Indo. J. Chem.

, 2007, 7 (1), 93 - 96 93

IDENTIFICATION OF THE ISOLATED COMPOUNDS FROM

Zingiber amaricans BL. RHIZOME

Sugeng Riyanto
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gadjah Mada University,
Yogyakarta, Indonesia 55281

Received 6 December 2006; Accepted 16 January 2007

ABSTRACT

Five extracts were obtained from extraction of rhizomes of Zingiber amaricans. Hexane, dichloromethane and
methanol extracts were obtained by maceration, while dichloromethane and acetone extracts the resulted of soxhlet
extraction. By column chromatography technique 2,6,9-humulantrien-9-one (zerumbone) was isolated as the major
constituent of the hexane, dichloromethane and methanol extracts. The minor constituents were phytosterol mixtures
isolated from hexane and dichloromethane extracts. The mixtures consisted cholesterol, campesterol, stigmasterol
and β-sitosterol. The structure elucidations of zerumbone was confirmed by spectroscopic method, whereas the
phytosterol mixtures was identified by gas chromatography-mass.

Keywords: zerumbone, phytosterol, zingiber amaricans, spectroscopy

INTRODUCTION Masuda et al. also studied Z. zerumbet collected

in Ryukyus, Japan [5]. Three new acetylated and one
Zingiber amaricans Bl.(Zingiberaceae) is locally known kaempferol glycosides were isolated from the
known in Central Java as “Lempuyang emprit”. It is fresh rhizomes. The molecular structures of the
distributed throughout Indonesia and resemble with aglycone were established as 3-O-(2-O-acetyl-α-L-
Zingiber zerumbet which is locally named “Lempuyang rhamnopyranoside), 3-O-(3-O-acetyl-α-L-rhamno-
Gajah”. Both plants are traditionally used as “jamu” and pyranoside), 3-O-(4-O-acetyl-α-L-rhamnopyranoside)
its can substitute each other. The differences between and 3-O-α-L-rhamno-pyranoside .
them are the size each parts of the plant, Zingiber Zerumbone was also isolated from rhizomes of Z.
amaricans is slightly smaller than Zingiber zerumbet and zerumbet collected from Thailand by Murakami et al.
the rhizomes of the former are harder and darker [1] [6]. The methanol extract was partitioned with CHCl3,
The juice of the fresh rhizome of Zingiber and the CHCl3 soluble fraction was subjected to silica
amaricans was used as an appetizer [1], and the juice gel column chromatography and eluted with EtOAc/n-
was constituent of beverage “Cabepuyang”. There was hexane mixtures.
no other publication on Zingiber amaricans but Zingiber This research aim to identify the constituents of Z.
zerumbet continued to become the subject for further amaricans rhizome because of the compounds content
investigations. hold the principal role on the foods or beverages safety
In 1980, Matthes at al. isolated one new and five if the plants are used as their constituents.
known compounds from Zingiber zerumbet rhizomes {2],
all of which showed cytotoxic activity on neoplasmatic rat EXPERIMENTAL SECTION
liver cells cultured strain. The molecular structure of the
new compound was established by spectral data and Material
chemical evidences as kaempferol-3-α-L-(3”,4”-O- Zingiber amaricans was purchased from central
diacetyl) rhamnopyranoside or 3”,4”-O-diacetyl afzelin. market Beringharjo, Yogyakarta, identified in
The known compounds were zerumbone and Pharmaceutical Biology Department, University of
zerumbone epoxide, whereas diferuloylmethane, Gadjah Mada and the voucher specimen was
feruloyl-p-coumaroylmethane and di-p-coumaroyl- deposited in the herbarium of that department.
methane have not previously been reported in Zingiber
zerumbet, but are known in Curcuma species. Instruments
The aqueous extract of Z. zerumbet showed the Melting points were determined on Kohfler
bronchodilator effects on isolated tracheal and ileum of melting points apparatus XSP-12 Model 500X
Guinea pig and antiinflamatory effect on mice equipped with microscope and were uncorrected.
experiment [3,4]. On the continuing study Asmawi et al. Ultraviolet spectra were recorded on Shimadzu UV-VIS
reported that the water extract of Z. zerumbet showed 160 in absolute ethanol or methanol. The IR spectra
the antifertility on the female, Sprague-Dawley rats. were recorded using KBr mini disc on Perkin Elmer

