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Food Chemistry 124 (2011) 514517

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Essential oils of Zingiber ofcinale var. rubrum Theilade and their antibacterial activities
Yasodha Sivasothy a, Wong Keng Chong b, Abdul Hamid b, Ibrahim M. Eldeen c,d, Shaida Fariza Sulaiman c, Khalijah Awang a,*

Department of Chemistry, Faculty of Science, University Malaya, 50603 Kuala Lumpur, Malaysia School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia d Faculty of Forestry, University of Khartoum, Shambat Campus, 13314 Khartoum Bahri, Sudan
b c

a r t i c l e

i n f o

a b s t r a c t
The essential oils obtained by hydrodistilation of the leaves and rhizomes of Zingiber ofcinale var. rubrum 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-caryophyllene (31.7%), while the oil from the rhizomes was predominantly monoterpenoid, with camphene (14.5%), geranial (14.3%), and geranyl acetate (13.7%) the three most abundant constituents. The evaluation of antibacterial activities using the micro-dilution technique revealed that both the leaf and rhizome oils were moderately active against the Gram-positive bacteria Bacillus licheniformis, Bacillus spizizenii and Staphylococcus aureus, and the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae and Pseudomonas stutzeri. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 23 February 2010 Received in revised form 4 May 2010 Accepted 16 June 2010

Keywords: Zingiber ofcinale var. rubrum Theilade Halia bara Essential oils Antibacterial activity b-Caryophyllene Camphene

1. Introduction The genus Zingiber comprises about 85 species of herbs mostly distributed in East Asia and tropical Australia. Many of these are used as food and for traditional treatment of a variety of ailments (Sabulal et al., 2006). The essential oil composition of many of these herbs and their biological activities has been the subject of numerous previous studies (Bordoloi, Sperkova, & Leclercq, 1999; Kami, Nakayama, & Hayashi, 1972; Prakash, Pant, & Mathela, 2006; Sabulal et al., 2006; Sacchetti et al., 2005; Srivastava, Srivastava, & Shah, 2002; Vahirua-Lechat, Menut, Lamaty, & Bessiere, 1996; Zhannan, Shiqiong, Quancai, Chao, & Zhengwen, 2009). Phytochemical investigation of the rhizomes of several Zingiber sp. has revealed the presence of bioactive compounds, such as gingerols, which are antibacterial agents and shogaols (Kim et al., 2008; Park, Bae, & Lee, 2008), diarylheptanoids (Zhou et al., 2007), phenylbutenoids (Jitoe, Masuda, & Nakatani, 1993), avanoids (Dae, Han, Park, Jhon, & Seo, 2004), diterpenoids (Akiyama et al., 2006), and sesquiterpenoids (Dae & Seo, 2005). Zingiber ofcinale var. rubrum Theilade is distributed mainly in Peninsular Malaysia, where it is known as halia bara. This herb is
* Corresponding author. Tel.: +60 3 79674064; fax: +60 3 79674193. E-mail address: (K. Awang). 0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.06.062

cultivated for its medicinal value. Its rhizome is a common ingredient in folk medicine (jamu) for treating stomach discomfort, tumours, relieving rheumatic pains, and as a post partum medicine (Ibrahim et al., 2008). Halia bara is morphologically similar to the common ginger (halia), but the rhizomes of this variant are smaller and more pungent, red on the outside with a yellow to pinkish cross-section, while the base of its leaf shoot is red. Unlike the common 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 this variety apart from a paper which reported the composition of the rhizome oil, which comprised mainly geranial, neral and geranyl acetate (Malek et al., 2005). Infectious diseases are the leading cause of death worldwide. The emergence of multidrug resistant pathogens threatened the clinical efcacy of many existing antibiotics. This situation fuels the ongoing research to discover antimicrobial agents from natural origins (Eldeen, Van Heerden, & Van Staden, 2010). The methanol extract of Zingiber ofcinale rhizomes was reported to possess signicant antibacterial activities against Escherichia coli, Salmonella enteriditis and Staphylococcus aureus during an in vitro investigation (Anbu Jeba Sunilson, Suraj, Rejitha, & Anandarajagopa, 2009). Hence, in continuation of our investigation on the essential oils of the Zingiberaceae family and their antimicrobial activities

