jfbc_258

835..851

DOI: 10.1111/j.1745-4514.2009.00258.x

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF SOME

ALPINA SPECIES
L.F. WONG, Y.Y. LIM1 and M. OMAR
School of Arts and Sciences, Monash University Sunway campus, Bandar Sunway,
46150 Petaling Jaya, Selangor, Malaysia
Accepted for Publication March 13, 2008

ABSTRACT
Methanol extracts of Alpinia galanga, Alpinia zerumbet, Alpinia zerumbet variegata and Alpinia purpurata were evaluated for total phenolic content
(TPC) and antioxidant activities (AOA). The AOA were investigated using
1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power (RP), ferrous ion
chelating as well as b-carotene bleaching assays. High antioxidant activities
shown in leaves of A. zerumbet, A. zerumbet variegata and A. purpurata by
using DPPH and RP assays were associated with high TPC values. In spite of
lower TPC values, A. galanga leaves and flowers showed highest chelating
and b-carotene bleaching abilities. The antimicrobial activities were screened
by using disc diffusion method. Extracts from A. galanga and A. purpurata
flowers showed the largest zone of inhibition of Micrococcus luteus. Only the
extract from A. galanga rhizome showed antifungal activity toward Aspergillus
niger. This work shows that leaves of A. zerumbet, A. zerumbet variegata and
A. purpurata may serve as potential dietary sources of natural antioxidants.

PRACTICAL APPLICATIONS
Alpinia species are widely used in traditional cures and as food ingredients. Alpinia galanga rhizome is used as spice and food flavoring agent, and its
leaves and inflorescence are consumed as vegetable. Its rhizome is also used to
treat diseases such as fungal skin infections, intestinal infections, Type II
diabetes, bronchitis and rheumatism. Alpinia zerumbet is used as ingredients in
traditional health supplement, as diuretic agent and hypertension control.
Alpinia zerumbet variegate and Alpinia purpurata are ornamental plants.

Corresponding author. TEL: +603 55146103; FAX: +603 55146099; EMAIL: lim.yau.yan@
sci.monash.edu.my

Journal of Food Biochemistry 33 (2009) 835851.

2009, The Author(s)
Journal compilation 2009, Wiley Periodicals, Inc.

835

836

L.F. WONG, Y.Y. LIM and M. OMAR

INTRODUCTION
The Zingiberaceae is a large diverse family comprising 1,200 species
belonging to 49 genera. A prominent member of this family is the Alpinia
genus. In Southeast Asia, Alpinia galanga is commonly known as the greater
galangal or languas (lengkuas). Rhizomes of this species of ginger are extensively used in spice and food flavoring products in beverage and food industry.
The rhizomes have been used locally in Malaysia to treat stomach ache,
rheumatism, diarrhea, vomiting, diabetes mellitus and respiratory diseases
(Matsuda et al. 2003). Studies conducted on the antioxidant activity of A.
galanga were focused on the ethanolic extracts of rhizomes (Juntachote and
Berghofer 2005). Essential oils such as mono-, sesquiterpene and cinnamate
derivatives were found (Jirovetz et al. 2003) in various parts of the plant. Its
use in sweet goods, dressings and personal care products is due to the presence
of pungent compound, 1-acetoxychavicol acetate (ACA) (Yang and Eilerman
1999). A pharmacological study has shown that the rhizome extract has a
hypoglycaemic activity (Akhtar et al. 2002). Other than the extensive applications of rhizomes, the young inflorescence and leaves are consumed as ulam
or salad, respectively, by the village folk. Infusions of leaves were reported to
have stimulant and anti-rheumatism properties (Raina et al. 2002).
Alpinia zerumbet is also known as Alpinia speciosa or Alpinia nutans
(Zoghbi et al. 1999). The plant extract is helpful in lowering blood pressure as
well as reducing atrium contractility (Habsah et al. 2003). It can behave as
diuretic, anti-platelet, bacteriostatic and fungistatic agent, as well as having a
depressor effect on COP9 signalosome function (Itokawa et al. 1984; Zoghbi
et al. 1999). For treatment of intestinal diseases, it is consumed as tea infusion.
The extracts were able to inhibit porphyrin photooxidative reaction and
showed antifungal properties (Leal-Cardoso et al. 2004).
Alpinia zerumbet variegata is a plant with greenyellow variegated
leaves and pinkish white flowers (Gillman 1999). It has a distinct spicy fragrance, and its attractive foliage and flowers make it a favorite as a landscape
plant. A. purpurata is an elegant ornamental plant because of its red bracts
(Chantrachit and Paull 1998). They are relatively new as ornamental or landscape plants. A. purpurata and A. zerumbet variegata are currently valued only
for aesthetic purposes.
Polyphenols such as kaempferol, quercetin, myricetin, isorhamnetin,
flavone C-glycosides and proanthocyanidins have been isolated from Alpinia
species (Williams and Harborne 1977). These polyphenols are antioxidants
acting as hydrogen donors, reducing agents and singlet oxygen quenchers.
These compounds are anti-allergic, anti-artherogenic, antiflammatory, antimicrobial, antithrombotic, cardioprotective and vasodilatory agents (Balasundram et al. 2006). They are also useful in the food and pharmaceutical

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALPINIA SPECIES

837

industries because they can be used as substitutes for the potentially carcinogenic synthetic antioxidants such as butylated hydroxyanisole and butylated
hydroxytoluene (Moure et al. 2001). Because A. galanga and A. zerumbet have
traditionally been used as herbs, it is of interest to evaluate and compare the
total phenolic content (TPC) and antioxidant activity of these two species, in
particular the leaf parts in which no reported work has been done. In addition,
this is the first study on the TPC and antioxidant activities (AOA) of different
Alpinia species, which are essentially ornamental plants. The potential of the
cultivated ornamental species, A. zerumbet variegata and A. purpurata as
potential sources of antioxidant compounds will be evaluated in this paper.

