G.

cardiopetalus
J. Essent. Oil Res., 18, 451-454 (July/August 2006)

The Composition and Antimicrobial Activity of Stem

Bark Essential Oil of Goniothalamus cardiopetalus
(Bl.) Hook.f. et Thoms.

Abdulkhader Hisham,* Nirmal Pathare and Salim Al-Saidi

Departments of Chemistry and Biology, College of Science, P.O.Box-36, Sultan Qaboos University, Al-Khode-123, Oman
G. Jayakumar and M.D. Ajitha Bhai
Department of Chemistry, M.G. College, Trivandrum, Kerala, India
B. Harikumar
Department of Chemistry, S.N. College, Kollam, Kerala, India

Abstract
A hydrodistilled oil obtained from the stem bark of Goniothalamus cardiopetalus (Annonaceae) was analyzed by
GC/MS. The oil showed a total of 60 components of which 40 compounds representing 67.1% of the oil were identified.
Linalool (11.7%), α-pinene (7.0%), trans-pinocarveol (5.2%), caryophyllene oxide (5.0%) α-terpineol (4.9%), guaiol
(4.4%) camphor (3.9%) and bornyl acetate (3.9%) were found to be the major individual constituents of the oil. The
antimicrobial activity of the oil was tested against a panel of 17 bacterial and six fungal strains by the disc diffusion
method. The oil inhibited the growth of all test organisms at varying levels and the minimal inhibitory concentrations
(MIC) were also determined.

Key Word Index

Goniothalamus cardiopetalus, Annonaceae, essential oil composition, antimicrobial activity.

Introduction species, we have studied the composition and antimicrobial

Goniothalamus is a genus belongs to the Annonaceae family
with approximately 115 species mostly distributed in Asia and
Australia (1). Phytochemical and pharmacological studies on
several Goniothalamus species have established the occurrence
of two important categories of bioactive natural products popu-
larly known as styryl lactones and annonaceous acetogenins.
Their isolation and bioactivities are well documented in the
recent review articles (2-4). Even though, many members of
Goniothalamus species are aromatic trees with a characteristic
smell, the reports on their volatile components are not available
in the literature except for only three species ie., G. australis
(5), G. uvariodes (6) and G. malayanus (7).
Goniothalamus cardiopetalus (Bl.) Hook.f. et Thoms. is a
rare, endemic, medium sized aromatic tree seen in the Palaruvi
forest region, Kerala, India (8). No reports on the use of this
plant in traditional medicine are available in literature. However,
our recent phytochemical investigations on the stem bark of
this plant resulted in the isolation and characterization of three
annonaceous acetogenins and eight styryllactones (9,10).
As part of our continuing studies on the constituents of this
activity of the essential oil obtained from the stem bark of this
plant and the results are reported here.
Experimental
The plant material used in this study was collected from
Palaruvi forest region, Kerala, India in March 2001 and au-
thenticated by N.Mohan, Scientist, Tropical Botanic Garden
and Research Institute (TBGRI), Kerala where a voucher
specimen has been deposited.
Oil isolation: A sample of pleasant smelling, colorless,
genuine oil was isolated by hydrodistillation of 150 g shade-
dried powdered stem bark using a Clevenger-type apparatus
(yield 0.5%).
Preliminary GC analysis: The preliminary GC analysis
was carried out by means of a HP 5890 gas chromatograph
equipped with a HP-1-cross-linked methylsilicone gum capil-
lary column (25 m x 0.32 mm x 0.52 µm film thickness), using
a FID detector.
GC/MS analysis: The oil was diluted with an appropri-
ate volume of dichloromethane and analyzed by GC/MS on a

*Address for correspondence Received: December 2004

Revised: June 2005
1041-2905/06/0004-0451$14.00/4­—© 2006 Allured Publishing Corp. Accepted: July 2005

