Majalah Farmasi Indonesia 11 (3).142-149, 2000 THE CONTENTS OF VOLATILE OIL ISOLATED FROM Kaempferia galanga RHIZOMES. MASS SPECTROSCOPIC APPROACH KANDUNGAN MINYAK ATSIRI YANG DIISOLASI DARI UMBI Kaempferia gaianga PENDEKATAN SECARA SPEKTROMETRIK MASSA Remo S. Sudibyo Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gadjah Mada University, Yogyakarta. Abstract Gas-chromatographic-mass-spectroscopic (GC-MS) analysis was carried out to volatile oil isolated from Kaempferia galanga rhizomes. It was found that eight components comprised the oil, in which five of them (compounds 3, 4, 6, 7, and 8 peaks) were the main components. The mass-spectroscopic analysis resulted that the four (compounds 3, 4, 6 and 8 peaks) of the five compounds were: P-phellandrene, « -terpineot, ethyl cinnamate, and dikydro-B-sesquiphellandrene respectively; whilst compound 7 was probably isododecylmethyl ketone or 4-butyl menthol, Compounds 1 and 2 were assumed as B-phellandrene derivatives, while compound 5 needs to be structurally elucidated using NMR-spectroscopic method in further. Keywords: GC-MS, Kaempferia galanga, p-phellandrene, a-terpineol, ethyl cinnamate, ditydro-f -sesquiphellandrene Abstrak Dilakukan analisig kandungan minyak atsiri dari rizom Kaempferia galanga secara khromatografi gas- spektroskopi massa (KG-SM). Didapatkan delapan komponen penyusun minyak atsiri tersebut, lima diantaranya (Senyawa dengan puncak 3, 4, 6, 7, dan 8) merupakan komponen pokok, Dari hasil analisis spektroskopi massa dinyatakan bahwa empal (senyawa dengan 3, 4, 6, dan 8) dari lima senyawa tersebut berturut-turut adalah; §- felandren, c-terpineol, etil sinnamat dan dihidroseskifelandren; sedangken senyawa 7 diduga isododesilmeti keton, atau derivat 4-butilmentol. Senyawal dan 2 diduga derivat B-felandren; sedangkan struktur senyawa 5 perlu dielusidasi lebih lanjut dengan metoda NMR-spektroskopi. Kata kunci: KG-SM, Kaempferia galanga, B-felandren, a-terpineol, et! sinnamat, dihidro-B-ses- Kifelandren. INTRODUCTION In the course of finding anticancer agents, some Yogyakarta local rhizomes known as kunir putih group had been investigated. Several species are grouped into the kwuir putih, such as: Curcuma zeodaria, C. mangga, Kaempferia rotunda and K. galanga. The comparison of the cytotoxic activity on the cancer fines, the secondary metabolite contents, protein content, and volatile oil contents of those species have been carried out by the group of Aunir putih, in TUC- Biotechnology-GMU, Yogyakarta. The analysis of the volatile oil contents using Gas Chromatographic-Mass Spectroscopic method (GC-MS) of K. galanga will be described in this paper. METHODS Material: K, galanga rhizomes were collected from Yogyakarta market. Instrument: Gas Chromatographic-Mass Spectrometer (GC-MS) QP 5000 (Shimadzu. Method: The isolation of volatile oil: Pre-washed rhizomes of K. galanga were chopped and steam distillated. The volatile oil obtained was collected. This volatile oil was run on the GC-MS to analyze its main Majalah Farmasi Indonesia 11(3),2000 142The Content of Volatile Oit coniponents. The GC-MS method: The GC-column uséd was 30 meter of DBI using first temperature 40°C for 5 tainutes and second temperature 280°C for 30 minutes, and 10 Kpa Helium as the carrier. The temperature of the injector and the detector was 280°C, while the ionizing chamber of the MS was using Electron Impact (ED) of 70 eV, The GC-MS spectra resulted was analyzed. RESULTS AND DISCUSSION ‘The gas chromatogram of the volatile oil isolated from X. galanga showed 8 