MATERIALS AND METHODS

Collection of Fresh Plant Materials
The leaves and flowers of A. galanga and leaves of A. zerumbet variegata
were harvested from Forest Research Institute Malaysia (FRIM), located
15 km from the Monash University Malaysia. In one sampling, leaves and
rhizomes of A. galangal were taken from the same plant in Bukit Maluri.
Rhizome of A. galanga was also purchased from a wet market. Leaves of A.
zerumbet were collected from Bukit Tinggi, an elevated location 20 km from
FRIM, while A. purpurata leaves and flowers were collected in the vicinity of
the Monash University Malaysia campus.
Chemicals and Reagents
TPC analysis: Folin-Ciocalteus phenol reagent (2N, Fluka, Steinheim,
France), gallic acid (Fluka, 98%), anhydrous sodium carbonate (Fluka, 99%);
diphenyl-2-picrylhydrazyl (DPPH) assay: 1,1-diphenyl-2-picrylhydrazyl
(90%, Sigma, St. Louis, MO); reducing power (RP) assay: ferric chloride
hexa-hydrate (100%, Fisher Scientific, Loughborough, UK), potassium ferricyanide (99%, Unilab, Auburn, Australia), trichloroacetic acid (99.8%, HmbG
Chemicals, Barcelona, Spain), potassium dihydrogen orthophosphate (99.5%,
Fisher Scientific), dipotassium hydrogen phosphate (99%, Merck, Darmstadt,
Germany); ferrous ion chelating (FIC) assay: ferrozine (98%, Acros Organics,
Morris Plains, NJ), ferrous sulphate hepta-hydrate (HmbG Chemicals); bcarotene bleaching (BCB) assay: b-carotene (Type 1: synthetic, Sigma), chloroform (100%, Fisher Scientific), linoleic acid (Fluka) and Tween 40 (Fluka).
Disc-diffusion assay: paper discs (6 mm, Schleicher & Schuell, Keene,
NH), Muller-Hinton agar (Merck), nutrient broth (Oxoid, Basingstoke, Hampshire, England), Sabouraud dextrose agar (Oxoid), tetracycline (Oxoid,

838

L.F. WONG, Y.Y. LIM and M. OMAR

30 mg), penicillin G (Oxoid, 2 units) and amphotericin B (40 mg, Merck

Calbiochem, Darmstadt, Germany) susceptibility discs.
Extraction of Fresh Plant Materials for Antioxidant Studies
Plant materials were thoroughly washed using deionized water, separated
into rhizomes, leaves and flowers, and mopped with tissue paper and air-dried
for approximately 1 h. One gram of the material was ground into fine powder
using liquid nitrogen in a mortar and extracted using 50 mL of methanol. The
powdery plant material was continuously swirled on a rotary orbital shaker for
1 h at room temperature. Extracts were filtered using vacuum filtration and the
filtrate was stored at -20C.
Extraction efficiency test was completed after the second and third extractions. The residue from the first extraction was transferred back into the flask
and extracted again with additional 50 mL solvents. To determine the best
organic solvent to be used in this study, 100% methanol, 100% acetone, 50%
methanol, 50% acetone and dichloromethane were used.
Extraction of Fresh Plant Materials for Antimicrobial Tests
About 10 g of plant materials (leaf, flower and rhizome) were sequentially extracted with acetone, methanol, dichloromethane and water extracts
for an hour. Each plant extract was filtered subsequently. The extracts were
combined and the solvents were rotary evaporated. The water extract in the
final stage was freeze-dried and the combined residue obtained was weighed.
The residues were redissolved in methanol.
TPC
The total phenolic contents of different parts of Alpinia samples were
determined using a modification of the FolinCiocalteu procedure used by
Kahkonen et al. (1999). Samples with 300 mL were mixed with 1.5 mL of
FolinCiocalteus reagent (diluted 10-fold) and 1.2 mL of 7.5% (w/v) sodium
carbonate. The mixtures were allowed to stand in the dark for 30 min before
absorption at 765 nm was measured with a U-1800 Hitachi spectrometer
(Tokyo, Japan). A calibration curve was made for gallic acid (range from 0 to
100 mg/L) and the results were determined from regression equation of this
calibration curve, which was expressed as gallic acid equivalent (GAE) in
mg/100 g material.
Determination of Antioxidant Activity
DPPH Scavenging Radical Activity. The scavenging activity of
extracts on DPPH radical was measured based on a slight modification of the

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALPINIA SPECIES

839

method of Bondet et al. (1997). One milliliter of various concentrations of the

plant extracts was added to 2.0 mL of DPPH solution (5.9 mg/100 mL methanol). All the tubes were allowed to stand for 30 min at room temperature
before the decrease in absorbance at 517 nm was measured. The AOA was
expressed in terms of IC50 (the concentration of extract that shows 50%
inhibition against DPPH). The result was expressed in % scavenging effect,
which was calculated in the following way:

