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Husain S Shahnaz1, Ali M1*, Panda BP2
1Phytochemistry Research Laboratory, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India.
2Microbial and Pharmaceutical Biotechnology Laboratory, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India.

ORIGINAL RESEARCH ARTICLE
Volume 2, Issue 2, Page 81-84, May-August 2014.

Article history
Received: 15 March 2014
Revised: 20 March 2014
Accepted: 20 April 2014
Early view: 28 April 2014

*Author for correspondence
E-mail: maliphyto@gmail.com
Mobile/ Tel.: +91 9968281082

ABSTRACT

Background: Cinnamomum tamala Nees et Eberm (Lauraceae), known as Tejpat, is a handsome, evergreen, medium sized tree, up to 8 m in height found in Asia Minor, north-western Himalayas, Sikkim, Assam, Mizoram, Meghalaya and southern Europe. The leaves have clove like taste and pepper like odor and are used to treat anorexia, bladder disorders, dryness of the mouth, coryza, diarrhea, nausea and spermatorrhea.
Materials and Methods: Hydrodistillation of the dried leaves in a Clavenger apparatus yielded a colorless volatile oil which was dried over anhydrous sodium sulphate. The gas chromatographic analysis of the volatile oil was carried out on Shimadzu 2010 Gas Chromatograph equipped with a flame ionization detector (FID) and ULBON HR-1 fused silica capillary column (60 m x 0.25 mm x 0.25 μm). The GC–MS analysis of the volatile constituents was performed on a silicon DB-1 fused silica column directly coupled to the MS.
Results: The leaf volatile oil was characterized by high amount of aromatic components (97.4 %) including eugenol (74.4 %), isoeugenol (21.1 %), acetyl eugenol (1.2 %) and ethyl cinnamate (0.7 %). There were two monoterpene hydrocarbons, α-thujene and α-pinene, present in trace amounts. Among five sesquiterpenes (2.1 %), β-elemene (1.1 %) and β-caryophyllene (0.2 %) were the hydrocarbons. There were two sesquiterpene alcohols and one sesquiterpene oxide, all occurring in trace amounts. The oil was devoid of aliphatic constituents. The volatile oil of the leaves showed significant antibacterial activity against Escherichia coli (NCTC-6571), Staphylococcus aureus (NCTC-10418) and Bacillus subtilis and antifungal effect against Aspergillus flavus, As. niger, As. fumigatus and Candida albicans.
Conclusion: The leaf volatile oil of C. tamala was characterized by a high amount of aromatic components including eugenol, isoeugenol and acetyl eugenol. It showed significant antimicrobial activity suggesting its use to control bacterial and fungal diseases.

Keywords: Cinnamomum tamala, Volatile oil, Antimicrobial.

INTRODUCTION

Cinnamomum tamala Nees et Eberm (Lauraceae), known as Tejpat, is a handsome, evergreen, medium sized tree, up to 8 m in height indigenous to Asia Minor and southern Europe. It is distributed in the north-western Himalayas, Sikkim, Assam, Mizoram and Meghalaya, tropical and subtropical Asia, Australia and Pacific region (Anonymous, 1992; Kirtikar and Basu, 2000). It is cultivated in Nainital (Uttarakhand), Kangra (Himachal Pradesh) and Tripura for its Tejpat leaves which are used in Indian cookery, as bay leaves in Europe, for flavoring food and in pharmaceutical preparations due to their hypoglycemic, stimulant and carminative properties (Anonymous, 1992). The leaves have clove-like taste and pepper-like odor and are used to treat anorexia, bladder disorders, dryness of the mouth, coryza, diarrhea, nausea and spermatorrhea (Kapoor, 2000). The essential oil of the leaves, famous as Tejpat oil, is medicinally used as a carminative, antiflatulent, diuretic and in cardiac diseases. The oil has pale lemon colour with a clove-like peppery odor and resembles the oil of Ceylon cinnamon leaves (Husain et al., 1988). Various chemical types of C. tamala exist, viz. eugenol-type, cinnamic aldehyde-type or cinnamic aldehyde-cum-linalool-type, named after the main constituents present in the oils from various regions, thus offering a wide scope of utilization of these aroma chemicals for flavor and perfumery purposes (Husain et al., 1988). Other constituents such as pinene, camphene, myrcene, limonene, 1,8-cineole, p-cymene, methyl eugenol and eugenol acetate have also been reported in Tejpat oil (Mir et al., 2004; Rema et al., 2005; Rana et al., 2012; Smith et al., 2002; Kapoor et al., 2009). The essential oil of Cinnamomum leaves showed inhibitory effect on bacteria (Minakshi et al., 1999) and Aspergillus species (Kapoor et al., 2009; Bisht et al., 2011). As a part of our investigation on the aromatic and medicinal plants of India, we describe in this communication the chemical composition and antimicrobial activity of the volatile oil of C. tamala leaves procured from Delhi, India.

