Antioxidant and Antimicrobial Activities of Some Alpinia

jfbc_258 835..851

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF SOMEALPINA 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 zerum-bet variegata and Alpinia purpurata were evaluated for total phenolic content(TPC) and antioxidant activities (AOA). The AOA were investigated using1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power (RP), ferrous ionchelating as well as b-carotene bleaching assays. High antioxidant activitiesshown in leaves of A. zerumbet, A. zerumbet variegata and A. purpurata byusing DPPH and RP assays were associated with high TPC values. In spite oflower TPC values, A. galanga leaves and flowers showed highest chelatingand b-carotene bleaching abilities. The antimicrobial activities were screenedby using disc diffusion method. Extracts from A. galanga and A. purpurataflowers showed the largest zone of inhibition of Micrococcus luteus. Only theextract from A. galanga rhizome showed antifungal activity toward Aspergillusniger. This work shows that leaves of A. zerumbet, A. zerumbet variegata andA. purpurata may serve as potential dietary sources of natural antioxidants.

PRACTICAL APPLICATIONS

Alpinia species are widely used in traditional cures and as food ingredi-ents. Alpinia galanga rhizome is used as spice and food flavoring agent, and itsleaves and inflorescence are consumed as vegetable. Its rhizome is also used totreat diseases such as fungal skin infections, intestinal infections, Type IIdiabetes, bronchitis and rheumatism. Alpinia zerumbet is used as ingredients intraditional health supplement, as diuretic agent and hypertension control.Alpinia zerumbet variegate and Alpinia purpurata are ornamental plants.

1 Corresponding author. TEL: +603 55146103; FAX: +603 55146099; EMAIL: [email protected]

DOI: 10.1111/j.1745-4514.2009.00258.x

Journal of Food Biochemistry 33 (2009) 835–851.© 2009, The Author(s)Journal compilation © 2009, Wiley Periodicals, Inc.

835

INTRODUCTION

The Zingiberaceae is a large diverse family comprising 1,200 speciesbelonging to 49 genera. A prominent member of this family is the Alpiniagenus. In Southeast Asia, Alpinia galanga is commonly known as the greatergalangal or languas (lengkuas). Rhizomes of this species of ginger are exten-sively 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 andBerghofer 2005). Essential oils such as mono-, sesquiterpene and cinnamatederivatives were found (Jirovetz et al. 2003) in various parts of the plant. Itsuse in sweet goods, dressings and personal care products is due to the presenceof pungent compound, 1′-acetoxychavicol acetate (ACA) (Yang and Eilerman1999). A pharmacological study has shown that the rhizome extract has ahypoglycaemic activity (Akhtar et al. 2002). Other than the extensive appli-cations of rhizomes, the young inflorescence and leaves are consumed as ulamor salad, respectively, by the village folk. Infusions of leaves were reported tohave 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 aswell as reducing atrium contractility (Habsah et al. 2003). It can behave asdiuretic, anti-platelet, bacteriostatic and fungistatic agent, as well as having adepressor effect on COP9 signalosome function (Itokawa et al. 1984; Zoghbiet al. 1999). For treatment of intestinal diseases, it is consumed as tea infusion.The extracts were able to inhibit porphyrin photooxidative reaction andshowed antifungal properties (Leal-Cardoso et al. 2004).

Alpinia zerumbet variegata is a plant with green–yellow variegatedleaves and pinkish white flowers (Gillman 1999). It has a distinct spicy fra-grance, and its attractive foliage and flowers make it a favorite as a landscapeplant. A. purpurata is an elegant ornamental plant because of its red bracts(Chantrachit and Paull 1998). They are relatively new as ornamental or land-scape plants. A. purpurata and A. zerumbet variegata are currently valued onlyfor aesthetic purposes.