Vol. 18, July/August 2006 Journal of Essential Oil Research/451

 Hisham et al.
Table I. Percentage composition of stem bark oil of
Goniothalamus cardiopetalus
Compound % Monoterpenes
tricyclene 0.1
α-thujene 0.1
α-pinene 7.0
camphene 2.2
β-pinene 0.3
p-cymene 0.4
limonene 0.2
1,8-cineole 0.7
cis-linalool oxide (furanoid) 0.4
trans-linalool oxide (furanoid) 0.3
linalool 11.7
hotrienol 0.2
α-fenchol 0.4
trans-pinocarveol 5.2
camphor 3.9
camphene hydrate 0.5
isoborneol 0.4
pinocarvone 1.8
borneol 1.2
terpinen-4-ol 2.9
p-cymen-8-ol 0.5
cis-p-mentha-1(7),8-dien-2-ol 0.6
α-terpineol 4.9
myrtenol 0.8
cis-sabinol 0.3
piperitol* 0.1
verbenone 0.2
trans-carveol 0.7
trans-p-mentha-1(7),8-dien-2-ol 0.3
bornyl acetate 3.9
carvacrol 0.5
Sesquiterpenes suspension containing 1 x 106 CFU/mL was added to 100 µL of
α-copaene 0.2
α-amorphene 0.6
cis-calamenene 1.0
spathulenol 1.5
caryophyllene oxide 5.0
β-copaen-4α-ol 0.6
guaiol 4.4
globulol 0.5
cadalene 0.6
Unidentified (20 peaks) 32.9
correct isomer not identified
*
Shimadzu model (GC-MS-QP/ 5050A) instrument equipped
with HP-5 (5%-phenyl)-methylpolysiloxane-nonpolar-capillary
column (30 m x 0.32 mm x 1.0 µm thickness) and interfaced
with a quadrupole mass spectrometer.
Analytical conditions: The injector and interface tem-
peratures were kept at 275°C and 300°C, respectively. The oven
temperature was programmed from 70°C to 270°C at a rate
of 3°C/min. Helium was used as the carrier gas with a linear
velocity of 74.6 cm/s and the total flow rate was 39.9 mL/min.
Mass spectra were continuously recorded from 40 to 500 m/z.
The MS operating parameters were: ionization voltage 70 eV,
scan rate 500 amu/s. The components of the oil were identified
by computer aided matching of their mass spectral data with
the reference spectra (Wiley 229, 2000) in the database.
452/Journal of Essential Oil Research Vol. 18, July/August 2006
Microbial cultures growth conditions: Test microorgan-
isms included the following standard antimicrobial susceptibility
strains: Staphylococcus aureus (NCTC 6571), Escherichia coli
(NCTC10418), Pseudomonas aeruginosa (NCTC10662) besides
a battery of Gram positive and Gram negative strains given in
Table II as per the NCCLS guidelines (11). Cultures of these
bacteria were grown in Mueller-Hinton broth (Difco) at 37°C
and maintained on slopes of nutrient agar (Difco) at 4°C.
Antimicrobial activity assay: The oil was tested for an-
timicrobial activity using the disc diffusion technique on solid
media as described by Bauer et al (12). Sterile, 6mm diameter
Whatman 41 discs [containing the filter-sterilized oil initially
diluted 4 mg/mL with ethylene glycol and further diluted to
achieve required disc concentrations with phosphate buffered
saline (w/v)] were placed on plates of Mueller-Hinton agar
(Difco), which had been surface spread with 0.1 mL of loga-
rithmic phase bacteria at a density adjusted to a 0.5 McFarland
turbidity standard (108 colony-forming units [CFU]/mL). The
plates were then incubated for 24 h at 37°C. The results were
recorded by measuring the average zones of growth inhibition
surrounding the discs. Discs containing 0.04 mL ethylene glycol
was placed in each plate as control. Standard antibiotic discs as
per the requirement of ‘laboratory control and antimicrobial
therapy’ (13) were used in parallel. Antifungal disc diffusion
assay was performed by employing above techniques utilizing
Sabouraud’s agar and 0.2 mL culture suspension. The plates
were incubated at 27οC for 48 h.
Minimal inhibitory concentration (MIC): The oil was
tested for antibacterial activity using the microbroth dilution
method in broth media Mueller-Hinton (Difco) as per the
NCCLS guidelines (14). In these experiments, 50 µL of a
susceptibility test broth containing serial twofold dilutions of the
oil in sterile ELISA plate wells. All these plates were incubated
at 37°C for 24 h before being read. The MIC was considered
the lowest concentration of the sample that prevented visible
growth. The minimum bactericidal concentrations (MBCs)
were determined by subculturing, 10 µL from each negative
well and from the positive growth control. MBCs were defined
as the lowest concentration yielding negative subcultures. All
the samples were examined in duplicate.
Results and Discussion
The chromatogram of oil exhibited 60 peaks of which 40
components representing 67.1% of the total oil was identified
by comparison of their mass spectral data with reference spectra
in the computer library. The identified compounds are given
in table I according to their increasing Rt values. Out of these,
31compounds constituting 52.7% were monoterpenes. Among
these, seven compounds comprising 10.3% were monoterpene
hydrocarbons whereas the remaining 24 compounds compris-
ing 42.3% of the oil were oxygenated monoterpenes. α-Pinene
(7.0%) was the dominant monoterpene hydrocarbon whereas
linalool (11.7%), trans-pinocarveol (5.2%), α-terpineol (4.9%),
camphor (3.9%), bornyl acetate (3.9%), terpinen-4-ol (2.9%),
pinocarvone (1.8%) and borneol (1.2%) were the major oxygen-
ated monoterpenes. All other compounds in the monoterpene
series were present in less than 1%.
 G. cardiopetalus