% Scavenging = ( ABlank ASample ) ABlank 100%

where ABlank refers to the absorbance of the control solution and ASample is the
absorbance of the sample solution. The result was also expressed in terms of
ascorbic acid equivalent antioxidant capacity (AEAC), which was calculated
as follows:

AEAC ( mg AA 100 g ) = IC50(ascorbic acid ) IC50(sample) 100,000

The IC50 of ascorbic acid used for calculation of AEAC was 0.00382
(0.00005) mg/mL.
RP. The RP assay was adapted from Juntachote and Berghofer (2005).
One milliliter sample solutions of various dilutions were mixed with 2.5 mL of
phosphate buffer (0.2 M, pH 6) and 2.5 mL of potassium ferricyanide (1%
w/v). These mixtures were incubated for 20 min at 50C. A total of 2.5 mL of
10% trichloroacetic acid was added into the mixture. The mixture was then
separated into aliquots of 2.5 mL and diluted with 2.5 mL of deionized water.
Five hundred microliters of 0.1% (w/v) FeCl3 was added into the solution. The
absorbance was measured at 700 nm after 30 min of equilibration. A calibration curve was made for gallic acid (range from 0 to 100 mg/L) and the results
were expressed as GAE in mg/g sample.
FIC Assay. Chelating ability was determined following the method
reported by Singh and Rajini (2004). Briefly, 2 mM of FeSO4 and 5 mM of
ferrozine were prepared. These reagents were diluted 20 times before use. A
series of extract dilutions (0, 0.2 mL, 0.5 mL and 1 mL) were prepared. One
milliliter of diluted FeSO4 was added into 1 mL of plant extract, followed by
1 mL of diluted ferrozine. The tubes were shaken well and equilibrated for
10 min at room temperature. The absorbances of the extracts were measured
against blank at 562 nm. To prepare the blank solution, 2 mL of ultra-pure
water was added into each diluted extract. The ability of sample to chelate
ferrous ion was calculated with the formula:

840

L.F. WONG, Y.Y. LIM and M. OMAR

Chelating effect (%) = (1 [ Asample Acontrol ]) 100%

where Acontrol is the absorbance of the solution of ferrous sulphate and ferrozine
without plant extract
BCB. The BCB assay was carried out according to the procedure of
Kumazawa et al. (2002) with slight modifications. Three milliliters of
b-carotene solution (5 mg/50 mL chloroform) was mixed with 40 mg of linoleic
acid and 400 mg of Tween 40 emulsifier in a conical flask. The chloroform was
evaporated with nitrogen gas. One hundred milliliters of oxygenated mili-Q
water was added to the flask and the mixture was stirred thoroughly. An initial
absorbance at 470 nm and 700 nm was immediately recorded.
Aliquots (3 mL) of b-carotene/linoleic acid emulsion were mixed with
10, 50 and 100 mL of extract, respectively. The test and control tubes were
capped and incubated in a 50C water bath. The absorbance of the emulsion at
470 and 700 nm was determined after 60 min with a double-beam spectrophotometer. The antioxidant activity was calculated using the formula:

Degradation rate ( DR ) of -carotene = Ln ([ Ainitial Asample ] 60 )

Antioxidant activity (%AOA ) = [( DRcontrol DRsample ) DRcontrol ] 100%
where Ainitial and Asample are absorbance of the b-carotene emulsion before and
after 1 h of incubation. Quercetin was used as a standard.
Antimicrobial Activity Test
Bacterial species Escherichia coli, Pseudomonas aeruginosa, Salmonella
typhi, Bacillus cereus, Micrococcus luteus, Staphylococcus aureus, and fungi
Candida albicans, Saccharomyces cerevisiae, Aspergillus niger and Penicillium albicans were used for antimicrobial activity test. The antimicrobial
activity of Alpinia species have been investigated by agar disc diffusion
method (Mackeen et al. 2000; Ficker et al. 2003).
Antimicrobial screening was performed using Nutrient agar and MullerHinton agar for bacteria while Sabouraud dextrose agar for yeast as well as
fungi (Ficker et al. 2003). Bacteria and yeasts were subcultured in Nutrient
broth and Sabouraud dextrose broth respectively. The turbidity of microorganisms was corrected by using saline until it matches McFarland turbidity
standard of 0.5 (bioMerieux, Lyon, France, approximately 1 to 2 108 cfu/
mL) (NCCLS 1999). Spore suspension was adjusted to 1 105 spores/mL with
sterile water using a Neubauer counting chamber.

