Chemical Composition and Antibacterial Activity of Rhizome Oils from Five Hedychium Species

Abstract

The essential oils from rhizomes of five Hedychium species, H. coronarium, H. neocarneum, H. flavescens, H. speciosum and H. stenopetalum (Zingiberaceae), were obtained by hydrodistillation and analyzed by capillary GC and GC/MS. Sixty components were identified and percentage oil yields from the fresh plants ranged from 0.06-0.17 % (v/w). All rhizome oils were rich in terpenes, especially monoterpenes (75.0-95.9 %). The most common compounds in the rhizome oils of Hedychium were beta-pinene, linalool and 1,8-cineole. The essential oils were tested against four bacterial strains. They showed moderate to weak activity against Gram-positive bacteria (inhibition zone 25-13 mm, MIC 0.3-8.3 mg/mL, MBC 0.6-8.3 mg/mL).

Full text

Chemical Composition and Antibacterial Activity of Rhizome Oils
from Five Hedychium Species
Ratchuporn Suksathana,d, Siriwoot Sookkheeb, Somboon Anuntalabhochaic and Sunee Chansakaowa,*
aDepartment of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200,
Thailand
bDepartment of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
cDepartment of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
dQueen Sirikit Botanic Garden, The Botanical Garden Organization, P.O. Box 7, Mae Rim, Chiang Mai 50180,
Thailand
chsunee@gmail.com, pmischns@chiangmai.ac.th
Received: January 24th, 2013; Accepted: February 25th, 2013
The essential oils from rhizomes of five Hedychium species, H. coronarium, H. neocarneum, H. flavescens, H. speciosum and H. stenopetalum
(Zingiberaceae), were obtained by hydrodistillation and analyzed by capillary GC and GC/MS. Sixty components were identified and percentage oil yields
from the fresh plants ranged from 0.06-0.17 % (v/w). All rhizome oils were rich in terpenes, especially monoterpenes (75.0-95.9 %). The most common
compounds in the rhizome oils of Hedychium were β-pinene, linalool and 1,8-cineole. The essential oils were tested against four bacterial strains. They showed
moderate to weak activity against Gram-positive bacteria (inhibition zone 25-13 mm, MIC 0.3-8.3 mg/mL, MBC 0.6-8.3 mg/mL).
Keywords: H. coronarium, H. neocarneum, H. flavescens, H. speciosum, H. stenopetalum, Essential oils, Monoterpene, Anti-bacterial activity.
Hedychium J. König species are perennial rhizomatous herbs, which
belong to the Zingiberaceae family. They are represented by eighty
species, approximately twenty-three of which exist in Thailand
[1a-1g]. Most species are fragrant and are widely cultivated for
ornamental purposes. Several species have been studied for their
pharmacological properties to expand their ethnobotanical uses.
Many Hedychium species are used in traditional medicine, i.e.
rhizome of H. acminatum (diarrhea, snake bite, liver complaints)
[2a]; H.coronarium: boiled leaves (Thailand: stiff and sore joints)
[2b]: rhizomes (body ache, stimulant and carminative) [2c-2e]; H.
spicatum (bactericide, fungicide, pain, stomach ailments,
inflammation) [2f].
The monoterpenes 1,8-cineole, β-pinene, linalool, terpin-4-ol, and
sabinene are most often found in the rhizome oil of Hedychium
species [2c,3a-3c]. There are several reports on the essential oil
composition of the rhizomes of Hedychium species, but none for H.
neocarneum, H. speciosum and H. stenopetalum. The present study
describes a detailed analysis of the oils from rhizomes of the
aforementioned Hedychium species found in Thailand and their
antibacterial activities compared with the worldwide-cultivated
species collected in Thailand, H. coronarium and H. flavescens.
The percentage oil yields from the plants ranged from 0.06-0.17%
(v/w) (Table 1). GC analysis revealed approximately 70
components. The identified constituents are presented in Table 2.
Seventeen compounds representing 81.1% of the rhizome oil were
identified in the essential oil of H. coronarium. The most abundant
compound was 1,8-cineole (33.8%), followed by β-pinene (13.6%)
and α-pinene (5.6%). Analysis of H. neocarneum resulted in
the identification of 39 volatile compounds representing 98.5% of
the oil. Linalool, 1,8-cineole, β-pinene and α-pinene were the
major components, amounting to 23.2%, 14.2%, 11.2% and 7.6%,
Table 1: Collection data for the investigated Hedychium species.