Sugeng Riyanto
94 Indo. J. Chem., 2007, 7 (1), 93 - 96

FTIR spectrophotometer model 1725X. Mass spectra Shimadzu GCMS-QP5050 mass spectrometer. A fused
were recorded on an AE1-MS 12 spectrometer with silica capillary column BPX-5 ( 30 m x 0.25 mm x 0.25
ionisation induced by electron impact at 70 eV. Proton mm), ionisation induced by electron impact at 70 eV
Nuclear Magnetic Resonance spectra were recorded on and ion source temperature of 170oC were applied.
either a JEOL FTNMR (600 MHz) or a Bruker DRX-500
(500 MHz) spectrometer with tetramethylsilane (TMS) as Extraction and Fractionation of Extract of
an internal standard. 13C-Nuclear Magnetic Resonance Rhizomes of Zingiber amaricans
spectra with off resonance decoupling and DEPT The fresh rhizomes was sliced thinly, dried at the
experiments were determined with either a JEOL room temperature and ground into powdered form. The
FTNMR or a Bruker DRX-500 spectrometer operating at sample (1.5 kg) was subsequently extracted with
150 and 125 MHz, respectively. The solvent used was hexane, dichloromethane and methanol. Follow-up
CDCl3 unless otherwise specified. The 1H-1H COSY, procedures gave corresponding hexane extract (33.1
NOESY, HMQC and HMBC NMR spectra were obtained g), dichloromethane extract (32.4 g) and methanol
with the usual pulse sequences. extract (43.3 g). Another portion of the sample (1.1 kg)
was extracted using Soxhlet apparatus. The solvent
Procedure used was dichloromethane, which was followed by
acetone. The extractions produced 64.8 g and 6.3 g of
Chromatography analysis extracts, respectively.
Vacuum column chromatography was performed
using Silica gel 60 PF254 MERCK 7749 as absorbent. Isolation and Spectroscopic Analysis of
Length and diameter of column are 40 cm and 8 cm, Compounds from Hexane Extract
respectively. The ratio of sample to the stationary phase The hexane extract (5 g) was fractionated using
was 1 part sample to 20 parts of stationary phase. The vacuum column chromatography from which collected
solvents or the mobile phases used were combination of 35 fractions. Fractions 10-12 were combined to give
hexane, petroleum ether, chloroform, dichloromethane, residue which then afforded compound A (2.5 g) while
ethyl acetate, acetone, and methanol. Water pump was fractions 26-27 give phytosterol mixtures (65 mg).
applied during the elution to absorb the pressure in the Follow-up procedure on dichloromethane and methanol
receiver flask. The gravity column chromatography was extract also resulted compound A, together with some
also used to further separate the fractions obtained from oily mixtures and unidentified in organic compound.
vacuum column chromatography. The column was Similar fractionation on dichloromethane fraction
prepared using silica gel 60 (70-230 mesh ASTM), obtained from Soxhlet apparatus extraction afforded
MERCK 7734. The diameter and length of the applied only compound A and phytosterol mixtures. In contrast,
column depend on the weight of sample. Generally, the further work on acetone extract did not lead to
ratio of sample to silica gel was 1 part of sample to 40 identification of any compounds.
part of silica gel. On the special cases, if the amounts of
samples are minimum, silica gel (230-400 mesh ASTM) RESULT AND DISCUSSION
MERCK 9385 was used.
Thin Layer chromatography (TLC) was used to Compound A appeared as colourless crystals
detect the various components present in crude extract with melting point 60-62oC. Infrared spectrum showed
and fractions collected during elution or to examine the peak at 3025 cm-1 indicating the olefinic C-H stretching,
impurities of the isolated compounds. TLC plastic sheets and peaks at 2964, 2924 and 2864 cm-1 correspond to
pre-coated with Silica gel 60 F254 (0.2 mm thickness, aliphatic C-H. A strong peak at 1653 cm-1 attributed to
20x20 cm) were used. The samples were spotted onto C=O group, while weak peaks at 1456 and 1264 cm-1
the proper width of TLC plate using glass capillary tubes were signal of C=C group and C-O bond, respectively.
and then developed in the chromatographic tank The mass spectrum showed molecular ion at m/z 218
containing saturated solution of the mobile phase. The which corresponds to the molecular formula of
spots on the plates were examined under UV lamp at λ C15H22O. 1H-NMR spectrum showed four singlets each
254 and 366 nm or kept in iodine tank. Analytical gas integrated to three protons at δ 1.07, 1.20, 1.54 and δ
chromatography (GC) were carried out on a Shimadzu 1.80 attributed to the four methyl groups, of which two
model GC-16A gas-chromatograph fitted with CBP-1 groups attached to C-3 and C-7, while other two groups
capillary column of dimension 30 m x 0.25 mm x 0.25 attached to C-11. The two doublets (J = 16.4 Hz)
μm and Helium was used as carrier gas. The GC oven observed at δ 1.90 and 2.35 were due to two
temperature was programmed from 80-265oC at nonequivalent protons of H-1, while methylene protons
3oC/minute with an initial hold 2 minute and final hold 10 of H-4 and H-5 exhibited multiplets around δ 2.19-2.36.
minute. GC-MS analyses were performed on the gas A doublet of doublet (J=16.4, 4.0 Hz) at δ 5.25 was due
chromatograph (Shimadzu GC-17A) coupled to a to the signal of H-2, which coupled to nonequivalent