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALPINIA SPECIES

841

The dissolved crude extracts (20 mL) were pipetted to sterile filter paper
discs five times, allowing for drying in between. A commercial antibacterial
test disc of Tetracycline (30 mg per disc), Penicillin G (2 IU per disc) and
Amphotericin B (40 mg per disc) were used as positive control. The disc
impregnated with methanol served as negative control.
Each of the discs was placed onto the inoculated agar surface and lightly
tapped to ensure adhesion and these plates were incubated overnight at 37 and
30C for bacteria and yeast, respectively. Fungi were incubated at room temperature for 1 week. Species with an inhibition zones were considered susceptible to samples while those without such a zone were considered resistant.
Statistical Analysis
All measurements were done in triplicate and expressed as
mean standard deviation (SD). Analysis of variance and Students t-test
were used to determine the differences among plants and plant parts. Differences at P < 0.05 were considered to be significant.
RESULTS AND DISCUSSION
Extraction Efficiency
In this study, preliminary experiments revealed that 100% methanol was
the best solvent for the extraction of phenolics from A. galanga leaf as it
yielded a maximum of 78.5 1.4% of compounds in the first extraction. The
second and third extraction yielded only 15.1 1.4% and 6.4 0.1% of
phenolics, respectively. The yields were in a decreasing order of methanol >
dichloromethane > 50% acetone 50% methanol > 100% acetone.
Methanol, besides having higher extraction efficiency, is more efficient in
cell wall degradation as compared with other four organic solvents (Lapornik
et al. 2005). A minimum of 70% methanol is needed to inactivate polyphenol
oxidases, which might produce qualitative and quantitative changes in the
phenolic content of fresh sample (Robards 2003).
TPC
The overall antioxidant activity in a plant sample is contributed mainly by
polyphenols. The TPC values of tested Alpinia species are shown in Table 1.
Among the leaves, A. zerumbet had the highest TPC (2012 289 mg GAE/
100 g) and A. galanga had the lowest (392 50 mg GAE/100 g). The TPC
values of A. purpurata flowers and leaves, and A. zerumbet variegata leaves
did not differ significantly. The TPC values of the four species reported here
are smaller than several Etlingera species reported earlier (Chan et al., 2007a).

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L.F. WONG, Y.Y. LIM and M. OMAR

TABLE 1.
ANTIOXIDANT ACTIVITIES OF DIFFERENT ALPINIA SPECIES
Sample

Part

TPC (GAE
mg/100 g)

AEAC (AA
mg/100 g)

IC50
(mg/mL)

RP
(GAE mg/g)

A. zerumbet
A. zerumbet variegata
A. purpurata

Leaf
Leaf
Leaf
Flower
Leaf
Flower
Rhizome

2012 289a
1154 41b
1189 174b
1338 204b
392 50c
302 91c
214 20d

1834 514a
1251 184a,b
1100 113b
1173 172b
90 36c
98 46c
168 13d

0.26 0.04
0.32 0.05
0.35 0.03
0.34 0.06
4.7 1.5
4.5 1.9
2.3 0.2

8.9 0.9a
6.7 1.0b
7.2 1.0b
7.4 0.9b
1.7 0.7c
1.0 0.3c
1.1 0.2c

A. galanga

Values are reported as mean standard deviation (n = 3). For each column, values followed by the
same letter (ad) are not statistically different at P < 0.05.
TPC, total phenolic content; GAE, gallic acid equivalent; AEAC, ascorbic acid equivalent antioxidant
capacity; RP, reducing power.

However, the TPC value of A. zerumbet leaf is much larger than that of
Phyllanthus amarus (TPC = 13001600 GAE mg/100 g), a herbal plant
widely used in traditional medicine in many Asian countries (Lim and
Murtijaya 2007). It is of interest to note that the TPC values of A. galanga
leaves and flowers were significantly larger than the rhizomes (P < 0.05). In
view of the fact these different parts of plant were from different sources, we
had also sampled the leaves and rhizomes from the same plant, and the results
(leaves, 366 15; rhizomes, 150 22 GAE mg/100 g) confirm the trend
observed. The result is in agreement with a comparative study on the TPC of
leaves and rhizomes of several other ginger species such as Etlingera elatior,
E. maingayi and Zingiber officinale (Chan et al. 2007b), in which both leaves
and rhizomes were taken from the same plants or locations.
Alpinia species is rich in flavonoids such as kaempferol, quercetin and
proanthocyanidins (Williams and Harborne 1977), which contribute toward the
TPC values reported in this work. There is no significant difference between leaf
and flower of A. purpurata and A. galanga. The relatively low phenolic content
and AOA in rhizome might be the result of low sunlight exposure as this part is
always embedded under the soil. The higher antioxidant potential of leaf
indicates that it has better stress resistance and nutritional quality (Lata et al.
2005). Therefore, leaf is a potential dietary food, which is easier to obtain than
rhizome, and which does not result in destructive harvesting of plants.
DPPH
DPPH is a stable radical that is used to screen free-radical-scavenging
ability of compounds. As shown in Table 1, high TPC was accompanied by
low IC50 or high AEAC. High DPPH radical scavenging activity was shown in