Code S
p
ecies Voucher s
p
ecimen number* Collection date RO
(
%
)
**
H.c. H. coronarium R. Spanuchat 09-1 August 2009 0.17
H.n. H. neocarneum M. Wongnak 1157 August 2009 0.06
H.f. H. flavescens M. Wongnak 175 August 2009 0.08
H.s. H. speciosum M. Wongnak 1150 August 2009 0.14
H.st. H. stenopetalum R. Spanuchat 09-2 August 2009 0.14
* Queen Sirikit Botanic Garden Herbarium (QBG)
** RO: rhizome oil [% oil yield, v/w]
respectively. Thirty-nine compounds were identified in the rhizome
oil of H. flavescens representing 97.6% of the total oil with
β-pinene (23.1%), linalool (21.0%), 1,8-cineole (17.9%) and
α-pinene (10.5%) as the main components. The oil of H. speciosum
contained 34 compounds representing 99.1% of the oil. The major
constituents were 1,8-cineole (32.0%), β-pinene (23.4%) and
nerolidol (17.0%). The essential oil from rhizomes of H.
stenopetalum showed 26 compounds making up 99.9% of the total
amount. Linalool (50.5%) was the main compound, followed by
β-pinene (12.4%), nerolidol (8.6%) and α-pinene (5.6%).
Monoterpenes represented the most commonly found group of
compounds in the essential oils from the rhizomes of the
investigated species: H. coronarium (75.0%); H. neocarneum
(89.4%); H. flavescens (95.9%); H. speciosum (79.1%); H.
stenopetalum (91.2%). A comparison of the main constituents of the
essential oils of these five species (Table 2) showed that terpenes
were found in the greatest amount and that each species had a
different set of dominant compounds. It has been reported that
terpenes have bacteriostatic and bactericidal effects [3c]. This is
true of monoterpenes especially oxygenated monoterpenes, which
were found in the essential oils from many kinds of popular
aromatic herbs (for example, lemongrass, basil, laurel and
cinnamon) [3d-3e], including species of Hedychium [2c,3a].
NPC Natural Product Communications
2013
Vol. 8
No. 4
519 - 522

520 Natural Product Communications Vol. 8 (4) 2013 Suksathan et al.
Table 2: Chemical composition (%) of rhizome oils from H. coronarium (H.c.), H.
neocarneum (H.n.), H. flavescens (H.f.), H. speciosum (H.s.) and H. stenopetalum (H.st.).
Compound KIa KIb H.c. H.n. H.f. H.s. H.st.
Tricyclene 927 927 - 0.1 - 0.1 -
α-Thujene 929 930 - 0.1 0.3 0.1 0.1
α-Pinene 938 939 5.6 7.6 10.5 6.4 5.6
Camphene 956 954 0.5 4.4 1.2 1.3 0.8
Sabinene 977 976 - 0.4 - 1.1 0.1
β-Pinene 982 980 13.6 11.2 23.1 23.4 12.4
β-Myrcene 992 991 - 0.6 0.8 0.7 0.8
1-Phellandrene 1008 1012 - 0.2 - - -
α-Phellandrene 1010 1003 - - 1.1 0.8 5.0
Δ-3 Carene 1013 1011 - 0.1 0.5 0.2 -
α-Terpinene 1021 1018 - 0.4 0.4 0.2 0.3
p-Cymene 1029 1026 1.4 5.8 4.3 0.1 0.8
Limonene 1034 1031 1.1 2.4 2.7 3.2 1.7
1,8-Cineole 1039 1039 33.8 14.2 17.9 32.0 3.3
cis-Ocimene 1041 1041 - - - - 0.4
γ-Terpinene 1063 1062 - 2.2 2.4 0.4 1.4
trans Sabinene hydrate 1076 1070 - - - 0.1 -
Linalool oxide cis 1077 1077 3.0 0.1 - - 0.4
α-Terpinolene 1089 1089 - 0.3 0.6 0.2 0.4
trans-Linalool oxide 1092 1097 - 0.1 0.1 - -
Methyl heptyl ketone 1097 1091 - - 0.2 - -
Linalool 1107 1098 2.4 23.2 21.0 2.6 50.5
Fenchyl alcohol 1127 1119 - 0.1 0.2 - 0.1