Polyphenols such as kaempferol, quercetin, myricetin, isorhamnetin,flavone C-glycosides and proanthocyanidins have been isolated from Alpiniaspecies (Williams and Harborne 1977). These polyphenols are antioxidantsacting as hydrogen donors, reducing agents and singlet oxygen quenchers.These compounds are anti-allergic, anti-artherogenic, antiflammatory, anti-microbial, antithrombotic, cardioprotective and vasodilatory agents (Balasun-dram et al. 2006). They are also useful in the food and pharmaceutical

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

industries because they can be used as substitutes for the potentially carcino-genic synthetic antioxidants such as butylated hydroxyanisole and butylatedhydroxytoluene (Moure et al. 2001). Because A. galanga and A. zerumbet havetraditionally been used as herbs, it is of interest to evaluate and compare thetotal phenolic content (TPC) and antioxidant activity of these two species, inparticular 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 differentAlpinia species, which are essentially ornamental plants. The potential of thecultivated ornamental species, A. zerumbet variegata and A. purpurata aspotential 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 variegatawere harvested from Forest Research Institute Malaysia (FRIM), located15 km from the Monash University Malaysia. In one sampling, leaves andrhizomes 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 fromFRIM, while A. purpurata leaves and flowers were collected in the vicinity ofthe Monash University Malaysia campus.

Chemicals and Reagents

TPC analysis: Folin-Ciocalteu’s 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 chloridehexa-hydrate (100%, Fisher Scientific, Loughborough, UK), potassium ferri-cyanide (99%, Unilab, Auburn, Australia), trichloroacetic acid (99.8%, HmbGChemicals, 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); b-carotene bleaching (BCB) assay: b-carotene (Type 1: synthetic, Sigma), chlo-roform (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, Hamp-shire, England), Sabouraud dextrose agar (Oxoid), tetracycline (Oxoid,

837ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALPINIA SPECIES

30 mg), penicillin G (Oxoid, 2 units) and amphotericin B (40 mg, MerckCalbiochem, Darmstadt, Germany) susceptibility discs.

Extraction of Fresh Plant Materials for Antioxidant Studies

Plant materials were thoroughly washed using deionized water, separatedinto rhizomes, leaves and flowers, and mopped with tissue paper and air-driedfor approximately 1 h. One gram of the material was ground into fine powderusing liquid nitrogen in a mortar and extracted using 50 mL of methanol. Thepowdery plant material was continuously swirled on a rotary orbital shaker for1 h at room temperature. Extracts were filtered using vacuum filtration and thefiltrate was stored at -20C.

Extraction efficiency test was completed after the second and third extrac-tions. The residue from the first extraction was transferred back into the flaskand extracted again with additional 50 mL solvents. To determine the bestorganic 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 sequen-tially extracted with acetone, methanol, dichloromethane and water extractsfor an hour. Each plant extract was filtered subsequently. The extracts werecombined and the solvents were rotary evaporated. The water extract in thefinal 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 weredetermined using a modification of the Folin–Ciocalteu procedure used byKahkonen et al. (1999). Samples with 300 mL were mixed with 1.5 mL ofFolin–Ciocalteu’s reagent (diluted 10-fold) and 1.2 mL of 7.5% (w/v) sodiumcarbonate. The mixtures were allowed to stand in the dark for 30 min beforeabsorption at 765 nm was measured with a U-1800 Hitachi spectrometer(Tokyo, Japan). A calibration curve was made for gallic acid (range from 0 to100 mg/L) and the results were determined from regression equation of thiscalibration curve, which was expressed as gallic acid equivalent (GAE) inmg/100 g material.

Determination of Antioxidant Activity

DPPH Scavenging Radical Activity. The scavenging activity ofextracts on DPPH radical was measured based on a slight modification of the


method of Bondet et al. (1997). One milliliter of various concentrations of theplant extracts was added to 2.0 mL of DPPH solution (5.9 mg/100 mL metha-nol). All the tubes were allowed to stand for 30 min at room temperaturebefore the decrease in absorbance at 517 nm was measured. The AOA wasexpressed 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 Blank Sample Blank= −( ) ×A A A 100

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

AEAC mg AA g IC IC ,ascorbic acid sample100 100 00050 50( ) = ×( ) ( )

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 ofphosphate 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 of10% trichloroacetic acid was added into the mixture. The mixture was thenseparated 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. Theabsorbance was measured at 700 nm after 30 min of equilibration. A calibra-tion curve was made for gallic acid (range from 0 to 100 mg/L) and the resultswere expressed as GAE in mg/g sample.