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALPINIA SPECIES

843

A. zerumbet, with low IC50 (0.26 0.04 mg/mL) as well as high AEAC values
(1,834 514 AA mg/100 g). The leaves of A. zerumbet variegata, A. purpurata and A. purpurata flower had high TPC values and similar IC50, which
ranged from 0.30.4 mg/mL. It was observed that A. galanga flowers
(4.5 1.9 mg/mL) or leaves (4.7 1.5 mg/mL) had less DPPH scavenging
ability than rhizome (2.3 0.2 mg/mL) in spite of higher TPC. The anomaly
may be due to kinetic factor, in which the 30-min incubation period may not
be sufficient for certain types of polyphenols to reach a steady state in their
reaction with DPPH radicals (Brand-Williams et al. 1995; Huang et al. 2005)
but this time is sufficient for these polyphenols in the extract to be completely
oxidized by Mo(VI) in the FolinCiocalteu reagent solution.
Although IC50 of A. galangal rhizome had been reported to be 0.42 mg
extract/mL (Juntachote and Berghofer 2005), our result cannot be directly
compared with it because as customarily practiced in our laboratory (Chan
et al. 2007a; Lim and Murtijaya 2007) our value was based on weight of fresh
plant material that was extracted by 1 mL of methanol. Another complicating
factor is the different incubation periods used for the DPPH quenching experiment. As mentioned earlier, the decrease in absorbance could depend on its
kinetic behavior, which depends on the structural conformation of the
polyphenols.
RP
The RP values of Alpinia samples are shown in Table 1. A. zerumbet leaf
had the strongest reducing power and A. galanga leaf, flower and rhizome had
the weakest reducing ability. No significant difference in reducing activity was
detected between the different parts of A. galanga. It is observed that IC50 and
RP are related. A low IC50 results in high RP and vice versa. These results are
consistent with Wong et al. (2006) who reported a strong correlation between
DPPH radical scavenging activity and ferric ion reducing activity in twenty
five edible tropical plants.
Chelating Activity on Fe2+ (FIC)
Chelating agents present in plant can bind to transition metal ions such as
Fe2+ or Cu2+. Transition metal ions are pro-oxidants that generate hydroxyl
radicals through Fenton reaction to cause lipid peroxidation in biological and
food system (Antolovich et al. 2002; Mira et al. 2002; Masarwa et al. 2005).
In this study, measurement of chelating activity on Fe2+ of Alpinia species
extracts was done using methanol extracts ranging from 1 to 7 mg per mL. The
results are illustrated in Fig. 1.
A. galanga flowers and leaves showed the best ferrous ion chelating
ability (89.6 and 87.3%, respectively, at 7 mg/mL) while other extracts showed

844

L.F. WONG, Y.Y. LIM and M. OMAR

100
90
80

Chelating (%)

70
60

A purpurata (F)
A zerumbet variegata (L)
A. zerumbet (L)
A galanga (L)
A galanga (R)
A purpurata (L)
A galanga (F)

50
40
30
20
10
0
1

Concentration (mg/mL)
FIG. 1. CHELATING ACTIVITY OF EXTRACTS OF DIFFERENT ALPINIA SPECIES

less metal chelating activity. This suggests that ligands in A. galanga flowers
and leaves compete well with ferrozine. There was a significant overlap of the
values for A. purpurata flowers and leaves and A. zerumbet leaves in 7 mg/mL
samples. The rhizome of A. galanga showed the lowest activity, which was
less than 10% at 7 mg/mL.
In spite of the relatively low TPC of A. galanga flower and leaf, they are
powerful secondary antioxidants that effectively retard ferrous iron catalyzed
lipid oxidation. Ligands in both parts of the plant effectively sequester ferrous
ions by intercepting all coordination sites of metal ions, thus suppressing the
formation of hydroxyl radical via Fenton reaction.
BCB Activity
In BCB assay, the antioxidant capacity to trap lipid peroxyl radicals was
determined. The linoleic acid radical formed upon the abstraction of a hydrogen atom from one of its methylene groups attacked the b-carotene molecules.
This causes loss of the double bonds in b-carotene and, therefore, loss of its
orange color. The greater the potential of the antioxidant compound, the lesser
the depletion of color and the higher the BCB inhibition activity.
The BCB activities of different Alpinia leaves, rhizome and flowers are
presented in a bar chart alongside quercetin in Fig. 2. Alpinia species exhibited
varying degrees of bleaching activity. The antioxidant activity of these extracts

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALPINIA SPECIES

845

0.1 mg/mL

100.0

0.3 mg/mL
0.7 mg/mL

90.0

Antioxidant activity (%)

80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
A. zerumbet A. zerumbet A. galanga
variegata (L)
(L)
(L)

A. purpurata A. purpurata
(L)
(F)

A. galanga A. galanga
(F)
(R)

Concentration (mg/mL)

Quercetin
0.3 1.7 3.3
(g/mL)

FIG. 2. b-CAROTENE BLEACHING ABILITY OF EXTRACTS OF DIFFERENT

ALPINIA SPECIES

increased with increasing concentration. Leaves of A. galanga and A. purpurata showed the highest antioxidant activity, and at 0.7 mg/mL, the activity
was comparable to quercetin at 3.3 mg/mL. The result shows that A. galanga
and A. purpurata leaves offer good protection against lipid-peroxidation
process. The result also shows that the BCB activity of extracts of the leaves
of A. zerumbet variegata and A. zerumbet, which did not differ significantly, is
inferior to that of A. galanga and A. purpurata.
At similar concentration of the extract, flowers and rhizomes of A.
galanga, A. purpurata had BCB activity significantly lower than that of the
leaves. The activity exhibited by A. galanga rhizome was similar at all three
concentrations.
As demonstrated in this study, there is no correlation between TPC,
AEAC and RP with the antioxidant effect measured by BCB. The leaf of A.
zerumbet has the highest TPC, AEAC and RP, but the BCB antioxidant activity
is the weakest. A. galanga leaf has low TPC but has the strongest BCB activity.
This could be due to the relatively high concentration of hydrophobic compounds present in A. galanga leaf and relatively low hydrophobic type in A.
zerumbet in spite of its high total antioxidant content.

