ru

id

MaiaiaSud

Theilade were analysed by capillary GC and GCMS. Forty-six constituents were identied in the leaf oil,while 54 were identied in the oil from the rhizomes. The leaf oil was clearly dominated by b-caryophyl-lene (31.7%), while the oil from the rhizomes was predominantly monoterpenoid, with camphene

which are antibacterial agents and shogaols (Kim et al., 2008; Park,Bae, & Lee, 2008), diarylheptanoids (Zhou et al., 2007), phenylbut-enoids (Jitoe, Masuda, & Nakatani, 1993), avanoids (Dae, Han,Park, Jhon, & Seo, 2004), diterpenoids (Akiyama et al., 2006), andsesquiterpenoids (Dae & Seo, 2005).

Zingiber ofcinale var. rubrum Theilade is distributed mainly inPeninsular Malaysia, where it is known as halia bara. This herb is

The emergence of multidrug resistant pathogens threatened theclinical efcacy of many existing antibiotics. This situation fuelsthe ongoing research to discover antimicrobial agents from naturalorigins (Eldeen, Van Heerden, & Van Staden, 2010). The methanolextract of Zingiber ofcinale rhizomes was reported to possess sig-nicant antibacterial activities against Escherichia coli, Salmonellaenteriditis and Staphylococcus aureus during an in vitro investigation(Anbu Jeba Sunilson, Suraj, Rejitha, & Anandarajagopa, 2009).Hence, in continuation of our investigation on the essential oilsof the Zingiberaceae family and their antimicrobial activities

* Corresponding author. Tel.: +60 3 79674064; fax: +60 3 79674193.

Food Chemistry 124 (2011) 514517

Contents lists availab

he

lseE-mail address: khalijah@um.edu.my (K. Awang).1. Introduction

The genus Zingiber comprises about 85 species of herbs mostlydistributed in East Asia and tropical Australia. Many of these areused as food and for traditional treatment of a variety of ailments(Sabulal et al., 2006). The essential oil composition of many ofthese herbs and their biological activities has been the subject ofnumerous previous studies (Bordoloi, Sperkova, & Leclercq, 1999;Kami, Nakayama, & Hayashi, 1972; Prakash, Pant, & Mathela,2006; Sabulal et al., 2006; Sacchetti et al., 2005; Srivastava, Srivast-ava, & Shah, 2002; Vahirua-Lechat, Menut, Lamaty, & Bessiere,1996; Zhannan, Shiqiong, Quancai, Chao, & Zhengwen, 2009). Phy-tochemical investigation of the rhizomes of several Zingiber sp. hasrevealed the presence of bioactive compounds, such as gingerols,

cultivated for its medicinal value. Its rhizome is a common ingredi-ent in folk medicine (jamu) for treating stomach discomfort,tumours, relieving rheumatic pains, and as a post partummedicine(Ibrahim et al., 2008). Halia bara is morphologically similar to thecommon ginger (halia), but the rhizomes of this variant are smallerand more pungent, red on the outside with a yellow to pinkishcross-section, while the base of its leaf shoot is red. Unlike thecommon ginger, the petiole of halia bara is reddish when young,and the lip is scarlet red mottled with cream (Ibrahim et al.,2008). Little is known about the chemical constituents of thisvariety apart from a paper which reported the composition of therhizome oil, which comprised mainly geranial, neral and geranylacetate (Malek et al., 2005).

Infectious diseases are the leading cause of death worldwide.Received 23 February 2010Received in revised form 4 May 2010Accepted 16 June 2010

Keywords:Zingiber ofcinale var. rubrum TheiladeHalia baraEssential oilsAntibacterial activityb-CaryophylleneCamphene0308-8146/$ - see front matter 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.06.062(14.5%), geranial (14.3%), and geranyl acetate (13.7%) the three most abundant constituents. The evalua-tion of antibacterial activities using the micro-dilution technique revealed that both the leaf and rhizomeoils were moderately active against the Gram-positive bacteria Bacillus licheniformis, Bacillus spizizenii andStaphylococcus aureus, and the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae and Pseudo-monas stutzeri.