FIC Assay. Chelating ability was determined following the methodreported by Singh and Rajini (2004). Briefly, 2 mM of FeSO4 and 5 mM offerrozine were prepared. These reagents were diluted 20 times before use. Aseries of extract dilutions (0, 0.2 mL, 0.5 mL and 1 mL) were prepared. Onemilliliter of diluted FeSO4 was added into 1 mL of plant extract, followed by1 mL of diluted ferrozine. The tubes were shaken well and equilibrated for10 min at room temperature. The absorbances of the extracts were measuredagainst blank at 562 nm. To prepare the blank solution, 2 mL of ultra-purewater was added into each diluted extract. The ability of sample to chelateferrous ion was calculated with the formula:


Chelating effect sample control% %( ) = − [ ]( ) ×1 100A A

where Acontrol is the absorbance of the solution of ferrous sulphate and ferrozinewithout plant extract

BCB. The BCB assay was carried out according to the procedure ofKumazawa et al. (2002) with slight modifications. Three milliliters ofb-carotene solution (5 mg/50 mLchloroform) was mixed with 40 mg of linoleicacid and 400 mg of Tween 40 emulsifier in a conical flask. The chloroform wasevaporated with nitrogen gas. One hundred milliliters of oxygenated mili-Qwater was added to the flask and the mixture was stirred thoroughly. An initialabsorbance at 470 nm and 700 nm was immediately recorded.

Aliquots (3 mL) of b-carotene/linoleic acid emulsion were mixed with10, 50 and 100 mL of extract, respectively. The test and control tubes werecapped and incubated in a 50C water bath. The absorbance of the emulsion at470 and 700 nm was determined after 60 min with a double-beam spectropho-tometer. The antioxidant activity was calculated using the formula:

Degradation rate DR of -carotene Ln initial sample( ) = [ ]( )β A A 60

Antioxidant activity AOA control sample control%( ) = −( )[ ] ×DR DR DR 1100%

where Ainitial and Asample are absorbance of the b-carotene emulsion before andafter 1 h of incubation. Quercetin was used as a standard.

Antimicrobial Activity Test

Bacterial species Escherichia coli, Pseudomonas aeruginosa, Salmonellatyphi, Bacillus cereus, Micrococcus luteus, Staphylococcus aureus, and fungiCandida albicans, Saccharomyces cerevisiae, Aspergillus niger and Penicil-lium albicans were used for antimicrobial activity test. The antimicrobialactivity of Alpinia species have been investigated by agar disc diffusionmethod (Mackeen et al. 2000; Ficker et al. 2003).

Antimicrobial screening was performed using Nutrient agar and Muller-Hinton agar for bacteria while Sabouraud dextrose agar for yeast as well asfungi (Ficker et al. 2003). Bacteria and yeasts were subcultured in Nutrientbroth and Sabouraud dextrose broth respectively. The turbidity of microorgan-isms was corrected by using saline until it matches McFarland turbiditystandard of 0.5 (bioMerieux, Lyon, France, approximately 1 to 2 ¥ 108 cfu/mL) (NCCLS 1999). Spore suspension was adjusted to 1 ¥ 105 spores/mL withsterile water using a Neubauer counting chamber.


The dissolved crude extracts (20 mL) were pipetted to sterile filter paperdiscs five times, allowing for drying in between. A commercial antibacterialtest disc of Tetracycline (30 mg per disc), Penicillin G (2 IU per disc) andAmphotericin B (40 mg per disc) were used as positive control. The discimpregnated with methanol served as negative control.

Each of the discs was placed onto the inoculated agar surface and lightlytapped to ensure adhesion and these plates were incubated overnight at 37 and30C for bacteria and yeast, respectively. Fungi were incubated at room tem-perature for 1 week. Species with an inhibition zones were considered sus-ceptible to samples while those without such a zone were considered resistant.

Statistical Analysis

All measurements were done in triplicate and expressed asmean � standard deviation (SD). Analysis of variance and Student’s t-testwere used to determine the differences among plants and plant parts. Differ-ences at P < 0.05 were considered to be significant.