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(Wong, Sivasothy, & Boey, 2006a, 2006b, 2006c; Ibrahim et al., 2009a, 2009b), this prompted us to investigate the chemical composition of the leaf and rhizome oils of halia bara in detail, and utilise these oils in an attempt to explore alternative sources of antibacterial agents to treat multidrug resistant pathogens that cause infections and food poisoning, which to the knowledge of the authors have not been previously reported. 2. Materials and methods 2.1. Plant material Fresh leaves and rhizomes of halia bara were purchased from a market in Negeri Sembilan in September, 2009, and a voucher specimen (KU 0107) has been deposited with the herbarium of University of Malaya. 2.2. Solvents and chemicals Pentane (GCMS grade) and ethanol (analytical grade) were purchased from Merck (Germany) and Systerm (Malaysia), respectively. The homologous series of n-alkanes (C6C30) was purchased from Dr. Ehrenstorfer Gmbh (Germany). Reference compounds were obtained from SigmaAldrich (USA). MuellerHinton broth was purchased from OXOID (England), and Tetracap 250 from Hovid (Malaysia). p-Iodonitrotetrazolium violet (INT) was obtained from SigmaAldrich (USA). 2.3. Isolation of essential oils Fresh leaves (45 g) and homogenised rhizomes (125 g) were separately hydrodistiled for 4 h in an all-glass apparatus similar to that described in the British Pharmacopoeia, using pentane as the collecting solvent. The solvent was carefully removed using a gentle stream of nitrogen gas, yielding yellow aromatic oils in each case. The oil yields (w/w) were 0.03% (leaves) and 0.02% (rhizomes), all on a fresh weight-basis. 2.4. Gas chromatography (GC) GC analysis was carried out using an Agilent 7890A GC System equipped with a FID and an Agilent 7683B Series auto-injector. HP-5MS UI (30 m 0.25 mm id, lm thickness 0.25 lm) fused-silica capillary column (J.W. Scientic) was employed. The operating conditions 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 at 4 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; split ratio, 50:1. The quantitative data were obtained electronically from FID area per cent without the use of correction factors. 2.5. Gas chromatographymass spectrometry (GCMS) GCMS analysis was performed using an Agilent 6890 N Network GC System equipped with an Agilent 7683B Series auto-injector, coupled to an Agilent 5975 Inert Mass Selective Detector and the same capillary GC conditions as described above. The carrier gas used was He at 1.0 min1. The signicant MS operating parameters were: ionisation voltage, 70 eV; ion source temperature 230 C; mass range 50600 U. 2.6. Identication of constituents The constituents were identied by comparison of their mass spectra with those of authentic compounds or with reference

spectra in the computer library (NIST 05), and conrmed by comparison of retention indices with those of authentic compounds, or with 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 to determine the minimum inhibitory concentration (MIC) of the essential oils for antibacterial activity. Two millilitres cultures of three Gram-positive bacterial strains, Bacillus licheniformis (ATCC12759), Bacillus spizizenii (ATCC6633) and Staphylococcus aureus (ATCC12600), and three Gram-negative bacterial strains, Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC13883) and Pseudomonas stutzeri (ATCC17588), were prepared and placed in an incubator overnight at 37 C. The overnight cultures were diluted with sterile MuellerHinton (MH) broth (1 ml bacteria/50 ml MH) to yield a density of bacterial cells between 106108 cells/ml. The extracts were resuspended to a concentration of 10 mg/ml with ethanol, to yield a nal concentration of 2.5 mg/ml in the assay. For each of the six bacteria used, 100 ll of redissolved extract were serially diluted twofold with 100 ll of sterile distilled water, in a sterile 96-well micro-plate. A similar twofold serial dilution of tetracycline (1 mg/ml) was used as a positive control against each bacterium. Ethanol (100%) was used as a negative control to yield a nal concentration of 25% in the assay. One hundred microlitres of each bacterial culture were added to each well. The plates were covered and incubated overnight at 37 C. To indicate bacterial growth, 50 ll of 0.2 mg/ml p-iodonitrotetrazolium violet (INT) were added to each well, and the plates were incubated at 37 C for 30 min. Bacterial growth in the wells was indicated by a red colour, whereas clear wells indicated the absence of active bacterial 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 rhizomes of halia bara, the relative GC peak areas of these constituents, and their experimental retention indices on the HP-5 MS UI column. Forty-six compounds, constituting 91.7% of the leaf oil of halia bara, were identied. Sesquiterpenoids (47.1%) and monoterpenoids (42.6%) were dominant, although these gures were largely due to b-caryophyllene (31.7%), geranial (13.1%), neral (9.8%) and caryophyllene oxide (6.3%). Other quantitatively signicant constituents included geraniol (4.4%), a-pinene (4.1%) and trans,trans-a-farnesene (3.2%). The most abundant component, b-caryophyllene, is known for its anti-inammatory and local anaesthetic activities. It is used in spice blends, citrus avours, soaps, detergents, creams and lotions and also in a variety of food products. It has also been identied as a volatile compound emitted by plants into the atmosphere in response to herbivore attack (Sabulal et al., 2006). The oil from the rhizomes of this variety yielded 54 identied constituents, accounting for 95.5% of the sample. The oil was very rich in monoterpenoids (81.9%), comprising mainly camphene (14.5%), geranyl acetate (13.7%), geranial (14.3%), neral (7.7%), geraniol (7.3%), and 1,8-cineole (5.0%). The presence of neral and geranial possibly contributed to the strong aroma reminiscent of lemon in the rhizomes similar to those of the common ginger from Australia (Wohlmuth, Smith, Brooks, Myers, & Leach, 2006). Borneol, bornyl acetate and 1,8-cineole, together, probably accounted for a trace of its camphoraceous odour (Bauer, Garbe, & Surburg,