2010 Elsevier Ltd. All rights reserved.Article history: The essential oils obtained by hydrodistilation of the leaves and rhizomes of Zingiber ofcinale var. rubrumEssential oils of Zingiber ofcinale var. rubantibacterial activities

Yasodha Sivasothy a, Wong Keng Chong b, Abdul HamKhalijah Awang a,*aDepartment of Chemistry, Faculty of Science, University Malaya, 50603 Kuala Lumpur,b School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysc School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysd Faculty of Forestry, University of Khartoum, Shambat Campus, 13314 Khartoum Bahri,

a r t i c l e i n f o a b s t r a c t

Food C

journal homepage: www.ell rights reserved.m Theilade and their

b, Ibrahim M. Eldeen c,d, Shaida Fariza Sulaiman c,

laysia

an

le at ScienceDirect

mistry

vier .com/locate / foodchem

growth, 50 ll of 0.2 mg/ml p-iodonitrotetrazolium violet (INT)

(14.5%), geranyl acetate (13.7%), geranial (14.3%), neral (7.7%),

hemi(Wong, Sivasothy, & Boey, 2006a, 2006b, 2006c; Ibrahim et al.,2009a, 2009b), this prompted us to investigate the chemical com-position of the leaf and rhizome oils of halia bara in detail, and uti-lise these oils in an attempt to explore alternative sources ofantibacterial agents to treat multidrug resistant pathogens thatcause infections and food poisoning, which to the knowledge ofthe authors have not been previously reported.

2. Materials and methods

2.1. Plant material

Fresh leaves and rhizomes of halia bara were purchased from amarket in Negeri Sembilan in September, 2009, and a voucherspecimen (KU 0107) has been deposited with the herbarium ofUniversity of Malaya.

2.2. Solvents and chemicals

Pentane (GCMS grade) and ethanol (analytical grade) werepurchased from Merck (Germany) and Systerm (Malaysia), respec-tively. The homologous series of n-alkanes (C6C30) was purchasedfrom Dr. Ehrenstorfer Gmbh (Germany). Reference compoundswere obtained from SigmaAldrich (USA). MuellerHinton brothwas purchased from OXOID (England), and Tetracap 250 from Ho-vid (Malaysia). p-Iodonitrotetrazolium violet (INT) was obtainedfrom SigmaAldrich (USA).

2.3. Isolation of essential oils

Fresh leaves (45 g) and homogenised rhizomes (125 g) wereseparately hydrodistiled for 4 h in an all-glass apparatus similarto that described in the British Pharmacopoeia, using pentane asthe collecting solvent. The solvent was carefully removed using agentle stream of nitrogen gas, yielding yellow aromatic oils in eachcase. The oil yields (w/w) were 0.03% (leaves) and 0.02% (rhi-zomes), all on a fresh weight-basis.

2.4. Gas chromatography (GC)

GC analysis was carried out using an Agilent 7890A GC Systemequipped with a FID and an Agilent 7683B Series auto-injector.HP-5MSUI (30 m 0.25 mmid, lm thickness 0.25 lm) fused-silicacapillary column (J.W. Scientic) was employed. The operating con-ditions were as follows: initial oven temperature, 50 C for 5 min,then to 150 C at 4 C min1 and held for 5 min, then to 250 C at4 C min1 and held for 10 min; injector and detector temperatures,275 C; carrier gas, 1.0 ml min1 N2; injection volume, 0.2 ll; splitratio, 50:1. The quantitative data were obtained electronically fromFID area per cent without the use of correction factors.

2.5. Gas chromatographymass spectrometry (GCMS)

GCMS analysis was performed using an Agilent 6890 N Net-work GC System equipped with an Agilent 7683B Series auto-injec-tor, coupled to an Agilent 5975 Inert Mass Selective Detector andthe same capillary GC conditions as described above. The carriergas used was He at 1.0 min1. The signicant MS operating param-eters were: ionisation voltage, 70 eV; ion source temperature230 C; mass range 50600 U.

