and methyl cycloheptane (3.45 %). DISCUSSION Natural products are considered as potential sources for drugs in several human diseases including cancer [12]. Many anticancer agents are plant-based compounds. Albertisia papuana is one of the endogenous plants in Dayak, East Kalimantan. Dayak people usually use A. papuana Becc for cancer treatment by boiling the root of the plant using water. In this study, the difference in solvents caused the difference of yield rendemen and cytotoxic activity of the extracts. The cytotoxic activity of the water extract was three time lower than the chloroform extract. Some alkaloids have been reported as heat labile compounds, so it is very possible that the alkaloids in the water extract were decomposed during the water extraction process [9]. In vitro cytotoxicity screening is often used to select the potential medicinal properties of a matter. This method was used to determine the inhibition of the growth of cells caused by an anticancer agent. According to The National Cancer Institute (NCI) USA, the IC50 values of 30 µg/mL is the upper limit of the crude extract which is qualified for further purification [12], so the chloroform root extract was promising for further purification because its IC50 value was 28.0 mg/mL. On other hand, to extract the root of the plant by boiling with water was not recommended. It showed that the extraction method is one of the crucial steps to obtain the active compound which have medicinal properties. The medicinal properties of plants are caused by the presence of active compounds of therapeutic value. The active compounds can be extracted from plants by an extraction method with certain solvents. The traditional medicines usually use water as a solvent, but modern medicines use various organic solvents in order to exploit the various compounds in herbal medicines [10]. The use of chloroform would extract non polar compounds, whereas water would extract polar compounds. The majority of compounds from the chloroform extract were organic acids, a long chain of saturated and an unsaturated hydrocarbon, or triterpene and sesquiterpene [11-14]. Based on the GC-MS chromatogram, it seems that the anticancer activity of chloroform root extract of A. papuana Becc. might be because of the action of the major compounds of this extract such asethyl linoleate, bicyclo (3.3.1) non-2-ene, ethyl palmitate, palmitic acid, and ethyl heptadecanoate. It has been reported that some of these compounds were also found in chloroform leaf extract of Finlaysonia obovata [12] and Acacia nilotica L [11]. Ethyl linoleate is a derivative of linoleic acid (LA). Various studies have reported that has biological beneficial effects, including anticancer activity. LA inhibits the initiation, promotion, and progression phases in mammary tumors [14].In addition, LA showed anticancer activity [16], especially by its antiproliferative activity [17] and by inducing apoptosis in breast cancer cells [18].LA was demonstrated to not only inhibit the growth of hepatoma cell but also induce apoptosis in colorectal cell line [19,20]. A study of human leukemic cells MOLT-4 showed that palmitic acid inhibited DNA topoisomerase I and induced apoptosis [20], however in human lung adenocarcinoma cell line A549, Table 1: Cytotoxicity ofA. papuana Becc. chloroform and water root extracts on T47D cell line Plant (part) Root Root - Solvent Chloroform Water - Code CE WE Doxorubicin (DX) IC50 (µg/mL) 28.0 ± 6.0 88.0 ± 5.5 8.5 ± 0.1 Trop J Pharm Res, May 2016; 15(5): 961 Kristiani et al Table 2: Compounds in chloroform root extract of A. papuana Becc analyzed using GC-MS No. 1 2 3 4 5 Retention