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L.F. WONG, Y.Y. LIM and M. OMAR

Antimicrobial Screening Test by Disc Diffusion Method

The inhibition zones measured by using agar disc diffusion method are
illustrated in Table 2. Nutrient agar (NA) and Muller-Hinton agar (MHA) were
used. The results showed they had similar trends. MHA medium is recommended for use in the standardized disk diffusion procedure for antibiotic
susceptibility testing by the Kirby-Bauer method. Such medium has low level
of thymine, thymidine, calcium ions and magnesium ions so as not to interfere
with susceptibility testing (EUCAST 2000; Lalitha 2004). NA is a medium
that support all nonfastidious bacteria.
All the methanol extracts evaluated, except A. galanga leaf, displayed
specific narrow spectrum antimicrobial activity against Bacillus cereus, Micrococcus luteus and Staphylococcus aureus. None of the extracts showed antibacterial activity against Gram negative bacteria tested. This is probably due to
poor penetration of the phenolics through bacterial outer membrane. This
study is in substantial agreement with a previous study (Habsah et al. 2000).
B. cereus and M. luteus were susceptible to A. galanga rhizome, A.
galanga flower and A. purpurata flower extracts. The study indicates the
possibility of using these plants as candidates for extraction of fragrances,
soaps and mouth gurgle as well as toothpaste. Leaf extracts that inhibited S.
aureus has the possibility of use as face wash or as food preservatives. Leaves
of A. zerumbet variegata, A. zerumbet and A. purpurata showed positive
results toward B. cereus, a common food as well as medical or pharmaceutical
materials contaminant. These ornamental plants possess a range of natural
products that may serve as food preservatives as well as eliminating the spore
germination in medical equipments (Prescott et al. 2003). The A. galanga
rhizome extract did not inhibit S. aureus. This result contradicted with the
study by Oonmetta-aree et al. (2006) who reported that S. aureus, B. cereus, E.
coli and S. cerevisiae were sensitive to galanga rhizome extract. This contradiction may be due to the different isolates of S. aureus used.
The rhizome extract showed antimicrobial effect against A. niger. As
stated by Ficker et al. (2003), A. galanga rhizome has a significant antifungal activity. In the search for more antifungal drugs, this observation is
encouraging. Rhizomes are used in traditional remedies either by direct
application to the affected area or as infusion for ingestion. Direct application is useful for skin diseases. The possible active compounds of the root
extract are ACA, p-coumaryl diacetate, palmitic acid, acetoxyeugenol
acetate, 9-octadecenoic acid, eugenol, b-bisabolene, b-farnesene and sesquiphellandrene (Oonmetta-aree et al. 2006). As suggested by Jitoe et al.
(1992) curcumin is also responsible for antimicrobial activity. The combination of these compounds was effective in inhibiting the germination of
spores (Cushnie and Lamb 2005).

 ()
()
()
()
()
()
()
()
()
()
()

Bacillus cereus
Micrococcus luteus
Staphylococcus aureus
Aspergillus niger
Escherichia coli
Pseudomonas aeruginosa
Salmonella typhi
Candida albicans
Saccharomyces cerevisiae
Penicillium albicans
Salmonella choleraesuis

12 (8.7)
14.6 (13.7)
(8.3)
()
()
()
()
()
()
()
()

A. zerumbet
variegata leaf
14.7 (14.3)
18 (16)
(11.7)
()
()
()
()
()
()
()
()

A. zerumbet
leaf
9 (10.7)
11 (10)
10 (9.5)
()
()
()
()
()
()
()
()

A. purpurata
leaf
10.3 (11.4)
14 (15)
()
11.1 (26.9)
()
()
()
()
()
()
()

A. galanga
rhizome

No inhibition zone; mean diameter of the zone of inhibition is in millimeters; average standard deviation is 15%.
Amount of crude extracts (100 mg per disc).
The values in the brackets are results shown using Muller-Hinton agar.

A. galanga
leaf

Samples
Microorganisms

13 (12)
14 (27.4)
()
()
()
()
()
()
()
()
()

A. galanga
flower

11 (10.3)
13 (22.2)
()
()
()
()
()
()
()
()
()

