pp.1-7

Suresh Kr. Sharma and Ramavtar Sharma*

Department of Botany, University of Rajasthan, Jaipur-302004, India

* Corresponding author E-mail: sharma_ra2007@yahoo.co.in

ABSTRACT

The chemical composition of essential oils obtained from Thuja orientalis L. (Cupressaceae) grown in Rajasthan was determined. Their essential oil was determined by hydro-distillation, analysed by GC/MS and GC-FID. The analyses of plant resulted in the identification of thirty two compounds, representing 88.5 % of the total oil. Out of 32 components, the major component was α-thujone (52.43 %) and other dominant components analyzed were β- Thujone (4.89 %), Camphor (4.36 %), Sabinene (4.21 %), Fenchone (3.57 %). Oil of T. orentalis exhibited the antibacterial (MIC values 1.32–1.83 mg/mL) as well as antifungal (MIC 1.86–2.87 mg/mL) activity. In all of the selected pathogens S. aureus bacterial strains (MIC 1.32 mg/mL) and A. alternate fungal strain (MIC 1.86 mg/mL) was found to be most sensitive to T. Orentalis essential oil. Isolated α- and β-Thujone from the plant also show high antibacterial (MIC values 0.064–0.091 mg/mL) as well as antifungal  activity  (MIC values 0.45–0.86 mg/mL).

Keywords: Thuja orientalis, essential oil, α- β thujone, Antimicrobial activity.

INTRODUCTION

Recently, multiple drug resistance has developed due to indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious diseases (Service, 1995) making it a global growing- problem. Isolation of microbial agents less susceptible to regular  antibiotics  and recovery of increasing resistant isolates during antibacterial therapy is rising throughout the world which highlights the needs of new principles. Natural products of higher plants may give a new source of antimicrobial agents with possibly novel mechanism of actions (Barbour et al, 2004; Hamil et al, 2003).Thuja   orientalis   L.   (Cupressaceae)   is   anevergreen species widely cultivated as a common ornamental plant (Assadi, 1998). It is widely distributed around China, Iran, Japan, America, Nagaland, Korea and India (Bucur, 1995, Asili et al., 2007;  Jaiswal  et al., 2011and Bansal et al., 2011).

1. orientalis  plant   is  commonly   used   in herbals   (Duke   and   Ayensu,   1985)   as   a haemostatic,  expectorant, and cough remedy (Kuo  and  Chen.,  1990).  The  stem  of  this plant  is  used  in  the  treatment  of  coughs, colds,  dysentery,  rheumatism  and  parasitic skin-diseases  (Nahed et al., 2010). The root bark  is  used  for  treatment   of  burns  and scalds,  and seeds  are used  internally  in  the treatment  of  palpitations,  insomnia,  nervous disorder   and   constipation      in   the   elderly (Duke and Ayensu, 1985). Dried leaves of P. orientalis  have  been used as a haemostatic, expectorant  and hypo tensor in Korean  folk medicine (Koo et al., 2002). Fresh leaves of that plant  are used as an anti  inflammatory drug (Pantonget al., 1986). A yellow dye is obtained  from the young branches  (Grieve, 1984).            Seeds   are   used         for   bronchitis, insomnia and as antitussive (Nishiama et al., 1995).  Thuja  is  also  occasionally  used  for treating                       diseases              of skin,  blood, gastrointestinal   tract,  kidney,   brain,  warty excrescences, spongy tumors (Biswas et al.,2011). It is used internally in the treatment of coughs, hemorrhages, excessive menstruation, bronchitis, asthma, skin infections, mumps, bacterial dysentery, arthritic pains and premature blandness (Bown, D. 1995 and Greenberg et al., 1978). The  chemical  constituents  of  P.  orientali such as terpenoids and flavonoids  showed the pharmacological or biological activities (Hassanzadeh et al., 2001). Many chemical components had been isolated by  the different parts ofP. orientalis like sesquiterpenoids and diterpenoids from the heartwood (Erdtman et al.,1956 ; Dev et al.,1964; Tomita et al.,1968 and Tomita et al., 1969), Flavonoids from leaves, mono and sesquiterpenoids in essential oils of different parts of the plant (Pelter et al., 1970 and Yan-hua et al.,2006), four bisnor and trinorlabdan type diterpenoids from seeds (Inoue et al.,1985), two monolognol derivatives from pollens  (Ohmoto  et al.,1988),  some labdane and isopimarane diterpenoids from pericarpes and  leaves (Kuo and Chen, 1990; Koo et al., 2002). The most prominent constituents of the oil are thujone, isothujone, fenchone and carnphor (Asili et al., 2007). Thujone was a weak inhibitor of acyl-CoA: lysophosphatidylcholine acyl-transferase activity in mouse brain synaptosomes compared to psychoactive cannabinoids (Greenberg et al., 1978). The essential oil which obtained by leaves is toxic. α-thujone is useful as an insecticide and an antihelminthic agent for the treatment of parasitic worms. However, α -thujone is a toxic substance that disrupts neurological signals in the brain. Ingestion of the essential oils of Thuja leaves can cause death (Hold et al., 2000).The chemical constituents of T. orientalis oil has not been much studied except for those of Chen et al. and Li et al. where they have reported α-pinene and α-cedrol as the major constituents of T. orientalis, respectively (Chen et al., 1984; Li and Liu, 1997).The aim of this study was to identify and evaluation of antimicrobial activity of essential oil constituents of the plant T. orientalis grown in Rajasthan.