Table II. Antimicrobial activity of stem bark oil of Goniothalamus cardiopetalus by the disc diffusion method and MIC

Diameter of zone of inhibition [mm] MIC

Bacterial strains Amox-Clav Cefotaxime Tetracycline Oil oil
Gram-positive [20 µg] [30 µg] [30 µg] [0.4 mg] [0.2 mg] [mg/mL]
# S. aureus * [NCTC6571] 13.0 6.5 9.0 20.0 16.5 1.0
◘S. aureus * 12.0 6.5 8.0 18.0 14.5 1.5
◊S. albus * 14.0 6.0 9.0 18.5 15.0 1.5
◘S. epidermidis* 11.0 6.0 9.0 16 12.5 1.0
◘Strept. mitis** 14.0 8.0 10.0 20.0 16.5 1.0
◊Strept. sanguis** 16.0 8.0 10.0 22.0 18.0 1.5
◊M. luteus*** 10.0 6.0 9.0 12.0 10.5 1.5
◊B. subtilis **** 7.0 2.0 11.0 14.5 12.0 1.0
◊B. cereus **** 6.0 2.0 9.0 16.5 14.0 1.5
Gram-negative
#E. coli ■ [NCTC10418] 6.5 10.0 6.5 15.0 13.5 1.5
◘E. coli ■ 7.5 10.0 5.5 9.5 7.0 1.5
◊Ent. aerogenes ■■ 3.5 5.5 5.0 11.5 8.5 3.0
◊Kleb. pneumoniae■■■ 2.5 6.5 4.0 13.0 10.5 1.5
◊Salmonella typhi 8.0 11.0 7.5 15.0 12.5 4.5
◊Proteus vulgaris 6.0 10.5 2.0 8.5 6.5 6.5
#Ps. aeruginosa■■■■
[NCTC10662] 0.0 2.5 0.0 12.0 9.0 5.0
◊Ps. aeruginosa ■■■■ 0.0 2.0 0.0 7.5 6.0 5.5
Fungal Strains Diameter of zone of inhibition [mm] Mycostatin
15 µg/µL 1.6 mg 0.8 mg 0.4 mg 0.2 mg
◊Candida albicans 6.0 4.0 2.0 1.5 5.5
◊Saccharomyces cerevisiae 6.5 4.5 2.5 2.0 6.0
◊Aspergillus flavus 6.0 4.5 3.0 2.0 10.0
◊Rhizopus stolonifer 8.0 5.5 3.5 2.0 4.5
◊Penicillium notatum 7.5 5.5 4.0 3.0 8.0
◊Fusarium oxysporum 5.5 3.5 2.0 1.0 4.0
N.B.-zone of inhibition values are the average of four determinations in four different directions; Whatman 41 (6 mm) discs were soaked with each sample tested at the given
concentration in phosphate buffered saline (w/v); initial stock dilution was done in ethylene glycol; Amox-Clav-Amoxicillin-Clavulanate; bacterial strains: S.* - Staphylococcus,
Strept. ** - Streptococcus, M. *** - Micrococcus, B. **** - Bacillus, E. - Escherichia, Ent. - Enterobacter, K. - Klebsiella, Ps. - Pseudomonas; #- the standard NCTC strains
recommended for standardization of antibiotic susceptibility test; - wild type strains isolated from human normal flora; ◊- wild type strains isolated from environment