A. purpurata
flower

TABLE 2.
ANTIMICROBIAL ACTIVITIES OF METHANOL EXTRACTS OF ALPINIA SPECIES USING NUTRIENT AND MULLER-HINTON AGAR

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALPINIA SPECIES

847

848

L.F. WONG, Y.Y. LIM and M. OMAR

The antimicrobial agent may inhibit nucleic acid synthesis by intercalating the nucleic bases via the hydroxyl group in the B ring. The phenolics act
synergistically to alter the fluidity of outer and inner membrane and resulting
in a release of cell materials in cytoplasm (Cushnie and Lamb 2005;
Oonmetta-aree et al. 2006; Wong and Kitts 2006). Phenol compounds also
inhibit the energy metabolism, disrupting the electron transport chain as well
as the metabolism in microorganisms. The antimicrobial activity might be the
synergic effects of various phenolic compounds such as flavanone and flavonols (Cushnie and Lamb 2005).
CONCLUSION
Methanol showed high extraction efficiency for A. galanga leaf. This
study indicates that the leaves of A. zerumbet, A. zerumbet variegata as well as
A. purpurata may serve as potential dietary sources of natural antioxidants for
improving human nutrition and health. A. zerumbet leaf has the highest natural
phenolic content and strongest DPPH radical scavenging and ferric reducing
activity among the four tested botanicals. The methanol fraction of crude
extracts exhibited antimicrobial activity toward Gram positive bacteria and
some fungi tested.
ACKNOWLEDGMENT
The authors wish to thank Monash University (Sunway campus) for
financial support.
REFERENCES
AKHTAR, M.S., KHAN, M.A. and MALIK, M.T. 2002. Hypoglycaemic
activity of Alpinia galangal rhizomes and its extracts in rabbits. Fitoterapia 73, 623628.
ANTOLOVICH, M., PRENZLER, P.D., PASTSALIDES, E., MCDONALD,
S. and ROBARDS, K. 2002. Methods for testing antioxidant activity.
Analyst 127, 183198.
BALASUNDRAM, N., SUNDRAM, K. and SAMMAN, S. 2006. Phenolic
compounds in plants and agri-industrial by-products: antioxidant activity,
occurrence, and potential uses. Food Chem. 99, 191203.
BONDET, V., BRAND-WILLIAMS, W. and BERSET, C. 1997. Kinetics and
mechanisms of antioxidant activity using the DPPH free radical method.
Lebensm. Wiss. Technol. 30, 609615.

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALPINIA SPECIES

849

BRAND-WILLIAMS, W., CUVELIER, M.E. and BERSET, C. 1995. Use of

a free radical method to evaluate antioxidant activity. Lebensm. Wiss.
Technol. 28, 2530.
CHAN, E.W.C., LIM, Y.Y. and OMAR, M. 2007a. Antioxidant and antibacterial activity of leaves of Etlingera species (Zingiberaceae) in Peninsular
Malaysia. Food Chem. 104, 15861593.
CHAN, E.W.C., LIM, Y.Y. and LIM, T.Y. 2007b. Total phenolic content and
antioxidant activity of leaves and rhizomes of some ginger species in
Peninsular Malaysia. Gardens Bull. Singapore 59, 4758.
CHANTRACHIT, T. and PAULL, R.E. 1998. Effect of hot water on red ginger
(Alpinia purpurata) inflorescence vase life. Postharvest Biol. Technol.
14, 7786.
CUSHNIE, T.P.T. and LAMB, A.J. 2005. Review: antimicrobial activity of
flavonoids. Int. J. Antimicrobial. Agents 26, 343356.
EUROPEAN COMMITTEE FOR ANTIMICROBIAL SUSCEPTIBILITY
TESTING (EUCAST). 2000. Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution. Eur. Soc. Clin.
Microbiol. Infect Dis. (ESCMID) 6, 509515.
FICKER, C.E., SMITH, M.L., SUSIARTI, S., LEAMAN, D.J., IRAWATI, C.
and ARNASON, J.T. 2003. Inhibition of human pathogenic fungi by
members of Zingiberaceae used by the Kenyah (Indonesian Borneo).
J. Ethnopharmacol. 85, 289293.
GILLMAN, E.F. 1999. Alpinia zerumbet Variegata. University of Florida,
Cooperative Extension Service, Fact Sheet FPS-36, October 1999.
HABSAH, M., AMRAN, M., MACKEEN, M.M., LAJIS, N.H., KIKUZAKI,
H., NAKATANI, N., RAHMAN, A.A. and ALI, A.M. 2000. Screening of
Zingiberaceae extracts for antimicrobial and antioxidant activities.
J. Ethnopharmacol. 72, 403410.
HABSAH, M., NORDIN, H., ALI, A.M., LAJIS, H., SUKARI, M.A., HIN,
Y.Y., KIKUZAKI, H. and NAKATANI, N. 2003. The antioxidative components from Alpinia nutans. Pharm. Biol. 41, 79.
HUANG, D., OU, B. and PRIOR, R.L. 2005. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 53, 18411856.
ITOKAWA, H., MORITA, M. and MIHASHI, S. 1984. Phenolic compounds
from the rhizomes of Alpinia speciosa. Phytochemistry 20, 2503
2506.
JIROVETZ, L., BUCHBAUER, G., SHAFF, M.P. and LEELA, N.K. 2003.
Analysis of the essential oils of the leaves, stems, rhizomes and roots of
the medicinal plant Alpinia galanga from southern India. Acta Pharm. 53,
7381.
JITOE, A., MASUDA, T., TENGAH, I.G.P., SUPRATA, D.N., GARA, I.W.
and NAKATANI, N. 1992. Antioxidant activity of tropical ginger extracts

