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8 (2011)
The essential oils from the leaves and rhizomes of Alpinia pahangensis Ridl., collected from Pahang,
Peninsular Malaysia, were obtained by hydrodistillation, and their chemical compositions were
determined by GC and GC/MS analyses. The major components of the rhizome oil were g-selinene
(11.60%), b-pinene (10.87%), (E,E)-farnesyl acetate (8.65%), and a-terpineol (6.38%), while those of
the leaf oil were b-pinene (39.61%), a-pinene (7.55%), and limonene (4.89%). The investigation of the
antimicrobial activity of the essential oils using the broth microdilution technique revealed that the
rhizome oil of A. pahangensis inhibited five Staphylococcus aureus strains with minimum inhibitory
concentration (MIC) values between 0.08 and 0.31 mg/ml, and four selected fungi with MIC values
between 1.25 and 2.50 mg/ml.
Introduction. – Alpinia is the largest genus in the family Zingiberaceae, with over
250 species in tropical Asia. The rhizomes of several Alpinia species are used in the
traditional medicine and the preparation and flavoring of food in many Asian countries.
For example, the rhizomes of A. galanga are useful in the treatment of cough, asthma,
rheumatoid arthritis, obesity, and diabetes and as cardiac stimulant and carminative. It
is also used as an ingredient of aphrodisiac preparations [1].
A. pahangensis is an endemic wild ginger from the state of Pahang, Peninsular
Malaysia. To the knowledge of the authors, no chemical studies have been reported on
this species. Although there is no report on the use of its rhizomes and leaves, the fresh
fruits are eaten by the local villagers. A. pahangensis is a moderately sized, perennial
aromatic herb with linear-oblong shaped leaves and an inflorescence borne terminally
on each frond. The flowers are orchid-like with attractive crimson colored lips. This
currently not well known species is unexploited and distributed mainly in the lowlands
of selected areas in Pahang, Peninsular Malaysia.
Here, the chemical compositions of the essential oils from the leaves and rhizomes
of A. pahangensis and their antimicrobial properties are reported.
Figure. Chemical structures of some of the lesser known compounds identified in the leaf and rhizome
essential oils of Alpinia pahangensis
In various previous studies, 1,8-cineole has been identified as the marker compound
of Alpinia species. Indeed, it was the most abundant compound in the rhizome and leaf
oils of several Alpinia species, such as A. calcarata [4], A. galanga [3] [5], A. conchigera
[6 – 8], and A. zerumbet [9]. However, in the present study, this compound was only
detected in the rhizome oil with a low concentration of 0.63% (Table 1). Moreover, b-
pinene, which was revealed as the most or second most abundant component in the leaf
and rhizome oils of A. pahangensis, respectively, has also been reported to be the most
dominant compound in some other Alpinia species such as A. carinata [10], A. allughas
670 CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Table 1. Composition of the Leaf and Rhizome Essential Oils of Alpinia pahangensis
Table 1 (cont.)
Compound RIexp a ) RIref b ) Composition [%]
Leaf oil Rhizome oil
a-Maaliene 1528 – – 0.67
a-Calacorene (7) 1547 1546 – 0.22
b-Gurjunene 1605 1434 – 0.35
g-Selinene 1663 1493 – 11.60
Heptadecane 1669 1700 – 4.49
b-Bisabolol 1675 1675 – 0.60
Phytone (8) 1787 – 0.48 –
( E,E )-Farnesyl acetate 1834 1844 c ) 0.40 8.65
Phytol 1916 1943 0.39 –
Isophytol 1959 1948 2.37 –
Total 86.91 75.28
a
) RIexp : Experimental Kovats retention indices determined on a CBP-5 capillary column. b ) RIref :
Reference RIs from [2], except for ( E,E )-farnesyl acetate. c ) Reference RI for ( E,E )-farnesyl acetate
from [3].
[11], and A. speciosa [12]. Thus, it may be suggested that b-pinene may be a common
monoterpene in many of the Alpinia species.
The essential oils obtained were tested for their minimum inhibitory concentration
(MIC) using the broth microdilution assay against five selected strains of Staph-
ylococcus aureus, viz., SA2 (ATCC 25923), SA3 (ATCC 33591), SA7 (ATCC 700699),
VISA24, and VRSA156, and four selected fungi, namely, Candida albicans (ATCC
10231), C. glabrata (ATCC 64677), Microsporum canis (ATCC 36299), and Tricho-
phyton rubrum (ATCC 28188). The MIC values of the essential oils are listed in
Table 2.
Table 2. Minimum Inhibitory Concentrations ( MICs) of the Leaf and Rhizome Essential Oils of Alpinia
pahangensis Against Five Staphylococcus aureus Strains and Four Fungi
The rhizome oil of A. pahangensis showed a strong inhibitory activity, with MIC
values below 1.0 mg/ml, against all strains of S. aureus tested. Against the strains SA7
(MIC 0.31 mg/ml) and VISA24 (MIC 0.16 mg/ml) it revealed even lower MIC values than
those obtained for the antibiotic oxacillin (0.63 and 0.31 mg/ml, resp.). Moreover,
against the strain SA3, the same MIC value (0.16 mg/ml) was obtained for both the
rhizome oil and oxacillin. However, the rhizome oil showed only moderate activity
against all fungi tested, with MIC values in the range of 1.25 to 2.50 mg/ml. The pinene-
type monoterpene hydrocarbons, such as a-pinene and b-pinene, have been reported to
possess only slight activity against several microorganisms, such as Bacillus subtilis,
Brevibacterium linens, Enterobacter aerogenes, Lactobacillus plantarum, and Leuco-
nostoc cremoris [13]. Hence, the strong to moderate antimicrobial activity of the
rhizome oil in this study could be due to the presence of other major components or the
synergism of the minor components present.
