Abstract
Essential oils have applications in folk medicine, food preservation, and as feed additives. The essential oils of Lantana camara Linn. (Verbenaceae), Ageratum houstonianum Mill. (Asteraceae) and Eupatorium adenophorum Spreng. (Asteraceae) were analyzed by Gas chromatography-mass spectrometry (GCMS). In L. camara oil, of the total identified (83.91%) volatile constituents, five constituents [3,7,11-trimethyl-1,6,10-dodecatriene (28.86%), β-caryophyllene (12.28%), zingiberene (7.63%), γ-curcumene (7.50%) and α-humulene (3.99%)] represented the major ones. In A. houstonianum oil, among the total identified volatile constituents (94.51%), three [precocene-II (52.64%), precocene-I (22.45%) and β-caryophyllene (9.66%)] represented the major ones. In E. adenophorum oil, of the total identified volatile constituents (84.95%), six [1-napthalenol (17.50%), α-bisabolol (9.53%), bornyl acetate (8.98%), β-bisabolene (6.16%), germacrene-D (5.74%) and α- phellandrene (3.85%)] represented the major ones. The antibacterial activity expressed as Minimum Bactericidal Concentration (MBC) (μg/mL) was determined by the broth dilution method. The essential oil of E. adenophorum had antibacterial activity against Arthrobacter protophormiae, Escherichia coli, Micrococcus luteus, Rhodococcus rhodochrous, and Staphylococcus aureus with MBC values of 200, 100, 100, 12.5, and 200, respectively. The essential oil of A. houstonianum showed antibacterial activity against M. luteus and R. rhodochrous with MBC of 100 and 12.5, but not against A. protophormiae, E. coli, and S. aureus. The essential oil of L. camara showed antibacterial activity against A. protophormiae, M. luteus, R. rhodochrous and S. aureus with MBC of 50, 25, 12.5, and 200, respectively, but not against E. coli. MBC was lowest for R. rhodochrous for all the three essential oils.
Introduction
Recently, plant secondary metabolites (PSM), particularly essential oils, have evoked considerable interest as food and feed additives and as an alternative to antibiotics and synthetic antioxidants (CitationWallace, 2004; CitationAcamovic & Brooker, 2005; CitationGreathead, 2003; CitationBurt, 2004; CitationKalema & Kunicka, 2003). Studies on PSM have become imperative in view of the legislation in the European Union to prohibit the use of growth promoting antibiotics in animal feeds as of January 2006 (EU 1831/2003). Plants and their extracts are compatible with the current thinking on the future of health care, agriculture, and food and consumer opinion that most “things natural” are better and safer (CitationGreathead, 2003). This has spurred research on PSM as antimicrobial agents and nutraceuticals. Essential oils, the odorous and volatile plant secondary metabolites, have a wide application in folk medicine, food flavoring, and preservation, and in the fragrance industry (CitationGreathead, 2003; CitationKalema & Kunicka, 2003). Essential oils are known to exhibit antimicrobial, anticancer, antioxidant activity, and sedative effects (CitationKalema & Kunicka, 2003; CitationYoo et al., 2005; CitationSylvestre et al., 2005; CitationRicci et al., 2005; CitationKordali et al., 2005; CitationCarvalho-Freitas & Costa, 2002; CitationWarnke et al., 2004). Antibacterial activity of essential oils has been successfully used to control malodor in cancer patients (CitationWarnke et al., 2004, Citation2005). Essential oils such as tea tree and eucalyptus oil have gained acceptance as safe and effective antiseptics (CitationWarnke et al., 2005; CitationCox et al., 2000). There is a search for plants with more potent PSM for applications in the feed and food industry and for medical applications (CitationGreathead, 2003; CitationBurt, 2004; CitationMessager et al., 2005; Citationde Sousa et al., 2004; CitationAllahverdiyev et al., 2004). Lantana camara Linn. (Verbenaceae) extracts are considered antiseptic, antispasmodic, carminative, and diaphoretic. In Ayurvedic medical practice the leaves are used to treat hemorrhage and diarrhea (CitationParrotta, 2001; CitationSastri, 1962). The plants of the various species of Ageratum are used in indigenous systems of medicine (CitationSharma & Sharma, 1995). Decoction or infusion of the plant is given in various stomach diseases such as diarrhea, dysentery, and intestinal colic and also in rheumatism and fever (CitationSharma & Sharma, 1995). Various species of Eupatorium have been used in the traditional system of medicine in different parts of the world (CitationSharma et al., 1998). E. adenophorum Spreng. (Asteraceae) is used in India as an antiseptic and blood coagulant. A decoction of the plant has been recommended to treat jaundice and ulcers (CitationSharma et al., 1998).