850

L.F. WONG, Y.Y. LIM and M. OMAR

and analysis of the contained curcuminoids. J. Agric. Food Chem. 40,

13371340.
JUNTACHOTE, T. and BERGHOFER, E. 2005. Antioxidative properties and
stability of ethanolic extracts of Holy Basil and Galangal. Food Chem.
92, 193202.
KAHKONEN, M.P., HOPIA, A.I., VUORELA, H.J., RAUHA, J.P.,
PIHLAJA, K., KUJALA, T.S. and HEINONEN, M. 1999. Antioxidant
activity of plant extracts containing phenolic compounds. J. Agric. Food
Chem. 47, 39543962.
KUMAZAWA, S., TANIGUCHI, M., SUZUKI, Y., SHIMURA, M., KWON,
M.S. and NAKAYAMA, T. 2002. Antioxidant activity of polyphenols in
Carob Pods. J. Agric. Food Chem. 50, 373377.
LALITHA, M.K. 2004. Manual on Antimicrobial susceptibility testing. http://
www.ijmm.org/Antimicrobial (accessed 25 December 2005).
LAPORNIK, B., PROSEK, M. and WONDRA, A.G. 2005. Comparison of
extracts prepared from plant by-products using different solvents and
extraction time. J. Food Eng. 71, 214222.
LATA, B., PREZERADZKA, M. and KOWSKA, M.B. 2005. Great differences in antioxidant properties exist between 56 apple cultivars and
vegetation seasons. J. Agric. Food Chem. 53, 89708978.
LEAL-CARDOSO, J.H., MOREIRA, M.R., PINTA DA CRUZ, G.M., DE
MORAIS, S.M., LAHLOU, M.S. and COELHO-DE-SOUZA, A.N.
2004. Effects of essential oil of Alpinia zerumbet on the compound action
potential of the rat sciatic nerve. Phytomedicine 11, 549553.
LIM, Y.Y. and MURTIJAYA, J. 2007. Antioxidant properties of Phyllanthus
amarus extracts as affected by different drying methods. Lebensm. Wiss.
Technol. 40, 16641669.
MACKEEN, M.M., ALI, A.M., LAJIS, N.H., KAWAZU, K., HASSAN, Z.,
AMRAN, M., HABSAH, M., MOOI, L.Y. and MOHAMED, S.M. 2000.
Antimicrobial, antioxidant, antitumour-promoting and cytotoxic activities of different plant part extracts of Garcinia atroviridis Griff. Ex T.
Andres. J. Ethnopharmacol. 72, 395402.
MASARWA, A., RACHMILOVICH-CALIS, S., MEYERSTEIN, N. and
MEYERSTEIN, D. 2005. Review: oxidation of organic substrates in
aerated aqueous solutions by the Fenton Reagent. Coord. Chem. Rev.
249, 19371943.
MATSUDA, H., PONPIRIYADACHA, Y., MORIKAWA, T., OCHI, M. and
YOSHIKAWA, M. 2003. Gastroprotective effects of phenylpropanoids
from the rhizomes of Alpinia galanga in rats: structural requirements and
mode of action. Eur. J. Pharmacol. 471, 5967.
MIRA, L., FERNANDEZ, M.T., SANTOS, M., ROCHA, R., FLORENCIO,
M.H. and JENNINGS, K.R. 2002. Interactions of flavonoids with iron

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALPINIA SPECIES

851

and copper ions: a mechanism for their antioxidant activity. Free Radic.
Res. 36, 11991208.
MOURE, A., CRUZ, J.M., FRANCO, D., DOMINGUEZ, J.M., SINEIRO, J.,
DOMINGUEZ, H., NUNEZ, M.J. and PARAJO, J.C. 2001. Natural
antioxidants from residual sources. Food Chem. 72, 145171.
NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARD
(NCCLS). 1999. Performance standards for antimicrobial susceptibility
testing: 8th Informal Supplement. Wayne, Pennsylvania: Document
M100-S9, Vol 19.
OONMETTA-AREE, J., SUZUKI, T., GASALUCK, P. and EUMKEB, G.
2006. Antimicrobial properties and action of galangal (Alpinia galanga
Linn.) on Staphylococcus aureus. Lebensm. Wiss. Technol. 39, 1214
1220.
PRESCOTT, L.M., HARLEY, J.P. and KLEIN, D.A. 2003. Microbiology, 5th
Ed., McGraw-Hill, New York.
RAINA, V.K., SRIVASTAVA, S.K. and SYAMSUNDER, K.V. 2002. The
essential oil of greater galangal Alpinia galangal (L.) Wild. from the
lower Himalayan region of India. Flavour Fragrance 17, 358360.
ROBARDS, K. 2003. Strategies for the determination of bioactive phenols in
plants, fruit and vegetables. J. Chromatogr. A 1000, 657691.
SINGH, N. and RAJINI, P.S. 2004. Free scavenging activity of an aqueous
extract of potato pell. Food Chem. 85, 611616.
WILLIAMS, C.A. and HARBORNE, J. 1977. The leaf flavonoids of the
Zingiberales. Biochem. Syst. Ecol. 5, 221229.
WONG, P.Y.Y. and KITTS, D.D. 2006. Studies on the dual antioxidant and
antibacterial properties of Parsley (Petroselinum crispum) and Cilantro
(Coriandrum sativum) Extracts. Food Chem. 97, 505515.
WONG, S.P., LEONG, L.P. and KOH, J.H.W. 2006. Antioxidant activities of
aqueous extracts of selected plants. Food Chem. 99, 775783.
YANG, X. and EILERMAN, R.G. 1999. Pungent principal of Alpinia galangal (L.) Swartz and its application. J. Agric. Food Chem. 47, 16571662.
ZOGHBI, M.G., ANDRADE, E.H. and MAIA, J.G. 1999. Volatile constituents from leaves and flowers of Alpinia speciosa K. Schum. and
A. purpurata (Viell.) Schum. J. Flavour Fragrance 14, 411414.

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