Sugeng Riyanto
 Indo. J. Chem., 2007, 7 (1), 93 - 96 95

13

O
5 7
4 6 8
3 9
2 10
12 HO HO
1 11 B C
14
15
A

HO HO

D E
Table 1. NMR spectroscopic data of compound A
1 13
C H-NMR C-NMR DEPT HMQC HMBC
number (ppm)
1 1.90, d.(J=13.2Hz) 42.4 CH H-1 H-14, H-15, H-10
2.35, d.(J=13.2Hz)
2 5.25, dd.(J=16.4,4.0Hz) 125.0 CH H-2 H-1, H-4, H-12
3 - 136.3 C - H-4, H-5, H-12
4 2.19-2.36, m 39.5 CH2 H-4 H-2,H-12
5 2.26, m 24.4 CH2 H-5 H-4
6 6.02, t.(J=13.2Hz) 148.8 CH H-6 H-13
7 - 137.9 C - H-6, H-13
8 - 204.4 C - H-9, H-10, H-13
9 5.97, d.(J=16.4Hz) 127.2 CH H-9 H-10
10 5.86, d.(J=16.4Hz) 160.8 CH H-10 H-9,H-14, H-15
11 - 37.9 C - H-9,H-14, H-15
12 1.54, s. 15.2 CH3 H-12 H-2,H-4
13 1.80, s. 11.8 CH3 H-13 -
14* 1.20, s. 24.2 CH3 H-14 H-15
15* 1.07, s. 29.4 CH3 H-15 H-14

Table 2. Chemical constituents of phytosterol mixtures

Number Retention Molecular Molecular Composition Compound
peaks Time (min) Weight Formula (%)
1 23.74 386 C27H46O 1.12 Cholesterol (E)
2 25.64 400 C28H48O 12.38 Campesterol (D)
3 26.47 412 C29H48O 30.16 Stigmasterol (B)
4 27.98 414 C29H50O 56.28 β-Sitosterol (C)

two protons of H-1. The triplet (J=13.2 Hz) observed at carbon signals at δ 39.5 (C-4) and 24.4 (C-5). The
δ 6.02 was due to H-6 which overlapped with doublet crosspeaks were also shown by signals of methyl
(J=16.4 Hz) at δ 5.97 attributed to H-9 and the later protons of C-13, C-12, C-14 and C-15 with their
correlated with H-10 to give another doublet (J=16.4 Hz) respective carbon signals.
at δ 5.87. Some of the long-range C-H correlations of the
13
C-NMR spectrum (BCM) exhibited fifteen signals compound are displayed by HMBC spectrum. The
for each of fifteen carbons present in the molecule. The positions of the methyl groups were confirmed by
DEPT spectrum showed that four methine carbons correlations of methyl proton signal at δ 1.54 (C-12)
appeared at124.9, 127.2, 148.8 ppm and 160.7 ppm. with carbon signal at δ 136.3 (C-3), methyl proton
HMQC spectrum confirmed the assignments of 1H and signal at δ 1.80 (C-13) with carbon signal at δ 137.9 (C-
13
C-NMR data. The spectrum revealed that resonances 7), and methyl proton signal at δ 1.20/1.07 (C-14/C-15)
for H-1 protons at δ 2.35 and 1.90 correlated with carbon with carbon signal at δ 37.9 (C-11). The presence of
signal for C-1 (δ 42.4). The signal of methylene protons carbonyl group was also confirmed by correlation of
of H-4 and H-5 (δ 2.20-2.50) showed correlation with carbon peak at δ 204.4 (C-8) with proton signals at δ