2.6. Identication of constituents

Y. Sivasothy et al. / Food CThe constituents were identied by comparison of their massspectra with those of authentic compounds or with referencegeraniol (7.3%), and 1,8-cineole (5.0%). The presence of neral andgeranial possibly contributed to the strong aroma reminiscent oflemon in the rhizomes similar to those of the common ginger fromwere added to each well, and the plates were incubated at 37 Cfor 30 min. Bacterial growth in the wells was indicated by a redcolour, whereas clear wells indicated the absence of active bacte-rial growth. The assay was repeated three times.

3. Results and discussion

3.1. Composition of the essential oils

Table 1 lists the oil constituents identied in the leaves and rhi-zomes of halia bara, the relative GC peak areas of these constitu-ents, and their experimental retention indices on the HP-5 MS UIcolumn.

Forty-six compounds, constituting 91.7% of the leaf oil of haliabara, were identied. Sesquiterpenoids (47.1%) and monoterpe-noids (42.6%) were dominant, although these gures were largelydue to b-caryophyllene (31.7%), geranial (13.1%), neral (9.8%) andcaryophyllene oxide (6.3%). Other quantitatively signicantconstituents included geraniol (4.4%), a-pinene (4.1%) and trans,-trans-a-farnesene (3.2%). The most abundant component, b-caryo-phyllene, is known for its anti-inammatory and local anaestheticactivities. It is used in spice blends, citrus avours, soaps, deter-gents, creams and lotions and also in a variety of food products.It has also been identied as a volatile compound emitted by plantsinto the atmosphere in response to herbivore attack (Sabulal et al.,2006).

The oil from the rhizomes of this variety yielded 54 identiedconstituents, accounting for 95.5% of the sample. The oil was veryrich in monoterpenoids (81.9%), comprising mainly camphenespectra in the computer library (NIST 05), and conrmed by com-parison of retention indices with those of authentic compounds, orwith data in the literature (Adams, 2001).

2.7. Micro-dilution antibacterial assay

The serial dilution technique by Eloff in 1998 (Eldeen, Elgorashi,& van Staden, 2005), using 96-well micro-plates was employed todetermine the minimum inhibitory concentration (MIC) of theessential oils for antibacterial activity. Two millilitres cultures ofthree Gram-positive bacterial strains, Bacillus licheniformis(ATCC12759), Bacillus spizizenii (ATCC6633) and Staphylococcusaureus (ATCC12600), and three Gram-negative bacterial strains,Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC13883)and Pseudomonas stutzeri (ATCC17588), were prepared and placedin an incubator overnight at 37 C. The overnight cultures were di-luted with sterile MuellerHinton (MH) broth (1 ml bacteria/50 mlMH) to yield a density of bacterial cells between 106108 cells/ml.The extracts were resuspended to a concentration of 10 mg/mlwith ethanol, to yield a nal concentration of 2.5 mg/ml in the as-say. For each of the six bacteria used, 100 ll of redissolved extractwere serially diluted twofold with 100 ll of sterile distilled water,in a sterile 96-well micro-plate. A similar twofold serial dilution oftetracycline (1 mg/ml) was used as a positive control against eachbacterium. Ethanol (100%) was used as a negative control to yield anal concentration of 25% in the assay. One hundred microlitres ofeach bacterial culture were added to each well. The plates werecovered and incubated overnight at 37 C. To indicate bacterial

stry 124 (2011) 514517 515Australia (Wohlmuth, Smith, Brooks, Myers, & Leach, 2006). Bor-neol, bornyl acetate and 1,8-cineole, together, probably accountedfor a trace of its camphoraceous odour (Bauer, Garbe, & Surburg,

emistry 124 (2011) 514517516 Y. Sivasothy et al. / Food Ch2001). It is interesting that b-caryophyllene, the most abundantconstituent of the leaf oil, was only found at a low level of 1.0%

in the oil from the rhizomes. A previous investigation of the rhi-zome oil of halia bara by Malek et al. (2005) also revealed a highcontent of monoterpenoids (64.6%). They, however, identied only19 constituents among which 17 were common to the presentstudy, and found much lower levels of camphene (1.8%) and gera-nyl acetate (8.8%). They did not detect geraniol but reported muchhigher levels of neral (14.2%), geranial (28.4%) and b-sesquiphel-landrene (9.9%). These marked differences in the composition of

bacteria (with MIC values of 0.310.63 mg/ml). This is in accor-

Table 1Constituents identied in the leaf and rhizome oils of Z. ofcinale var. rubrum Thielade(halia bara).