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Table 1 Constituents identied in the leaf and rhizome oils of Z. ofcinale var. rubrum Thielade (halia bara). Constituent RI (HP-5MS UI) Area (%)a Leaves 2-Heptanol Tricyclene a-Pineneb Campheneb Sabinene b-Pineneb 6-Methyl-5-hepten-2-one Myrceneb a-Phellandreneb d-3-Carene p-Cymene Limonene b-Phellandrene 1,8-Cineole cis-b-Ocimene 2-Heptyl acetate Trans-b-ocimene c-Terpinene Terpinolene 2-Nonanone Linaloolb Trans-sabinene hydrate Camphorb Camphene hydrate Citronellal Isoborneol Borneolb Terpinen-4-ol a-Terpineolb Myrtenal Linalyl formate b-Citronellol Neralb Geraniol Trans-2-decenal Geranialb Bornyl acetateb 2-Undecanone Myrtenyl acetate Neryl acetate a-Copaene Geranyl acetateb b-Elemene Isocaryophyllene b-Caryophyllene a-Humuleneb Allo-aromadendrene Ar-curcumeneb Germacrene D a-Zingibereneb a-Selinene a-Muurolene Trans,trans-a-farnesene b-Sesquiphellandreneb d-Cadinene a-Elemol Trans-nerolidol Caryophyllene oxide c-Eudesmol Caryophyllenedienol I b-Eudesmol a-Bisabolol Cis,cis-farnesol Trans,trans-farnesol Trans,trans-farnesal Phytol 902 922 934 949 974 977 988 992 1004 1010 1025 1029 1030 1032 1039 1044 1049 1060 1089 1093 1100 1122 1147 1151 1155 1160 1168 1180 1193 1199 1216 1229 1243 1256 1259 1273 1289 1295 1329 1366 1380 1384 1395 1411 1424 1458 1465 1485 1486 1495 1496 1503 1508 1524 1525 1549 1561 1587 1653 1641 1652 1690 1717 1723 1730 2058 0.9 4.1 t 0.2 2.0 0.2 1.3 0.2 0.1 0.1 2.6 0.7 0.1 0.2 0.2 0.1 1.1 0.1 0.3 0.4 0.2 0.5 9.8 4.4 0.3 13.1 0.2 0.6 1.0 0.1 31.7 0.9 0.3 0.2 0.3 1.1 3.2 0.3 0.6 6.3 0.4 0.3 0.3 0.2 0.3 0.2 91.7%
a b