METHODOLOGY

Plant material: Plant T. orientalis was collected from the University botanical garden and shade dried. Specimen (No.)  of the plant was deposited at the herbarium of Department of Botany, University of Rajasthan, Jaipur.

ISOLATION OF THE ESSENTIAL OIL

Shade dried fresh plant parts of T. orientalis were subjected to hydro distillation. The distillate was then extracted by using petroleum ether as solvent. The resulting extract was dried over anhydrous sodium sulfate. Petroleum ether was removed carefully under vacuum and a pale yellow color essential oil was obtained. Further the essential oil was used for antimicrobial activity.

GAS CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS

The  extract  and  the  standard  samples  were analyzed   by  GC-MS   of   Hewlett-Packard 6890/5973  operating  at  1000  eV  ionization energy,        equipped          with     using    Agilent 7890A/5975C  GC  HP-5.  Capillary  column (phenyl methyl siloxane, 25 m×0.25 mm i.d) with Helium (He) was used as the carrier gas with  split  ratio  1:5.  Oven  temperature  was 100 °C (3 min) to 280 °C at 1 to 40 °C/min; detector temperature, 250 to 280°C; carrier gas, He (0.9  mL/min).  Retention  indices were determined by using retention times of samples that were injected under the same chromatographic   conditions.   The components of the standard and plant samples were identified by comparison of their mass spectra and retention time with those given in literature and by comparison with the mass spectra of the Wiley library or with the published mass spectra.

ISOLATION OF TERPENOIDS ANTIMICROBIAL ACTIVITY ASSAYS

Microorganisms: All the test organisms, bacterial isolates of Escheriachia coli, Bacillus subtilius,  Staphylococcus  aureus and S. epidermidis and fungal isolates viz Candida spp., Aspergillus niger, Penicillium spp. and Alternaria alternate were obtained from Division of Biosciences, Seminal Applied Sciences Pvt Ltd, Jaipur, Rajasthan. Antimicrobial activity of the essential oils and the isolated compounds was determined using the agar dilution technique (Janssen et al., 1987). Standard antibiotics Streptomycin and Ketokenozol were used in  order  to control the tested bacteria and fungi. For each strain, the growth conditions and the sterility of the medium were checked and the plates were incubated at 37 °C and the MICs were determined as  the  lowest concentrations preventing visible growth.