p-Menth-2-en-1-ol 1133 1121 - 0.1 0.1 - 0.1
α-Campholene- 1135 1126 0.8 0.1 0.1 - -
aldehyde
trans-Pinocarveol 1150 1145 2.6 0.1 0.2 0.1 -
Terpinene-1-ol 1152 1147 - - - - 0.1
Camphor 1158 1143 - 0.1 - - -
Pinocarvone 1172 1165 2.1 0.1 0.1 0.1 -
endo-Borneol 1181 1169 1.3 6.8 1.4 2.1 2.0
3-Pinanone 1184 1170 - - 0.1 - -
Terpinene-4-ol 1188 1177 0.2 2.2 2.2 0.8 1.3
p-Cymene-8-ol 1195 1183 - 0.1 - - -
α-Terpineol 1199 1189 - 5.4 4.6 2.2 2.7
β-Fenchyl alcohol 1199 - 5.0 - - - -
a-Phellandrene epoxide 1210 - - - 0.1 - -
trans-Carveol 1227 1217 0.3 - - - -
p-Menthan-2-ol, 1,8- 1235 1227 1.2 - - - -
epoxy
cis-Geraniol 1232 1232 - 0.1 - - 0.5
Citronellol 1237 1228 - 0.5 - - 0.1
Geraniol 1259 1259 - 0.2 - - -
Bornyl acetate 1289 1289 - 0.7 0.2 2.1 0.3
Thymol 1307 1290 - - 0.1 - -
N/A 1328 - 6.1 - - - -
δ-Elemene 1338 1338 - - 0.1 - -
p-Menth-8-ene-1,2-diol 1355 1321 0.2 - - - -
N/A 1373 - - 0.1 - - -
trans-Caryophyllene 1420 1419 - - 0.1 0.2 -
Aristolen 1442 1450 - - 0.1 - -
N/A 1449 - - - 0.1 - -
trans-Isoeugenol 1452 1449 - 0.2 - - -
1,6,10-Dodecatriene, 1453 1459 - - 0.1 - -
7,11-dimethyl-3-
methylene
Alloaromadendrene 1459 1459 - - - 0.2 -
β-Himachalene 1479 1490 - 0.1 - - -
Germacrene D 1481 1480 - - - 0.1 -
Curcumene 1483 1486 - 0.2 - - -
Cadinene 1489 1516 - - 0.1 - -
(-)-α-Selinene 1497 1497 - - 0.1 - -
Bicyclogermacrene 1499 1499 - - - 0.2 -
δ-Cadinene 1505 1523 - - 0.1 - -
Farnesene 1506 1508 - - - 0.2 -
N/A 1512 - - 0.2 - - -
(-)-α-Panasinsen 1521 - - - 0.1 0.1 -
Sesquisabinene hydrate 1563 1547 - 0.1 - - -
Germacrene B 1567 1561 - - 0.2 - -
Nerolidol 1571 1566 - 7.1 - 17.0 8.6
Spathulenol 1590 1591 - 0.2 0.3 0.7 -
(-)-Caryophyllene 1595 1581 - - - 0.1 -
oxide
Veridiflorol 1607 1593 - - 0.1 0.1 -
α-Cadinol 1656 1650 - 0.2 - 0.1 -
N/A 1886 - - - - - 0.1
Monoterpene 56.0 49.5 65.2 69.0 33.2
Oxygenated monoterpene 19.0 39.9 30.7 10.1 57.9
Sesquiterpene - 0.4 0.8 0.9 0.0
Oxygenated sesquiterpene - 7.6 0.4 17.9 8.6
Others - 0.2 - - -
Unidentified compound (N/A) 6.1 0.9 0.4 1.2 0.2
Total 81.1 98.5 97.6 99.1 99.9
a: Kovats retention index relative to C8-C20 n-alkanes on DB-5 MS column; b: Kovats
retention index from literature data [2f, 5a-5h]; N/A:Unidentified compound
Table 3: Antibacterial screening of the rhizome oils of H.c.: H. coronarium, H.n.: H.
neocarneum, H.f.: H. flavescens, H.s.: H. speciosum, and H.st.: H. stenopetalum, in
comparison with G: gentamicin (1 mg/mL), C: chloramphenicol (1 mg/mL).
Bacteria Zone of Inhibition (mm)
H.c. H.n. H.f. H.s. H.st. G C
Staphylococcus aureus 13
b
24a 25a 14
b
23a 28 13
Bacillus subtilis 15d 25a 17c 18c 23
b
29 15
Escherichia coli 9
b
8
c 9
b
7
c 13a 27 20
Pseudomonas aeruginosa - - - - - 22 -
* The letter in the same row is not significantly different (P<0.05)
The essential oils of the investigated Hedychium species exhibited
significant antibacterial activity against Gram-positive bacteria
(inhibition zone 25-13 mm). The rhizome oil from H. neocarneum
and H. flavescens showed maximum zones of inhibition (25 mm)
against Bacillus subtilis and Staphylococcus aureus,, respectively.