RESULTS AND DISCUSSION

Extraction Efficiency

In this study, preliminary experiments revealed that 100% methanol wasthe best solvent for the extraction of phenolics from A. galanga leaf as ityielded a maximum of 78.5 � 1.4% of compounds in the first extraction. Thesecond and third extraction yielded only 15.1 � 1.4% and 6.4 � 0.1% ofphenolics, respectively. The yields were in a decreasing order of methanol >dichloromethane > 50% acetone ª 50% methanol > 100% acetone.

Methanol, besides having higher extraction efficiency, is more efficient incell wall degradation as compared with other four organic solvents (Laporniket al. 2005). A minimum of 70% methanol is needed to inactivate polyphenoloxidases, which might produce qualitative and quantitative changes in thephenolic content of fresh sample (Robards 2003).

TPC

The overall antioxidant activity in a plant sample is contributed mainly bypolyphenols. 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 TPCvalues of A. purpurata flowers and leaves, and A. zerumbet variegata leavesdid not differ significantly. The TPC values of the four species reported hereare smaller than several Etlingera species reported earlier (Chan et al., 2007a).


However, the TPC value of A. zerumbet leaf is much larger than that ofPhyllanthus amarus (TPC = 1300–1600 GAE mg/100 g), a herbal plantwidely used in traditional medicine in many Asian countries (Lim andMurtijaya 2007). It is of interest to note that the TPC values of A. galangaleaves and flowers were significantly larger than the rhizomes (P < 0.05). Inview of the fact these different parts of plant were from different sources, wehad 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 trendobserved. The result is in agreement with a comparative study on the TPC ofleaves and rhizomes of several other ginger species such as Etlingera elatior,E. maingayi and Zingiber officinale (Chan et al. 2007b), in which both leavesand rhizomes were taken from the same plants or locations.

Alpinia species is rich in flavonoids such as kaempferol, quercetin andproanthocyanidins (Williams and Harborne 1977), which contribute toward theTPC values reported in this work. There is no significant difference between leafand flower of A. purpurata and A. galanga. The relatively low phenolic contentand AOA in rhizome might be the result of low sunlight exposure as this part isalways embedded under the soil. The higher antioxidant potential of leafindicates 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 thanrhizome, and which does not result in destructive harvesting of plants.

DPPH

DPPH is a stable radical that is used to screen free-radical-scavengingability of compounds. As shown in Table 1, high TPC was accompanied bylow IC50 or high AEAC. High DPPH radical scavenging activity was shown in

TABLE 1.ANTIOXIDANT ACTIVITIES OF DIFFERENT ALPINIA SPECIES

Sample Part TPC (GAEmg/100 g)

AEAC (AAmg/100 g)

IC50

(mg/mL)RP(GAE mg/g)

A. zerumbet Leaf 2012 � 289a 1834 � 514a 0.26 � 0.04 8.9 � 0.9a

A. zerumbet variegata Leaf 1154 � 41b 1251 � 184a,b 0.32 � 0.05 6.7 � 1.0b

A. purpurata Leaf 1189 � 174b 1100 � 113b 0.35 � 0.03 7.2 � 1.0b

Flower 1338 � 204b 1173 � 172b 0.34 � 0.06 7.4 � 0.9b

A. galanga Leaf 392 � 50c 90 � 36c 4.7 � 1.5 1.7 � 0.7c

Flower 302 � 91c 98 � 46c 4.5 � 1.9 1.0 � 0.3c

Rhizome 214 � 20d 168 � 13d 2.3 � 0.2 1.1 � 0.2c

Values are reported as mean � standard deviation (n = 3). For each column, values followed by thesame letter (a–d) are not statistically different at P < 0.05.TPC, total phenolic content; GAE, gallic acid equivalent; AEAC, ascorbic acid equivalent antioxidantcapacity; RP, reducing power.


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. purpu-rata and A. purpurata flower had high TPC values and similar IC50, whichranged from 0.3–0.4 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 scavengingability than rhizome (2.3 � 0.2 mg/mL) in spite of higher TPC. The anomalymay be due to kinetic factor, in which the 30-min incubation period may notbe sufficient for certain types of polyphenols to reach a steady state in theirreaction 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 completelyoxidized by Mo(VI) in the Folin–Ciocalteu reagent solution.