Among the sesquiterpene series, nine compounds were
identified, of which four were sesquiterpene hydrocarbons (total
2.5%), while the remaining five were oxygenated sesquiterpenes
(total 12.0%). Caryophyllene oxide (5.0%) and guaiol (4.4%)
were the major oxygenated compounds. The identification of
20 peaks comprising 32.9% of the total oil was unsuccessful due
to the absence of reference spectrum in the library. However,
the mass spectral data of these compounds suggested that they
were oxygenated sesquiterpenes.
The antibacterial activity of the oil was tested against a panel
of 17 bacterial and six fungal strains by disc diffusion method.
The MICs were also determined and the experimental results
are summarized in Table II.
The oil inhibited the growth of all test organisms in varying
levels thus exhibiting broad spectrum antimicrobial activity.
Most of the Gram positive strains of saprophytic Bacillus spe-
cies as well as pathogenic species of Staphylococci and Strep-
tococci exhibited higher susceptibility to the oil. Amongst these,
Streptococci were found to be most sensitive. Gram negative
organisms were also found to be sensitive to the oil; higher
susceptibility being exhibited by enteric strains belonging to
E. coli and Salmonella species. The oil retained its activity up
to 0.2 mg concentration in the disc diffusion assay. The MIC
range varied from 1.0-1.5 mg/mL for Gram + organisms and
1.5-6.5 mg/mL for Gram – organisms. The oil also showed
moderate antifungal activity against yeasts and molds; however
the inhibitory oil concentrations were higher compared to the
inhibitory concentrations required for bacterial species.
Goniothalamus cardiopetalus bark oil was found to con-
tain both mono- and sesquiterpene compounds in significant
amounts, whereas the bark oil of a related species, G. malaya-
nus, was rich in sesquiterpenoids particularly two components,
β-eudesmol (32.2%) and γ-eudesmol (16.0%) (7). The bark oil
of another related species, G. uvariodes, was also distinct from
G. cardiopetalus but similar to G. malayanus in sesquiterpene
contents such as β-eudesmol (31.5%), γ-eudesmol (16.0%),
hedycaryol (13.6%) and α-eudesmol (5.6%) (5). Finally, G.
australis possessed a leaf oil containing both mono- and ses-
quiterpenes with pinocarvone (10%) and trans-pinocarveol
(17%) as the major components. Therefore G. cardiopetalus
bark oil is at least similar to G. australis leaf oil in its mono-
and sequiterpene content.
Acknowledgments
Authors G.J and M.D.A.B thank the Science Technology and
Environment Department, Goverment of Kerala, India for financial
support for the research.

Vol. 18, July/August 2006 Journal of Essential Oil Research/453

 Hisham et al.
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