peaks of retention times as shown in figure 1 and table 1. These peaks showed that there were 8 components (compounds 1 to 8) contained in the volatile oil, in which compounds 3, 4, 6, 7, and 8-~were the main components, while the rest (compounds 1, 2, and 5) were the small components, Figure 1.: The gas chronatogram of volatile oil isolated from Kaempferia galanga chizomes. Table I. The gas chromatogram-peaks, retention times, molecular ions and fragment ions of 8 compounds contained in the volatile oil of Kaempferia galanga, Lome. Petention | Contain | Mol. fon ‘Fragment Tons Substance io,__ [Time (Sec) | (%) (ve) (m/z) 1 8505 [2.09 136 _ | 121, 107, 53 Gp) 79, 67, 53, 41, B-phellandrene derivative? 2 9.160 | 2.92 136 | 121, 93 (bp), 79, 69, 53, 41. B-phellandrene . derivative? oa 3 9989 [1294 (136 | 121, 105, 93 op), 77, 67, 53, 41. B-phellandrene : 4 10.338 | 21.04 154} 139, 125, 108, 96,93, 81, 71, 55,43 (bp). | a-Terpineol 5 12945 | 291 139 | 136, 121, 110, 95 (bp), 67, 55, 41. 2 6 17431 | 21.76 ‘176 | 158, 148, 131 (bp), 103, 91,77, 51. Ethyl cinnamate 7 18.135 | 625 212 | 184, 127, 113, 99, 85, 71, 57 (bp),43. | Isododecylmethylketone or 41 ? 8 21.017 | 30.17 206 | 177, 161 (bp), 147, 134, 118, 103, 89, 77, teaeoee 63,51. phellandrene ‘Compound 3, which had molecular ion m/z 136 and fragment ions as shown in Table 1, most probably as f-phellandrene (Figure 2). This molecular ion under an o-cleavage produced a fragment ion m/z 121, which Mojalah Farmasi Indonesia 11 (3), 2000 143Sudibyo, R. then underwent a 1,2+H shift rearrangement resulted a tropylium-like ion (McLafferty, 1980; Silverstein ey 1991; Williams and Fleming, 1995; De Hoffmann ef al., 1996). a, Hs cu, 1” EL -cuy of Mev © cleavage « cH B ms cH en, uy cr, ° (8-Phactlandrene Mt 16 me idl HC SCH, | -CHy O- oH ry o Owes Figure 2.A Mass-spectrogram of compound 3 contained in K galanga volatile oil. B, The fragmentation pattem of compound 3. By releasing a neutral molecule of ethylene from the tropylium-like ion resulted a fragment ion cyclopentene carbonium (m/z 93) which was the base peak. This last ion then again could refease neutl molecules: methane or methylenecyclopropene. By releasing the methane the cyclopentene carbonium would produce fragment ion of cyclopentadiene carbonium (m/z 77); while the releasing methylenecyclopropene would produce fragment ion of cyclopropane carbonium (m/z 41). Majalah Farmasi indonesat 1 1(3),2000The Content of Volatile Oi! ~ Data Mass Peak : 47, Ret Time 10, 342 Scan8 :762-B.G, Scan 8: 822 Base Peak 43.00_( 11095683) ‘OH Hen acto, 4 2 mis 108 P { kon of 7 Figure 3. A. Mass-spectrogram of compound 4 contained in K. galanga volatile oil. B. The fragmentation pattem of compound 4. Majalah Farmast Indonesia 11 (3), 2000 145Sudibyo, R. The tropylium-like ion could also release either a methane 10 give a fragment ion of m/t 105 oF release g methylene carbene to give a ftagment ion of m/s 107. It is known that tropylium ion easily breaks up and releases an acetylene (De Hoffmann ef al., 1996). This case occurred to the tropylium-like ion by releasing carbene it produced cyclopentane carbonium with m/= 69. Based on the pattem of fragmentation it was confirmed that the molecular ion (m/z 136) was B- phellandrene (Figure 2). Similar fragmentation pattem of j. phellandrene of was indicated in the mass-spectrogram of Curcuma mangga volatile oil (Sudibyo, 2001), Compound 4, which had molecular ion méz 1$4 and fragment ions as shown in Table 1, was assigned as a-terpineol (Figure 3). The patter of the fragmentation was found mostly to follow the Karni-Mandelbaum Rule (KMR) (MeLafferty, 1982; Williams and Fleming, 1995), ct-Cleavage of the molecular ion (M* 154) resulted in an odd fragment ion of m/c 139. This odd fragment ion, following the KMR, then released a methylene carbene to produce another even fragment ion of m/z 125. Unkmown Spectrum Data : Keneur B DOL ‘Mass Peak =33, Ret. Time 17.433 ‘Sean 8 31613 B.G, Sean 8; 3020 Base Peak; 231.10 (12247507. 