L. camara, A. houstonianum, and E. adenophorum grow as weeds on vast expanses in many parts of the world and are an attractive source of bioactive natural products (CitationSharma & Sharma, 1989, Citation1995; CitationSharma et al., 1998; CitationJames et al., 2004). There are some previous reports on the antimicrobial activity of the oils of some species of Lantana, Ageratum, and Eupatorium (CitationHernandez et al., 2005; CitationJuliani et al., 2002; CitationDeena & Thoppil, 2000; CitationPattnaik et al., 1996; CitationHabtemariam & MacPherson, 2000; CitationGupta et al., 2002; CitationSasikumar et al., 2005). However, the natural products profile and consequently the bioactivity is known to vary with the climate and geographic location of the plants (CitationBurt, 2004). The bioactivity of the essential oils of L. camara, A. houstonianum, and E. adenophorum (syn: Ageratina adenophora (Spreng.) King and H.E. Robins) in the sub-Himalayan region of India has not been investigated. We report here the chemical composition and antibacterial activity of the essential oils extracted from L. camara, A. houstonianum, and E. adenophorum samples collected from Kangra Valley, Himachal Pradesh, India.
Materials and methods
Plant material
L. camara, A. houstonianum, and E. adenophorum samples were collected in September 2007 from Palampur (latitude 32° 07′, longitude 76° 31′, elevation 1275 m) H.P. India, and identified by Brij Lal, Division of Biodiversity, Institute of Himalayan Bioresource Technology (IHBT), and Palampur. Voucher specimens were deposited in the Herbarium of IHBT: Lantana camara var. aculeata no. PLP 3491; Eupatorium adenophorum no. PLP 3492; Ageratum houstonianum no. PLP-3493.
Extraction of essential oil
The samples were fresh leaves of L. camara and E. adenophorum and fresh aerial parts of A. houstonianum. Essential oil was extracted by hydrodistillation using Clevenger-type apparatus, collected, dried using sodium sulfate and stored at 4°C. The yield (%) of oil (on fresh weight) was 0.05, 0.1, and 0.15 for the samples of L. camara, A. houstonianum and E. adenophorum, respectively.
Chemical composition
The analysis of volatile oil of L. camara, A. houstonianum, and E. adenophorum was carried by GCMS analysis recorded on Shimadzu QP2010 GCMS fitted with BP-20 polar fused and silica capillary column 30 m, 0.25 mm i.d., film thickness 0.25 μm. The operating conditions were: injector and detector temperatures as 250°C, carrier gas He, flow rate 1.1 mL/min, oven temperature isothermal at 70°C increase at 4°C/m up to 220°C, finally held isothermal for 5 min, sample split ratio 1:50, injection volume 0.2 μL.
Antibacterial assay
Arthrobacter protophormiae, MTCC no. 2682, Escherichia coli MTCC no. 739, Micrococcus luteus, MTCC no. 106, Rhodococcus rhodochrous MTCC no. 265 and Staphylococcus aureus MTCC no. 26 procured from the Institute of Microbial Technology, Chandigarh, were kindly provided by M.K. Gupta Department of Microbiology, CSK HP Agricultural University, Palampur, India. Essential oils are volatile, insoluble in water, viscous and complex mixtures. So, the common tests such as disc/well diffusion do not give reproducible results for determination of antibacterial activity of essential oils (CitationHood et al., 2003). Therefore, the validated protocol of broth dilution method for assay of antibacterial activity of essential oils was used in this study (CitationHood et al., 2003). The data are presented as minimum bactericidal concentration (MBC).