time 5.949 6.053 6.164 8.069 8.179 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 8.589 8.706 9.784 8.920 9.044 10.578 10.643 11.039 11.215 11.345 11.429 11.657 12.684 13.250 13.497 13.653 13.822 14.394 14.576 15.323 15.570 15.830 15.882 15.999 16.136 16.357 16.981 17.033 17.189 17.351 17.644 17.988 18.463 39 40 41 42 43 44 45 46 18.749 19.484 20.199 20.888 21.577 22.006 24.398 25.094 Compound Formula C11H22 C7H10O2 C11H22 C14H30 C15H22O Mol wt 154 154 128 198 218 Area (%) 0.17 0.15 0.26 0.07 0.06 7-Methyl-2-decene 2-Acetylcyclopentanone 4-Methyl-2-decene 2,3,5,8-Tetramethyldecane 2,6-Di-tert-butyl-4-methylene-2,5-cyclo hexadiene-1one 2,4-Bis(1,1-Dimethylethyl)-phenol 1-Cyclopentylethanone 1-(Dodecyloxy)ethanol 1,2,4-Trimethylcyclohexane 1,1’-Oxybis decane n-Heptadecane 2,6,11,15-Tetramethyl hexadecane 1,3,5-Trimethylcyclohexane Tetratetracontane 1,2,4-Trimethylcyclohexane 1-Bromohexadecane n-Octadecane Nonadecane Hexadecenoic acid Palmitic acid Ethyl palmitate Diethylmethylborane Heptadecanoic acid Ethyl heptadecanoate Ethyl linoleate Bicyclo (3.3.1) non-2-ene (Cyclohex-2-enyl)acetic acid Methyl-6-octadecynoate (Cyclohex-2-enyl) acetic acid 2-Methyl-Z,Z-3,13-octadecadienol Ethyl nonadecanoate 2-Methylene-cyclododecanone Tetradec-13-en-11-yn-1-ol Ethyl heptadecanoate 9,12,15-Octadecatrienoic acid 2-Methyl-Z,Z-3,13-octadecadienol 1-Methyltridecyl methoxyacetate 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester 1-Chlorooctadecane Eicosane Ethyl palmitate Ethyl heptadecanoate 7h-dibenzo-c,g-carbazole N-(3-methoxyphenyl)-2,2-dimethylpropanamide Beta-stigmasterol Gamma-sitosterol C14H22O C7H12O C14H30O2 C9H18 C20H42O C17H36 C20H42 C9H18 C44H90 C9H18 C16H33Br C18H38 C19H40 C16H30O C16H32O2 C18H36O2 C5H13B C17H34O2 C19H38O2 C20H36O2 C9H14 C8H1202 C19H36O2 C8H1202 C19H36O C21H42O2 C13H22O C14H24O C19H38O2 C18H30O2 C19H36O C17H34O3 C16H22O4 206 112 230 126 298 240 282 126 619 126 305 254 268 254 256 284 84 270 298 308 122 140 296 140 280 326 194 208 298 278 280 286 278 0.42 0.11 0.10 0.13 0.11 0.10 0.10 0.07 0.25 0.36 0.18 0.36 0.25 0.44 3.67 5.06 0.13 0.35 0.58 49.68 29.29 0.36 0.43 0.42 0.25 0.55 0.80 0.52 1.57 0.39 0.18 0.21 0.13 C18H37Cl C20H42 C18H36O2 C19H38O2 C20H13N C12H17NO2 C29H48O C29H50O 288 282 284 298 281 207 412 414 0.22 0.16 0.29 0.22 0.20 0.21 0.18 0.19 Mol wt 88 112 178 Content (%) 15.58 3.45 80.96 Table 3: Compounds in water root extract of A. papuana Becc. analyzed using GC-MS No. 1 2 3 Retention time 14.524 14.758 16.513 Compound Formula Butanoic acid Methylcycloheptane Methyl 2-O-methylpentofuranoside C4H8O2 C8H16 C7H114O5 palmitic acid only inhibited DNA topoisomerase I without inducing apoptosis [21]. Palmitic acid, an active compound from Marthasterias glacialis L. showed apoptotic activity in neuroblastoma cell line by a ceramideindependent mechanism [22].In this study, a minor compound of water root extract of A. papuana Becc., butanoic acid was reported to have anticancer activity. Previous studies Trop J Pharm Res, May 2016; 15(5): 962 Kristiani et al demonstrate that butyric acid induced apoptosis in some cancers cells [23]. According to this study, the volatile compounds in both of the extracts were had anticancer activity. The chloroform extract contains forty six compounds, some of which have been known to have cytotoxic activity. While, water extract contains three compounds and only one compound has cytotoxic activity. Therefore, the water extract was less toxic than the chloroform extract. CONCLUSION The chloroform root extract of A. papuana Becc. has a fairly potent anticancer activity with some promise. 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