The rhizome oils showed low inhibitory activity against the Gram-
negative bacterium, Escherichia coli (inhibition zone 13-7 mm),
and none of essential oils showed activity against Pseudomonas
aeruginosa. The zone of inhibition values are summarized in Table
3.
The Gram-positive bacterial strains were further tested at different
concentrations to determine the minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC) values. All
of the Hedychium oils showed activity against the Gram-positive
bacteria, S. aureus and B. subtilis, with MIC values ranging from
0.3-8.3 mg/mL and MBC values from 0.6-8.3 mg/mL.
H. flavescens rhizome oil showed the strongest activity against S.
aureus (inhibition zone 25 mm, MIC/MBC: 0.3/0.6 mg/mL),
followed by H. neocarneum (clear zone 24 mm, MIC/MBC: 0.4/0.7
mg/mL) and H. stenopetalum (inhibition zone 23 mm, MIC/MBC:
0.5/0.8 mg/mL), while H. neocarneum showed the best activity
against B. subtilis (inhibition zone 25 mm, MIC/MBC: 0.4/0.7
mg/mL), followed by H. stenopetalum (inhibition zone 23 mm,
MIC/MBC: 0.5/0.8 mg/mL) and H. speciosum (inhibition zone 18
mm, MIC/MBC: 1.2/2.4 mg/mL). The rhizome oil of H.
coronarium exhibited the lowest activity against both organisms
with the highest MIC and MBC values. These activities however,
were moderate to weak compared with the positive controls. The
results are summarized in Table 4.
Table 4: MIC/MBC values (mg/mL) of the rhizome oils of H. coronarium, H.
neocarneum, H. flavescens, H. speciosum and H. stenopetalum, in comparison with G:
gentamicin (µg/mL), C: chloramphenicol (µg/mL) using microdilution method.
Bacteria S. aureus B. subtilis
MIC MBC* MIC MBC*
H. coronarium 4.2 8.3 8.3 8.3
H. neocarneum 0.4 0.7 0.4 0.7
H. flavescens 0.3 0.6 1.3 2.5
H. speciosum 4.8 4.8 1.2 2.4
H. stenopetalum 0.5 0.8 0.5 0.8
Gentamicin (µg/mL) 2.0 4.0 2.0 4.0
Chloramphenicol (µg/mL) 6.3 12.5 6.3 12.5
* MBC was the lowest concentration at which the bacteria were killed as at ≥ 99.9%
For the overall analysis of the inhibition zone, the broadest activity
against all four bacterial strains was shown by rhizome oils from H.
stenopetalum, H. neocarneum and H. flavescens, with no significant
difference between them (P<0.05) (data not shown). These results
could be ascribed to them containing monoterpenes as their main
components, especially the oxygenated monoterpenes, 1,8-cineole
and linalool (37.4-53.8%), which were previously observed to be
compounds with high antibacterial properties [3d-3e]. These results
were congruent with several reports about the relationship between
monoterpene content and the antimicrobial property of Hedychium
oils [2f, 4], especially against S. aureus. The results from the other

Rhizome oils from five Hedychium species Natural Product Communications Vol. 8 (4) 2013 521
bacterial strains were not as definitive (Table 3), which could be
explained by the synergistic effect of the different constituents of
the essential oils [3e].
The Hedychium essential oils possessed greater activity against the
Gram-positive than Gram-negative bacteria. The chemical profiles
of the investigated Hedychium oils and their antibacterial properties
confirm the usefulness of these plants as supplementary natural
pharmaceutical ingredients for application for the treatment of
infectious Gram-positive bacterial diseases, for example, skin
diseases, and sore throat, and as alternative natural products to
substitute for synthetic antibacterial agents.
Experimental
Plant materials: Rhizomes of five Hedychium species were
collected from the living collection at Queen Sirikit Botanic
Garden, Chiang Mai, Thailand by random sampling. The plants
were identified by Dr Piyakaset Suksathan and voucher specimens
have been deposited at the Queen Sirikit Botanic Garden Herbarium
(QBG) as shown in Table 1.
Extraction of essential oil: Fresh rhizomes (1 Kg) were subjected
to hydrodistillation for 5 h using a Clevenger-type apparatus. The
obtained oils were dried over anhydrous sodium sulfate and stored
in sealed vials at 4°C in the dark until analyzed. The oils were
transparent with a faint yellow color.