Constituent RI (HP-5MS UI) Area (%)a

Leaves Rhizomes

2-Heptanol 902 0.9 0.1Tricyclene 922 0.2a-Pineneb 934 4.1 3.6Campheneb 949 t 14.5Sabinene 974 0.2 0.1b-Pineneb 977 2.0 0.66-Methyl-5-hepten-2-one 988 0.2 0.2Myrceneb 992 1.3 2.0a-Phellandreneb 1004 0.2 0.2d-3-Carene 1010 0.1 0.1p-Cymene 1025 0.1 0.1Limonene 1029 2.5b-Phellandrene 1030 2.6 1,8-Cineole 1032 0.7 5.0cis-b-Ocimene 1039 0.1 2-Heptyl acetate 1044 0.2 0.3Trans-b-ocimene 1049 0.2 c-Terpinene 1060 0.1 0.1Terpinolene 1089 0.42-Nonanone 1093 0.2Linaloolb 1100 1.1 2.3Trans-sabinene hydrate 1122 0.1Camphorb 1147 0.3Camphene hydrate 1151 0.2Citronellal 1155 0.1 0.1Isoborneol 1160 0.1Borneolb 1168 2.9Terpinen-4-ol 1180 0.3 0.3a-Terpineolb 1193 0.4 1.1Myrtenal 1199 0.2 0.1Linalyl formate 1216 0.5 b-Citronellol 1229 0.4Neralb 1243 9.8 7.7Geraniol 1256 4.4 7.3Trans-2-decenal 1259 0.3 Geranialb 1273 13.1 14.3Bornyl acetateb 1289 1.42-Undecanone 1295 0.2 0.1Myrtenyl acetate 1329 0.1Neryl acetate 1366 0.1a-Copaene 1380 0.6 0.2Geranyl acetateb 1384 1.0 13.7b-Elemene 1395 0.2Isocaryophyllene 1411 0.1 b-Caryophyllene 1424 31.7 1.0a-Humuleneb 1458 0.9 1.1Allo-aromadendrene 1465 0.3 0.2Ar-curcumeneb 1485 1.0Germacrene D 1486 0.2 a-Zingibereneb 1495 3.2a-Selinene 1496 0.3 a-Muurolene 1503 1.1 0.3Trans,trans-a-farnesene 1508 3.2 1.8b-Sesquiphellandreneb 1524 1.6d-Cadinene 1525 0.3 a-Elemol 1549 0.6Trans-nerolidol 1561 0.6 0.1Caryophyllene oxide 1587 6.3 0.2c-Eudesmol 1653 0.6Caryophyllenedienol I 1641 0.4 b-Eudesmol 1652 0.3 0.1a-Bisabolol 1690 0.3Cis,cis-farnesol 1717 0.3 0.1Trans,trans-farnesol 1723 0.2 Trans,trans-farnesal 1730 0.3 0.1Phytol 2058 0.2

91.7% 95.5%

a Percentage of total FID area obtained on HP-5 MS UI column, t = ( B. spizizenii > P. stutzeri > K. pneumoniae >E. coli, while for the rhizome oil, the order is: B. licheniformis > B.spizizenii > S. aureus = E. coli > K. pneumoniae > P. stutzeri. S. aureus,E. coli and Bacillus sp. are agents of food poisoning (Kivrak et al.,2009). The low MIC values of the leaf, rhizome and root oils seenhere against B. licheniformis, B. spizizenii, S. aureus and E. coli wouldsuggest that the oils from halia bara could possibly be used as nat-ural preservatives against food-borne pathogens or for delaying

Table 2Antibacterial activity (MIC) of the leaf and rhizome oils of Z. ofcinale var. rubrumThielade (halia bara) as determined by the micro-dilution assay.

Bacterial Strains MIC (mg/ml)

Leaf oil Rhizome oil Tetracycline

Bacillus licheniformis (ATCC12759) 0.16 0.16 1.0 103Bacillus spizizenii (ATCC6633) 0.24 0.24 1.8 103Staphylococcus aureus (ATCC12600) 0.16 0.31 7.7 103Escherichia coli (ATCC25922) 0.63 0.31 15.6 103the rhizome oil determined by Malek et al. (2005) from that ofthe present study could be attributed to the source, cultivation,vegetative stage and growing season of the plant under investiga-tion (Sari et al., 2006).