STATISTICAL ANALYSIS

Results of the analyses were compared  by one way analysis of  variance  (ANOVA). The significance between pairs of variable means were analysed using least significant difference (LSD) test at 5 % level of significance (Gomez and Gomez, 1984).

RESULTS AND DISCUSSION

The hydrodistillation of the shade dry parts of the plant T. orientalis gave a pale yellow oil with 0.89% yield. The essential oils components of the studied plant with their percentages and their retention indices are listed in Table 1, while the antibacterial and antifungal activities of the essential oils and their components are given in Table 2. As a result of GC analyses,  32 components were identified representing 88.4% of the total oil. The plant T. orientalis was found to have α-thujone (52.43 %), β-Thujone  (4.89%), Camphor (4.36 %), Sabinene (4.21 %), Fenchone (3.57 %) as major constituents and followed    by  27 other essential  oil constituents (i.e. 19 %). (Table 1)Antimicrobial activity of T. Orentalis essential oil and isolated compounds shown inhibitory activity against selected bacterial and fungal strains and comparable to control (streptomycin sulphate and ketokenozol). (Table 2) In the screening of antimicrobial activity, the oil of T. orentalis exhibited the antibacterial (MIC values 1.32–1.83 mg/mL) as well as antifungal (MIC 1.86–2.87 mg/mL) activity. In all of the selected bacterial strains S. aureus (MIC 1.32 mg/mL) was found to be most sensitive to T. Orentalis essential  oil and followed by B. subtilius (MIC 1.36 mg/mL), S. epidermidis (MIC 1.44 mg/mL) and E. coli(MIC 1.83 mg/mL). Fungi A. alternate (MIC 1.86 mg/mL) was most sensitive among all fungal strains and followed by A. niger(MIC 2.13 mg/mL), Penicillium spp. (MIC 2.45 mg/mL) and Candida spp. (MIC 2.87 mg/mL).The antimicrobial activity of isolated α- and β-Thujone from the plant also high antibacterial (MIC values 0.064–0.091 mg/mL) as well as antifungal activity (MIC values 0.45–0.86 mg/mL).

1. aureus (MIC 0.064 mg/mL) was found to have minimum MIC value    and      most sensitive to the compounds and followed by
2. epidermidis (MIC 0.074 mg/mL), E. coli (MIC 0.082 mg/mL) and B. subtilius (MIC0.091  mg/mL).  Fungi  Candida  spp.  (MIC mg/mL)  was   most   sensitive    and followed by Penicillium spp. (MIC 0.76 mg/mL), A. alternate (MIC 0.78 mg/mL) and A. niger (MIC 0.86 mg/mL). The most prominent constituents of  the  oil are thujone, isothujone, fenchone and carnphor (Asili et al., 2007). High antibacterial and antifungal activity is due to high content of α- and β-thujone, which also exhibited strong microbial activities against the selected pathogens  and  are  well  known as the main active compounds in many essential oils from the plant having similar antimicrobial activity (Baser et al, 2002; Sivropoulou et al., 1997). The  present  study  reveals  that  the  plant Thuja  occidentalis  is found  to have  strongantibacterial and antifungal activity. The chemosystematic value of the total ketone content, especially of thujone isomers, is confirmed from this study. Pathogens are economically damaging human and animal health, agriculture and food. Thus, an approach towards the discovery of lead compounds has been made using an essential oil from T. orientalis. Sensitivity of pathogens towards active constituents of T. orientalis has indicated the need for the application of natural biocides in the  field and during post-harvest storage. Present findings could support the potential use of plant essential oils, which could be of economical benefit.

Table 1. Components (%) of the volatiles of Thuja Orentalis essential oils

Kovats Indices (KI)

Table 2. Antimicrobial activities (MIC mg/mL) of T. Orentalis essential oils and their main components

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