Although IC50 of A. galangal rhizome had been reported to be 0.42 mgextract/mL (Juntachote and Berghofer 2005), our result cannot be directlycompared with it because as customarily practiced in our laboratory (Chanet al. 2007a; Lim and Murtijaya 2007) our value was based on weight of freshplant material that was extracted by 1 mL of methanol. Another complicatingfactor is the different incubation periods used for the DPPH quenching experi-ment. As mentioned earlier, the decrease in absorbance could depend on itskinetic behavior, which depends on the structural conformation of thepolyphenols.

RP

The RP values of Alpinia samples are shown in Table 1. A. zerumbet leafhad the strongest reducing power and A. galanga leaf, flower and rhizome hadthe weakest reducing ability. No significant difference in reducing activity wasdetected between the different parts of A. galanga. It is observed that IC50 andRP are related. A low IC50 results in high RP and vice versa. These results areconsistent with Wong et al. (2006) who reported a strong correlation betweenDPPH radical scavenging activity and ferric ion reducing activity in twentyfive edible tropical plants.

Chelating Activity on Fe2+ (FIC)

Chelating agents present in plant can bind to transition metal ions such asFe2+ or Cu2+. Transition metal ions are pro-oxidants that generate hydroxylradicals through Fenton reaction to cause lipid peroxidation in biological andfood system (Antolovich et al. 2002; Mira et al. 2002; Masarwa et al. 2005).In this study, measurement of chelating activity on Fe2+ of Alpinia speciesextracts was done using methanol extracts ranging from 1 to 7 mg per mL. Theresults are illustrated in Fig. 1.

A. galanga flowers and leaves showed the best ferrous ion chelatingability (89.6 and 87.3%, respectively, at 7 mg/mL) while other extracts showed


less metal chelating activity. This suggests that ligands in A. galanga flowersand leaves compete well with ferrozine. There was a significant overlap of thevalues for A. purpurata flowers and leaves and A. zerumbet leaves in 7 mg/mLsamples. The rhizome of A. galanga showed the lowest activity, which wasless than 10% at 7 mg/mL.

In spite of the relatively low TPC of A. galanga flower and leaf, they arepowerful secondary antioxidants that effectively retard ferrous iron catalyzedlipid oxidation. Ligands in both parts of the plant effectively sequester ferrousions by intercepting all coordination sites of metal ions, thus suppressing theformation of hydroxyl radical via Fenton reaction.

BCB Activity

In BCB assay, the antioxidant capacity to trap lipid peroxyl radicals wasdetermined. The linoleic acid radical formed upon the abstraction of a hydro-gen 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 itsorange color. The greater the potential of the antioxidant compound, the lesserthe depletion of color and the higher the BCB inhibition activity.

The BCB activities of different Alpinia leaves, rhizome and flowers arepresented in a bar chart alongside quercetin in Fig. 2. Alpinia species exhibitedvarying degrees of bleaching activity. The antioxidant activity of these extracts

0

10

20

30

40

50

60

70

80

90

100

731

Concentration (mg/mL)

Ch

elat

ing

(%

)

A purpurata (F)

A zerumbet variegata (L)

A. zerumbet (L)

A galanga (L)

A galanga (R)

A purpurata (L)

A galanga (F)

FIG. 1. CHELATING ACTIVITY OF EXTRACTS OF DIFFERENT ALPINIA SPECIES


increased with increasing concentration. Leaves of A. galanga and A. purpu-rata showed the highest antioxidant activity, and at 0.7 mg/mL, the activitywas comparable to quercetin at 3.3 mg/mL. The result shows that A. galangaand A. purpurata leaves offer good protection against lipid-peroxidationprocess. The result also shows that the BCB activity of extracts of the leavesof A. zerumbet variegata and A. zerumbet, which did not differ significantly, isinferior 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 theleaves. The activity exhibited by A. galanga rhizome was similar at all threeconcentrations.

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 activityis 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 com-pounds present in A. galanga leaf and relatively low hydrophobic type in A.zerumbet in spite of its high total antioxidant content.