There are also signicant differences when the present resultsare compared with the composition of the rhizome oil of the com-mon ginger collected from various locations, as the rhizome oil ofthe common ginger is typically characterised by high percentagesof sesquiterpene hydrocarbons, in particular, a-zingiberene, ar-curcumene b-bisabolene and b-sesquiphellandrene (Norajit, Lao-hakunjit, & Kerdchoechuen, 2007; Onyenekwe & Hashimoto,1999; Pino, Marbot, Rosado, & Batista, 2004; Singh, Maurya, Cata-lan, & de Lampasona, 2005; Wohlmuth et al., 2006).

3.2. Antibacterial activity of the essential oils

The leaf and rhizome oils were tested against three Gram-posi-tive (B. licheniformis,B. spizizenii,S. aureus) and three Gram-negative(E. coli, K. pneumoniae, P. stutzeri) bacteria. The results (Table 2)from the bioassays revealed that the leaf oil and the oil from therhizomes of halia bara possessed moderate antibacterial activity(MIC values of 0.160.63 mg/ml) against all tested bacterial strains,which could have resulted from the presence of caryophylleneoxide, a-pinene, a-terpineol, linalool, 1,8-cineole and geraniol,compounds that are known to possess antibacterial activity.Although present in low concentrations, the aforementioned con-stituents could have imparted a signicant effect on the antibacte-rial activities of the oils via a synergistic effect (Giles, Zhao, An, &Agboola, 2010; Vagionas, Graikou, Ngassapa, Runyoro, & Chinou,2007a; Vagionas et al., 2007b). In general, both oils exhibited bet-ter antibacterial activity against the Gram-positive bacteria (withMIC values of 0.160.31 mg/ml) than against the Gram-negativeKlebsiella pneumoniae (ATCC13883) 0.47 0.47 3.7 103Pseudomonas stutzeri (ATCC17588) 0.31 0.63 8.1 103

food spoilage. However, further investigation on the activities of Ibrahim, H., Awang, K., Ali, N.A.M., Malek, S.N.A., Jantan, I., & Syamsir, D.R. (2008).

Y. Sivasothy et al. / Food Chemistry 124 (2011) 514517 517these oils against other food-borne pathogens (Listeria sp., Salmo-nella sp.) should be conducted.

4. Conclusions

The leaf oil and the oil from the rhizomes of halia bara differedin chemical composition, the former comprising mainly sesquit-erpenoids and monoterpenoids in approximately equal amounts,while the latter was dominated by monoterpenoids. Antimicrobialassays of these oils have demonstrated that the Gram-positive bac-teria in this study were more sensitive to both the oils compared tothe Gram-negative bacteria. Toward the leaf oil,B. licheniformis andS. aureus were the most sensitive strains, while E. coli was the mostresistant strain. B. licheniformis and P. stutzeri were the most sensi-tive and resistant strains, respectively, toward the oil of the rhi-zomes. The sensitivity of the B. licheniformis, B. spizizenii, S.aureus and E. coli strains toward both oils would suggest that theseoils may be promising natural preservatives in the food industry.However, it is commendable that further analysis should be carriedout on other food poisoning agents, such as Listeria sp., Salmonellasp.

Acknowledgements

The authors acknowledge the research grant 5702031007 (Sci-ence Fund) provided by the French Government that has resultedin this article. The authors wish to thank Mr. Teo, Mr. Ray andMr. Din in assisting with the preparation of the voucher specimen,and Ms. Saripah in assisting with the GCMS analysis.

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Essential oils of Zingiber officinale var. rubrum Theilade and their antibacterial activitiesIntroductionMaterials and methodsPlant materialSolvents and chemicalsIsolation of essential oilsGas chromatography (GC)Gas chromatographymass spectrometry (GCMS)Identification of constituentsMicro-dilution antibacterial assay

Results and discussionComposition of the essential oilsAntibacterial activity of the essential oils

ConclusionsAcknowledgementsReferences