Results and discussion
The chemical composition of the essential oils of L. camara, A. houstonianum and E. adenophorum is given in . GCMS analysis of essential oil of Lantana camara revealed the presence of 3,7,11-trimethyl-1,6,10-dodecatriene (28.86%), β-caryophyllene (12.28%), zingiberene (7.63%), γ-curcumene (7.50%) and α-humulene (3.99) as major constituents. The oil was represented with lone monoterpene hydrocarbon β-phellandrene (0.34%) but was mainly dominated by sesquiterpene hydrocarbons. The earlier workers also observed that essential oil of L. camara leaves is dominated by sesquiterpenes (CitationMisra & Laatsch, 2000; CitationWeyerstahl et al., 1999). However, there is marked difference in the nature and relative content of different constituents in the previous reports (CitationMisra & Laatsch, 2000; CitationWeyerstahl et al., 1999) and the data on . The sample used in this study and those in the previous studies might be representing different chemotypes. Recently, CitationMarongiu et al. (2007) reported the composition and antibacterial activity of the essential oil extracted from L. camara leaves by supercritical carbon dioxide. The major constituents were: ar-curcumene (38.7%), α-humulene (9.6%), α-zingiberene (7.8%), (E)-caryophyllene (7.8%), γ-curcumene (7.6%), allo-aromadendrene (4.8%), β-curcumene (3.2%), (E)-farnesene (2.5%), 7-epi-selinene (2.4%) and β-sesquiphellandrene (2.4%).
Table 1. Chemical composition (% amount in parenthesis) of essential oils of Lantana camara, Ageratum houstonianum and Eupatorium adenophorum.
GCMS analysis of A. houstonianum essential oil () revealed that the oil was dominated by chromenes, precocene-I (22.45%) and precocene-II (52.64%). In addition two more chromene derivatives, desmethoxyencecalin (0.78%) and androencecalinol (0.3%) were also identified. The oil was devoid of any monoterpene hydrocarbons but sesquiterpene hydrocarbons were dominating (15.97%) among which β-caryophyllene was the major constituent (). The oxygenated monoterpenes were represented only by linalool and isobornyl formate while the sesquiterpene alcohols were represented only by α-murrolol, cubenol, thujopsane-2-ol and longipinanol (). There is only one earlier report on the chemical composition of essential oil of A. houstonianum (CitationChandra et al., 1996). The major constituents were precocene-II (43.99%), precocene-I (23.34%) and β-caryophyllene (9.16%). The chemical composition of the solvents extracts of A. houstonianum from different locations has been investigated by earlier workers as well (CitationSharma & Sharma, 1995). The predominant constituents in these extracts also were precocene I and precocene-II. Hitherto, potential application of isolated precocenes and Ageratum essential oil is insecticidal activity (CitationSharma & Sharma, 1995).
GCMS analysis of the essential oil of E. adenophorum leaves revealed the presence of 1-napthalenol (17.50%), α-bisabolol (9.53), bornyl acetate (8.98), β-bisabolene (6.16), germacrene-D (5.74) and α-phellandrene (3.85) as major constituents (). This is the first report on the essential oil of the leaves of E. adenophorum. CitationWeyerstahl and co-workers (1997) reported the composition of the essential oil of flowers of E. adenophorum. The major constituents were α-phellandrene (15.3%), camphene (12.2%), bornyl acetate (10.6%), p-cymene (8.5%), γ-curcumene (4.5%) and 2-carene. CitationPala-Paul and co-workers (2002) reported the analysis of the essential oil of the aerial parts of E. adenophorum. The major constituents were p-cymene (11.6%), α-phellandrene (5.7%), γ-curcumene (5.0%), δ-2-carene (5.0%), camphene (4.8%), and endo-bornyl acetate (4.4%).