2 a0 as L oa pt ase L he Teo 10 6 ar A to: 9° If &, och ce Soa cr wie Ethyl cingumae (base petky = | B Figure 4.: A. Mass-spectrogram of compound 6 contained in K. galanga volatile oil B. The fragmentation pattem of compound 6. The later fragment ion then underwent twice single-bond breaking released a radical hydroxy! and resulted an odd fragment ion m/s 108. After breaking a radical methyl, this ion yielded even fragment ion m/2 93. Beside that, the even fragment ion of m/: 125 again following the KMR released a methylene carbene to product another even fragment ion of m/z 111. This last even ion again underwent twice KMR resulted other evet fragments ions of subsequently m/z 97 and 71 by releasing methylene carbene and acetylene respectively. The even fragment ion m/z 111 could also release a water molecule and yielded fragment ion m/z 93. The fragment jon m/z 97 lost a radical hydrogen and yielded a fragment ion of m/z 96 that in further released respectively methylene carbene and radical hydrogen, and resulted subsequently fragment ions w/z 82 (not observed) and 81. Majalah Farmasi Indonesai 11(3).2000 46The Content of Volatile Oil ‘The fragment ions of m/z 59, 55 and 43 (the base peak) were respectively isopropanol, isobutene, and isopropane earboriums those could be derived from the breaking of the molecular ion, Base on the analysis of the fagmentation pattern, it was concluded that the motecular ion m/= 154 was a-terpineo! (Figure 3). Compound 6, which had molecular ion m/z 176 and fragment ions as shown in Table 1, was assigned as ashy] cinnamate (Figure 4). Releasing a radical ethoxy group via at-cleavage from the molecular ion produced gaement ion m= 131 which was the base peak. The fragment ion then released a neutral molecule of carbon monoxide to produce a fragment ion m/z 103. Twice loosing of acetylenes from the ion yielded fragment ions at iz 77 and 51 respectively. Fragment ions m/: 158 and 148 were formed directly from the molecular ion by releasing neutral molecules of water and carbon monoxide. It was concluded then that the molecular ion of m/z 116 was ethyl cinnamate. ‘Compound 7, which had molecular ion m/z 212 and fragment ions as shown in Table 1, was not easy to be assigned. However the end of the ffagmentation releasing mostly neutral molecules (seven times) with molecular weight of 14 that most probably was a methylene group. This indicated that the molecular ion was an Unknown Spectrum Dota: KencurB DOL Mass Peak =29, Ret Time 18.142 Scan8 — : 1698 B.G. Sean8: 2624 Bese Peak : $7.10 _ 6272639 st ici Cth cH 8 Isododecytmethylketone Mol. 1282 4-Burhylmenthol ‘MoLWe 212 Figure 5.: A. Mass-spectrogram of compound 7 contained in K. galanga volatile cil. B, The fragmentation pattem of compound 7. akane. In the first fragmentation, the ‘molecular ion releasing a molecule with molecular weight of 28 and yielded a fragment ion which then fragmented and released a molecular weight of 43. The first molecule released by the molecular ion could be a carbon monoxide, while the second molecule released was probably a propane. Base