Rhizomes 0.1 0.2 3.6 14.5 0.1 0.6 0.2 2.0 0.2 0.1 0.1 2.5 5.0 0.3 0.1 0.4 0.2 2.3 0.1 0.3 0.2 0.1 0.1 2.9 0.3 1.1 0.1 0.4 7.7 7.3 14.3 1.4 0.1 0.1 0.1 0.2 13.7 0.2 1.0 1.1 0.2 1.0 3.2 0.3 1.8 1.6 0.6 0.1 0.2 0.6 0.1 0.3 0.1 0.1 95.5%

in the oil from the rhizomes. A previous investigation of the rhizome oil of halia bara by Malek et al. (2005) also revealed a high content of monoterpenoids (64.6%). They, however, identied only 19 constituents among which 17 were common to the present study, and found much lower levels of camphene (1.8%) and geranyl acetate (8.8%). They did not detect geraniol but reported much higher levels of neral (14.2%), geranial (28.4%) and b-sesquiphellandrene (9.9%). These marked differences in the composition of the rhizome oil determined by Malek et al. (2005) from that of the present study could be attributed to the source, cultivation, vegetative stage and growing season of the plant under investigation (Sari et al., 2006). There are also signicant differences when the present results are compared with the composition of the rhizome oil of the common ginger collected from various locations, as the rhizome oil of the common ginger is typically characterised by high percentages of sesquiterpene hydrocarbons, in particular, a-zingiberene, arcurcumene b-bisabolene and b-sesquiphellandrene (Norajit, Laohakunjit, & Kerdchoechuen, 2007; Onyenekwe & Hashimoto, 1999; Pino, Marbot, Rosado, & Batista, 2004; Singh, Maurya, Catalan, & 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-positive (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 the rhizomes 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 caryophyllene oxide, 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 constituents could have imparted a signicant effect on the antibacterial 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 better antibacterial activity against the Gram-positive bacteria (with MIC values of 0.160.31 mg/ml) than against the Gram-negative bacteria (with MIC values of 0.310.63 mg/ml). This is in accordance with previous reports indicating that Gram-negative bacteria are more resistant to essential oils compared to Grampositive bacteria (Kivrak et al., 2009; Oyedeji, Lawal, Shode, & Oyediji, 2009; Sari et al., 2006). Based on our ndings, the sensitivity of the bacterial strains to the leaf oil decreases in the order B. licheniformis = S. aureus > 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 seen here against B. licheniformis, B. spizizenii, S. aureus and E. coli would suggest that the oils from halia bara could possibly be used as natural preservatives against food-borne pathogens or for delaying
Table 2 Antibacterial activity (MIC) of the leaf and rhizome oils of Z. ofcinale var. rubrum Thielade (halia bara) as determined by the micro-dilution assay. Bacterial Strains MIC (mg/ml) Leaf oil Bacillus licheniformis (ATCC12759) Bacillus spizizenii (ATCC6633) Staphylococcus aureus (ATCC12600) Escherichia coli (ATCC25922) Klebsiella pneumoniae (ATCC13883) Pseudomonas stutzeri (ATCC17588) 0.16 0.24 0.16 0.63 0.47 0.31 Rhizome oil 0.16 0.24 0.31 0.31 0.47 0.63 Tetracycline 1.0 103 1.8 103 7.7 103 15.6 103 3.7 103 8.1 103

Percentage of total FID area obtained on HP-5 MS UI column, t = (<0.05%). Previously reported by Malek et al. (2005).

2001). It is interesting that b-caryophyllene, the most abundant constituent of the leaf oil, was only found at a low level of 1.0%

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food spoilage. However, further investigation on the activities of these oils against other food-borne pathogens (Listeria sp., Salmonella sp.) should be conducted. 4. Conclusions The leaf oil and the oil from the rhizomes of halia bara differed in chemical composition, the former comprising mainly sesquiterpenoids and monoterpenoids in approximately equal amounts, while the latter was dominated by monoterpenoids. Antimicrobial assays of these oils have demonstrated that the Gram-positive bacteria in this study were more sensitive to both the oils compared to the Gram-negative bacteria. Toward the leaf oil, B. licheniformis and S. aureus were the most sensitive strains, while E. coli was the most resistant strain. B. licheniformis and P. stutzeri were the most sensitive and resistant strains, respectively, toward the oil of the rhizomes. The sensitivity of the B. licheniformis, B. spizizenii, S. aureus and E. coli strains toward both oils would suggest that these oils may be promising natural preservatives in the food industry. However, it is commendable that further analysis should be carried out on other food poisoning agents, such as Listeria sp., Salmonella sp. Acknowledgements The authors acknowledge the research grant 5702031007 (Science Fund) provided by the French Government that has resulted in this article. The authors wish to thank Mr. Teo, Mr. Ray and Mr. Din in assisting with the preparation of the voucher specimen, and Ms. Saripah in assisting with the GCMS analysis. References
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