Antibacterial activity of essential oils of L. camara, A. houstonianum and E. adenophorum is shown in . Essential oil of E. adenophorum leaves showed antibacterial activity against all five organisms tested (). The growth of S. aureus was not inhibited by the essential oil of A. houstonianum and the MBC for the oil of L. camara and E. adenophorum was rather high for this organism. R. rhodochrous was the most susceptible organism with an MBC value of 12.5 μL/mL for the essential oil of all three plants. The growth of E. coli was not inhibited by essential oil of L. camara and A. houstonianum. CitationBhattacharjee and co-workers (2005) also observed that essential oil of Cestrum diurnum (L.) did not inhibit the growth of E. coli. The essential oil of Abies balsamea was also found to be inactive against E. coli and active against S. aureus (CitationPichette et al., 2006). Antibacterial effect of essential oils is, generally, less against Gram-negative bacteria than against Gram-positive bacteria (CitationBurt, 2004). The E. coli strain was resistant to tetracycline as well (). CitationDeena and Thoppil (2000) observed that essential oil of L. camara had antimicrobial activity towards both Gram-positive as well as negative bacteria. More recently, CitationMarongiu et al. (2007) observed that essential oil of L. camara extracted using supercritical carbon dioxide inhibited the growth of S. aureus and S. epidermis. The assay of antibacterial activity in both these studies was done by the filter paper disc diffusion method. CitationHood et al. (2003) observed that disc diffusion, well diffusion and agar dilution methods are unreliable and give inconsistent results for the assay of antimicrobial activity of essential oils. The composition, organoleptic profile and antimicrobial activity of essential oils prepared by steam distillation, carbon dioxide under low pressure and solvents like hexane, vary (CitationBurt, 2004). Our report is the first one on the antibacterial activity of the essential oils of A. houstonianum and E. adenophorum. Earlier, A. houstonianum essential oil has been reported to have acaricidal activity (CitationTedonkeng Pamo et al., 2005). A foam soap containing essential oil of A. houstonianum leaves was tested on Rhipicephalus lunulatus collected from West African dwarf goats. The LD50 of the foam soap containing essential oil of this plant was 0.0259 and 0.0173 mL/g on day 2 after treatment, in the laboratory and on the farm, respectively (CitationTedonkeng Pamo et al., 2005). Essential oils comprise a large number of components and it is likely that their mode of action involves several targets in the bacterial cell (CitationBurt, 2004). Some locations and mechanisms of the antibacterial action of the essential oil components are: degradation of the cell wall, damage to cytoplasmic membrane, damage to membrane proteins, leakage of cell contents, coagulation of cytoplasm and depletion of the proton motive force (CitationBurt, 2004). Carvacrol, an important constituent of the essential oils of oregano and thyme stimulates E. coli O157:H7 to produce Hsp60 and prevents the synthesis of flagellin, causing cells to be aflagellate and therefore non-motile (CitationBurt et al., 2007). The hydrophobicity of essential oils enables them to partition in the lipids of the cell membrane and mitochondria, rendering them permeable and leading to leakage of cell contents (CitationBurt, 2004; CitationCristani et al., 2007). In general, several constituents appear to contribute to the overall antibacterial action of essential oils (CitationKoutsoudaki et al., 2005). In view of the antibacterial activity and the abundance of the raw material, there is a potential for further investigations on the use of the essential oils of L. camara, A. houstonianum and E. adenophorum in various applications in food, feed, animal health, and in palliative care medicine (CitationAcamovic & Brooker, 2005; CitationGreathead, 2003; CitationBurt, 2004; CitationKalema & Kunicka, 2003; CitationYoo et al., 2005).
Table 2. Antibacterial activity of essential oils of Lantana camara, Ageratum houstonianum and Eupatorium adenophorum.
Acknowledgement
We thank Ms Jyoti Dhar, Technical Assistant, for excellent technical support in these studies.
Declaration of interest
The authors report no conflict of interest. The authors are responsible for the content and writing of the paper.
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