on those assumptions, the molecular ion might be an isoalkane ketone that most probably was isododecylmethyt ketone; or its cyclic derivative of 4-butylmenthol (Figure 5). To decide which one was true between these two, NMR-spectrometric analysis should be carried out to the pure Majaleh Farmast Indonesia 11 (3), 2000 147Some of its fragment ions (Table 1) were also 2 lager than those of B-sesquiphellandrene (Sudibyo, 2000). analyzing the fragmentation pattern and comparing it to that of B-sesquiphellandrene, the compound 8 ya, assigned as dihydro-B-sesquiphellandrene (Figure 6). Unknown Date Spectra : Kencu B DOL Mass Peak : $4, Ret Time 21.01792 Scan8 :2043 BG. Scan8: 2278 Base Peak : 161.10 : 11150405) Sudibyo, R. The molecular ion of compound 8 was m/z 206 that was 2 larger than that of B-sesquiphellandreng phellandrene ‘Mol We 206 -cH, Q= =O= 2 ‘ , on eu tos nev a ="CH ~icH, "e ne an, ne" Ae wou omyurp. sesqui- MI 206 ms 19 meiTT gr ms 8 mw be ave 138 . mi (base peaky | B Figure 6.: A. Mass-spectrogram of compound 8 conteined in K. galanga volatile oil, B. The fragmentation pattern of compound 8. Again the KMR occurred in the fragmentation process of compound 8. The even fragmention of m/z 191 following the KMR fragmented info another even fragment ion m/z 177 by releasing a neutral molecule, methylene carbene. The even ion resulted again following the rule released a methane and produced anothet even ion m/z 161 that was the base peak, This later ion once again followed the rule to produce even ion m/s 135 Majatah Farmasi Indonesai 11(3),2000 18The Content of Volatile Oil afer releasing acetylene. The last even ion then fragmented into ion m/s 134 and 118 after releasing 2 radical n and methane group respectively. Based on the fragmentation pattem, compound 8 was concluded 2s dinydro-B-sesquiphellandrene (Figure 6). The small compounds 1 and 2 had identical molecular ion and similar fragmentat-ion pattern to that of compound 3 (B-phellandrene, m/z 136), (Table 1). These two compounds could be the isomers of O- phellandrene; and their fragmentation pattems were proposed as shown in figure 7. Compound 5, that had lecular ion m 139, was difficult to be assigned from its mass-spectrogram. Other analytical method like .. NMR-spectroscopy is necessary to carry out to structurally elucidate this compound. CONCLUSION ‘There were five main compounds contained in volatile oil isolated from X. galanga, four of those were P-phellandrenea-terpineol, ethyl cinnamate, dihydro-B-sesqui-phellandrene respectively, whilst the other one ‘was probably isododecylmethylketone or 4-butylmenthol. ACKNOWLEDGEMENT The author thanks very much to Prof. Dr. Umar A. Jenie for his discussion and helping her on analyzing the mass-spectrogram data. The author really appreciated to Nurkhasanah for her technical assistance. REFERENCES De Hoffmann, E.; J. Charette; and V. Stroobant, 1996, Mass Spectrometry. Principles and Applications, John Wiley & Sons, Chichester-Singapore, Masson, Paris-Barcelone, 187. a MeLafferty, F.W., 1980, Interpretation of Mass Spectra (3"* Ed), University Science Books, Mill Valey- California, 45-74. Silverstein, RML, G.C. Bessler, and T.C. Mortil, 1991, Spectrometric Identification of Organic Compounds, 5 Ed., John Wiley and Sons Inc., New York, Singapore, 13-15. Sudibyo, RS., 2001, Gas Chromatographic-Mass Spectrometric Analysis of the Main Content of Volatile Oil Isolated from Curcuma mangga, Journal Mathemattc and Natural Sciences, (in Press). 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