Sugeng Riyanto
96 Indo. J. Chem., 2007, 7 (1), 93 - 96

5.97 (H-9) and 1.80 (13-CH3). The protons signal at CONCLUSION

around δ 2.19-2.36 (H-4) showed crosspeaks with
carbon signals at δ 24.4 (C-5), δ 136.3 (C-3), δ 15.2 (C- The major constituent of Z. amaricans rhizome
12), and δ 125.0 (C-2), while proton signal at δ 2.35 (H- was 2,6,9-humulatrien-8-one (zerumbone), and the
1) showed correlation with carbon signals at δ 125.0 (C- minor constituent was a mixture of phytosterol
2) and δ 136.3 (C-3). Other HMBC correlations and other consisted β-sitosterol, cholesterol, campesterol and
NMR data are summarized in Table 1. stigmasterol.
Based on the spectroscopic data obtained and
comparison to the data reported previously [2], ACKNOWLEDGEMENT
compound A was elucidated as 2,6,9-humulatrien-8-one
(zerumbone). The mass fragmentation pattern of the I would like to express my appreciation to Ass
compound supported the assignment of the Prof. Mohd Aspollah Sukari and Prof. Mawardi
sesquiterpene. Rahmani as the supervisors when I studied at the
The phytosterol mixtures was washed with acetone Faculty of Science and Environmental Studies,
and followed by recrystallization using hexane to give University Putra Malaysia.
needles shape crystals with melting point of 138-140 oC.
GC – MS analysis showed that the substance consisted REFFERENCES
four component. The major components were
stigmasterol (B)(30.16%), β-sitosterol (C) (56.28%), 1. Heyne,K. 1987, Tumbuhan Berguna Indonesia,
campesterol (D) (12.38%) and cholesterol(E) (1.12%). Jilid I, Badan Litbang Kehutanan, Jakarta.
Their retention time were obtained from the 2. Matthes, H.W.D., Luu, B. and Ourisson, G., 1980,
chromatogram and their structures were identified based Phytochem., 19, 2643-2650.
on the similarities of their mass spectra patterns with the 3. Asmawi, M.Z., Sadikun, A., Jiu, L.L., and Rahman,
library data in the GC-MS system. The results of GC-MS A.A. 1987, The Bronchodilator and Antiinflamatory
analysis are listed in Table 2. β-sitosterol (C); mass effects of Zingiber zerumbet (Lo.) Sm Rhizome,
spectrum m/z (% intensity) : 412(M+, 81), 400(25), eds. Mohd. Shohimi Mustapha, Proceeding, of
396(20), 369(18), 351(20), 314(28), 300(45), 271(60), Seventh Annual Scientific Meeting of Maspet, 12-
255(65), 231(18), 213(36), 163(31), 159(51), 145(55), 13 June 1987
107(58), 81(80), 55(100). Stigmasterol (B); mass 4. Asmawi, M.Z. and Pauzi, M.A. 1989. Antifertility
spectrum m/z (% intensity): 412(M+, 81), 400(25), effect of Lempuyang Gajah (Zingiber zerumbet
396(20), 369(18), 351(20), 314(28), 300(45), 271(60), Lo.), Eight Scientific Meeting of the Malaysian
255(65), 231(18), 213(36), 163(31), 159(51), 145(55), Society of Pharmacology and Physiology 21-22th
107(58), 81(80), 55(100). Campesterol (D); mass July 1989,p 26. Kualalumpur.
spectrum m/z (% intensity) : 400(M+,53 ), 382 (26), 367 5. Masuda, T., Jitoe, A., Kato, S. and Nakatani, N.
(23), 315 (47 ), 289 (48 ), 273 (27 ), 255 (33), 231 (31), 1991, Phytochem. 30(7), 2391-2392.
213 (60), 173 ( 33), 159 (63),145 (84), 133 (53), 119 6. Murakami, A., Takahashi, M., Jiwajinda, S.,
(64), 105 (98), 91 (86), 81 (82), 55(100). Cholesterol (E); Koshimizu, K., and Ohigashi, H. 1999, Biosci.
mass spectrum m/z (% intensity) : 386 (M+,47), 368 (23), Biotechnol. Biochem, 63 (10),1811-1812.
353 (22) , 301 (46), 275 (59), 255 (29), 231 (23), 213
(47), 159 (59), 145 (81), 119 (64) , 105 (97), 91 (91), 55
(100).

Sugeng Riyanto