On the other hand, except for the strong activity against the S. aureus strain
VRSA156 (MIC 0.63 mg/ml), the leaf oil showed only moderate activity against all other
S. aureus strains (MIC 1.25 – 2.50 mg/ml) and all fungi (MIC 2.50 mg/ml) tested. The lower
antimicrobial activity of the leaf oil compared to the rhizome oil may be due to the
absence of some compounds in the leaf oil that were present in the rhizome oil or vice
versa.
This project was funded by grants of the Dr. Siti Hasmah Mohd. Ali Chair and the Sciencefund (12-
02-03-2070). We thank Prof. Datin Dr. Sri Nurestri Abdul Malik, Mr. Ahmad Nazif Aziz, Mr. Lee Guan
Serm, Mr. Din Mohd. Nor, Mr. Abu Said Ahmad, and Mrs. Mazurah Mohd. Isa for their technical and
field assistance.
Experimental Part
Plant Material. The fresh leaves and rhizomes of Alpinia pahangensis were collected from Pahang
and authenticated by H. I. Voucher specimens were deposited with the Herbarium of the Chemistry
Department, University of Malaya (No. KU 001). The fresh samples of the leaves and rhizomes were
immediately washed, cut into small pieces, and air dried for 3 d. Then, the samples were grinded and
hydrodistilled for 8 h using a Clevenger-type apparatus.
GC Analysis. GC Analysis was performed with a Shimadzu GC-2010 apparatus equipped with a
flame ionization detector (FID) and a CBP-5 fused-silica cap. column (25 m 0.22 mm i.d., film
thickness 0.25 mm). The oven temp. was programmed isothermal at 608 for 10 min, rising from 60 to 2308
at 38/min, and then held isothermal at 2308 for 10 min; injector temp., 2508; detector temp., 2508; carrier
gas, He (linear velocity of 50 cm3/min). The samples were dissolved in hexane and injected in the split
mode.
GC/MS Analysis. The essential oils were analyzed with a 6890N Network GC System equipped with a
7683 Series auto-injector and a 5975 inert mass-selective detector and the same CBP-5 fused-silica cap.
column as described above. The GC-anal. conditions were as described above (see GC Analysis) and the
MS operating conditions were: ionization voltage, 70 eV; ion source temp. 2308; mass range 50 – 600 amu.
The components of the oils were identified by matching their mass spectral data with those of the NIST
(National Institute of Standards and Technology) mass spectral library and confirmed by comparison of
their retention indices (RI) with literature data [2] [3].
Microbial Strains. The Staphylococcus aureus strains SA2 (ATCC 25923), SA3 (ATCC 33591), and
SA7 (ATCC 700699) were grown and maintained on MuellerHinton agar (MHA), the S. aureus strains
VISA24 and VRSA156 on tryptic soy agar (TSA), and the fungi Candida albicans (ATCC 10231), C.
glabrata (ATCC 64677), Microsporum canis (ATCC 36299), and Trichophyton rubrum (ATCC 28188)
were grown and maintained in potato dextrose agar (PDA) slants. They were stored at 48 under aerobic
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011) 673
conditions. The S. aureus strains SA2, SA3, and SA7 were then cultured in MuellerHinton broth (MHB)
and the S. aureus strains VISA24 and VRSA156 in tryptic soy broth (TSB) overnight (24 h) at 378, while
the fungi were cultured in TSB overnight at 268. For further use, the inocula were adjusted to a turbidity
comparable to that of McFarland standard tube No. 0.5 [14].
Antimicrobial Activity. Media were sterilized by autoclaving at 1208 for 15 min, and all subsequent
manipulations were carried out in a class 2 laminar flow cabinet. The antibacterial activities of the
essential oils were quantified in liquid media using the microdilution method in 96-well microtiter plates.
Aliquots (10 ml) of the essential oil stock solutions (50 mg/ml) in DMSO ( < 10% of the total volume in
well A) and 90 ml of broth were added to the wells labeled as A. Aliquots of 50 ml of broth were added to
the wells B – H. The oils and broth in wells A were mixed thoroughly before transferring 50 ml of the
resultant mixture to wells B. The same procedure was repeated for the sample mixtures in wells B – H,
thus performing a serial dilution of the oils. Aliquots (50 ml) of the inocula were added to wells A – H, and
the microtiter plates were then incubated at 378 for 24 h. Oxacillin and cycloheximide (50 mg/ml) were
used as the standard antibiotics to compare the antibacterial and antifungal activities of the essential oils,
and DMSO served as negative control. The turbidity was taken as an indication of growth, the lowest
concentration that remained clear after macroscopic evaluation being taken as the minimum inhibitory
concentration (MIC). The MIC values were given as means of triplicate determinations, and the
activities were categorized as weak (MIC 5 mg/ml), moderate (1.0 mg/ml < MIC < 4.9 mg/ml), or strong
(MIC 1.0 mg/ml).
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