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

Concentration (mg/mL)

An

tio

xid

ant

acti

vity

(%

)

0.1 mg/mL

0.3 mg/mL

0.7 mg/mL

A. zerumbet variegata (L)

A. zerumbet (L)

A. galanga (L)

A. purpurata (L)

A. purpurata (F)

A. galanga (F)

A. galanga (R)

Quercetin 0.3 1.7 3.3 (µg/mL)

FIG. 2. b-CAROTENE BLEACHING ABILITY OF EXTRACTS OF DIFFERENTALPINIA SPECIES


Antimicrobial Screening Test by Disc Diffusion Method

The inhibition zones measured by using agar disc diffusion method areillustrated in Table 2. Nutrient agar (NA) and Muller-Hinton agar (MHA) wereused. The results showed they had similar trends. MHA medium is recom-mended for use in the standardized disk diffusion procedure for antibioticsusceptibility testing by the Kirby-Bauer method. Such medium has low levelof thymine, thymidine, calcium ions and magnesium ions so as not to interferewith susceptibility testing (EUCAST 2000; Lalitha 2004). NA is a mediumthat support all nonfastidious bacteria.

All the methanol extracts evaluated, except A. galanga leaf, displayedspecific narrow spectrum antimicrobial activity against Bacillus cereus, Micro-coccus luteus and Staphylococcus aureus. None of the extracts showed anti-bacterial activity against Gram negative bacteria tested. This is probably due topoor penetration of the phenolics through bacterial outer membrane. Thisstudy 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 thepossibility 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. Leavesof A. zerumbet variegata, A. zerumbet and A. purpurata showed positiveresults toward B. cereus, a common food as well as medical or pharmaceuticalmaterials contaminant. These ornamental plants possess a range of naturalproducts that may serve as food preservatives as well as eliminating the sporegermination in medical equipments (Prescott et al. 2003). The A. galangarhizome extract did not inhibit S. aureus. This result contradicted with thestudy 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 contra-diction may be due to the different isolates of S. aureus used.

The rhizome extract showed antimicrobial effect against A. niger. Asstated by Ficker et al. (2003), A. galanga rhizome has a significant antifun-gal activity. In the search for more antifungal drugs, this observation isencouraging. Rhizomes are used in traditional remedies either by directapplication to the affected area or as infusion for ingestion. Direct applica-tion is useful for skin diseases. The possible active compounds of the rootextract are ACA, p-coumaryl diacetate, palmitic acid, acetoxyeugenolacetate, 9-octadecenoic acid, eugenol, b-bisabolene, b-farnesene and ses-quiphellandrene (Oonmetta-aree et al. 2006). As suggested by Jitoe et al.(1992) curcumin is also responsible for antimicrobial activity. The combi-nation of these compounds was effective in inhibiting the germination ofspores (Cushnie and Lamb 2005).


TAB

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leaf

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leaf

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10.3

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11(1

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Mic

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(–)

14.6

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11(1

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13(2

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Stap

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The antimicrobial agent may inhibit nucleic acid synthesis by intercalat-ing the nucleic bases via the hydroxyl group in the B ring. The phenolics actsynergistically to alter the fluidity of outer and inner membrane and resultingin a release of cell materials in cytoplasm (Cushnie and Lamb 2005;Oonmetta-aree et al. 2006; Wong and Kitts 2006). Phenol compounds alsoinhibit the energy metabolism, disrupting the electron transport chain as wellas the metabolism in microorganisms. The antimicrobial activity might be thesynergic effects of various phenolic compounds such as flavanone and fla-vonols (Cushnie and Lamb 2005).

CONCLUSION

Methanol showed high extraction efficiency for A. galanga leaf. Thisstudy indicates that the leaves of A. zerumbet, A. zerumbet variegata as well asA. purpurata may serve as potential dietary sources of natural antioxidants forimproving human nutrition and health. A. zerumbet leaf has the highest naturalphenolic content and strongest DPPH radical scavenging and ferric reducingactivity among the four tested botanicals. The methanol fraction of crudeextracts exhibited antimicrobial activity toward Gram positive bacteria andsome fungi tested.

ACKNOWLEDGMENT

The authors wish to thank Monash University (Sunway campus) forfinancial support.

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Antioxidant and Antimicrobial Activities of Some Alpinia