GC-GC/MS analysis: A Shimadzu GCMS-QP 2010 Plus system
was used, with a mass-selective detector with electron impact
ionization. The samples were separated using a DB-5 MS capillary
column (5% phenylmethylpolysiloxane, 30 m x 0.25 mm, 0.25 μm
film thickness) with helium as the carrier gas (0.99 mL/min). The
temperature program used for analysis was as follows: initial
temperature was 60°C and programmed to 140°C at a rate of 5
°C/min for 16 min, ramp to 155°C at a rate of 1°C/min and then
ramp to 230°C at a rate 10°C/min and kept constant for 5 min. The
split flow ratio was 1: 100. The injection temperature was 160°C
and the FID detector temperature 250°C.
Identification of components: The identification of volatile
components was based on computer matching with WILEY 7 and
the NIST 2005 Library, as well as by comparison of the mass
spectra and Kovat’s retention indices (KI) with a series of n-alkanes
(C8-C20) and MS literature data [2f,5a-5h].
Antibacterial assay
Microbial culture: The following bacterial species were used:
Gram-positive; Staphylococcus aureus (ATCC 25923) Bacillus
subtilis (ATCC 6633); Gram-negative: Escherichia coli (ATCC
25922), Pseudomonas aeruginosa (ATCC 9027).
The antibacterial assays were carried out by the agar disc-diffusion
method modified from the Bauer et al. method [6], and the
microdilution method modified from Konaté et al. [7]. These assays
were used in order to determine the antibacterial activity of the oils
against human pathogenic bacteria.
Disc-diffusion test: Bacteria were cultured overnight at 37°C in
Tryptic soy broth (TSB) and then adjusted to be equivalent to the
0.5 McFarland standard (1.0 × 108 CFU/mL). The inocula were
prepared daily and stored at 4°C until use. Dilutions of the inocula
were cultured on solid medium to verify the absence of
contamination and to check the validity of the inoculum.
Inoculation of the plate with the test organisms was completed by
streaking the swab and placing the discs impregnated with the oils
on the surface of a 2 mL/agar plate of solid Tryptic soy agar (TSA).
The essential oils (25.0 μL/disc) were investigated by the disc
diffusion method using 6 mm sterile filter discs (Whatman No.1).
After 24 h of incubation at 37°C, the diameters of the growth
inhibition zones were measured. The positive controls were 20
μL/disc of 1 mg/mL gentamicin (Vesco Pharmaceutical Co., Ltd.)
and 1 mg/mL chloramphenicol (Atlantic Laboratories Corp. Ltd.).
The diameters of inhibition zones, including the disc diameter, were
measured in mm. Tests were performed in triplicate.
Microdilution test: The minimum inhibitory and bactericidal
concentrations (MICs and MBCs) were determined using 96-well
microtiter plates. The bacterial suspension was adjusted with sterile
TSB to be equivalent to the 0.5 McFarland standard (1.0 × 108
CFU/ml). The inocula were prepared daily and stored at 4°C until
used. Dilutions of the inocula were cultured on solid medium to
verify the absence of contamination and to check the validity of the
inoculum. It was observed that 50% DMSO (DMSO from Sigma)
did not affect the investigated bacterial strains. The essential oils to
be investigated were dissolved in 50% DMSO (100 μL) and were
diluted two-fold to the wanted concentrations with TSB. The
microorganism suspension (1.0 × 105 CFU per well) of 50 µL was
added to the broth dilutions. The microplates were incubated for 24
h at 37 °C. The lowest concentrations without visible growth were
defined as concentrations that completely inhibited bacterial growth
(MICs). The wells used in the MIC studies that did not show any
turbidity were determined for MBCs. An aliquot of the suspension
(0.02 mL) was spread onto TSA and further incubated at 37°C for
24 h. The MBC was the lowest concentration at which the initial
inoculums were killed (99.9% or more). Gentamicin (1 mg/mL) and
chloramphenicol (1 mg/mL) were used as positive controls. Three
replicates were used for each sample.
Statistical analysis of disc-diffusion test: All experiments were
conducted in triplicate (n = 3). Analysis of variance (Anova and Q
Cochran’s) was performed (P<0.05) for each bacterial species and
overall activity of each sample. The differences between means
were determined by Duncan’s test (P<0.05). SPSS 15.0 statistical
software, Chicago, IL, USA was used for the analysis [8].
Acknowledgments - This work was supported by grants from the
Department of Pharmaceutical Science, the Faculty of Pharmacy
and the Graduate School, Chiang Mai University, Chiang Mai,
Thailand and NUI-RC (NSTDA University Industry Research
Collaboration) in the National Science and Technology
Development Agency (NSTDA). Queen Sirikit Botanic Garden,
Chiang Mai, Thailand is thanked for providing the plant samples
and Mr Methee Wongnak for preparing the plant materials.
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