MATERIALS AND METHODS

Plant material
The dried leaves of C. tamala were purchased from the local market of Ghonda Maujpur, Delhi-110053, India. The plant material was identified by Dr. SR Mir, Asst. Prof., Department of Pharmacognosy and Phytochemistry, Jamia Hamdard, New Delhi. A voucher specimen No. PRL/JH/09/17 is preserved in the herbarium of the Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India.
Isolation of the volatile oil
The dried leaves (2 kg) of C. tamala were crushed and hydrodistilled in an all glass Clavenger apparatus according to the method recommended in the British Pharmacopoeia, 1988. The colorless volatile oil was dried over anhydrous sodium sulphate and stored at 4 °C in the dark. The yield was 2.31 % based on the dried weight of the plant material.
GC analysis
The gas chromatographic analysis of the volatile oil was carried out on Shimadzu 2010 Gas Chromatograph (Japan) equipped with a flame ionization detector (FID) and ULBON HR-1 fused silica capillary column (60 m x 0.25 mm x 0.25 μm). The injector and detector (FID) temperatures were maintain at 250 and 270 °C, respectively. The carrier gas used was nitrogen at a flow rate of 1.21 mL/min with column pressure of 155.1 kPa. The sample (0.2μl) was injected into the column with a split ratio of 80:1. Component separation was achieved following a linear temperature programmed from 60 to 230 °C at a rate of 3° C/min and then held at 230° C for 9 min, with a total run time of 55.14 min. Percentage of the constituents were calculated by electronic integration of FID peak areas.
GC-MS analysis
The GC–MS analysis of the volatile constituents was performed on a silicon DB-1 fused silica column directly coupled to the MS. The carrier gas was helium with a flow rate of 1.21 mL/min. Oven temperature was programmed as 50˚C for 1 min and subsequently held isothermal for 2 min., injector port: 250˚C, detector: 280˚C, split ratio 1:50, volume injected: 1μL of the oil. The recording was performed at 70 eV, scan time 1.5 s; mass range 40-750 amu. Software adopted to handle mass spectra and chromatograph was a Chem station.
Identification of compounds
The individual compounds were identified by comparing their retention indices (RI) of the peaks on ULBON HR-1 fused silica capillary column with literature values, matching against the standard library spectra, built up using pure substances and components of known essential oils. Further identification was carried out by comparison of fragmentation pattern of the mass spectra obtained by GC-MS analysis with those stored in the spectrometer database of NBS 54 K.L, WILEY8 libraries and published literature (Adams et al., 2001; Ali, 2001; Joulain and Konig 1998; McLafferty 1994). Relative amounts of identical components were based on peak areas obtained without FID response factor correction.
Antimicrobial activity
Test organisms and inoculums

The bacterial strains Escherichia coli (NCTC-6571), Staphylococcus aureus (NCTC-10418) and Bacillus subtilis were obtained from the Division of Biotechnology, Faculty of Pharmacy, Jamia Hamdard, New Delhi. The fungal strains Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus and Candida albicans were procured from the Institute of Genomics and Integrative Biology (CSIR), New Delhi, India.
Antimicrobial standard
Tetracycline solution with specific activity of 50 g/ml was prepared in dimethyl sulphoxide (DMSO) solution (antibacterial). Fluconazole with specific activity of 50 g/ml was prepared in DMSO solution (antifungal).
Media
Dehydrated nutrient agar media was prepared in distilled deionized water. The media (g/100 ml) was composed of peptone (5.1 g), sodium chloride (5.0 g), beef extract (1.5 g), yeast extract (1.5 g) and agar (1.5 g).
Preparation of media
Dehydrated nutrient agar medium (28 g) was accurately weighed and suspended in 1000 ml of distilled water in a conical flask. It was heated on a water bath to dissolve the medium completely. Direct heating was avoided as it may lead to charring of the medium components and render it useless for the purpose.
Sterilization of media
The conical flask containing the nutrient agar medium was plugged with the help of a non-absorbent cotton plug. The mouths of the conical flask and the cotton bung were properly covered with aluminum foil. The medium was then sterilized by autoclaving at 15-lbs/in2 pressure for 20 minutes.
Preparation of test organisms
The test organisms were maintained on slants of medium and transferred to a fresh slant once a week. The slants were incubated at 37 °C for 24 hours. Using 3 ml of saline solution, the organisms were washed from the agar slant on to a large agar surface (medium) and incubated for 24 hours at 37 ± 2 °C. The growth from the nutrient surface was washed using 50 ml of distilled water. A dilution factor was determined which gave 25% light transmission at 530 nm. The amount of suspension to be added to each 100 ml agar or nutrient broth was determined by use of test plates or test broth. The test organisms were stored under refrigeration.
Temperature control
Thermostatic control is required in several stages of a microbial assay when culturing a micro-organism and preparing its inoculums and during inoculation in a plate assay.
Cup and plate method
A previously liquefied and sterilized medium was poured into plastic petri-plates of 100 mm size. Required plates were prepared and kept for solidifying. Six holes were made in each plate with a stainless steel borer having 6 mm i.d. Different dilutions of the volatile oil of C. tamala were made having concentration of 3 µl/ml, 5 µl/ml, 7 µl/ml and 9 µl/ml of solution. Tetracycline and fluconazole solutions were used as standards. The plates were labeled as Co (control), S (standard), X (B. subtilis), Y (S. aureus), Z (E .coli) with four or five different holes, labeled as 3, 5, 7 and 9 for different concentrations. All dilutions were made in DMSO solvent, which were used in experiment. Micropipette was used to deliver the solutions into the holes. The plates were then left for standing for 1 h for proper diffusion of the drug solutions. They were incubated for about 24 h at 32  2 °C. After 24 h the plates were examined and the diameter of zones of inhibition was accurately measured. Antifungal activity was determined against Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus and Candida albicans similar to antibacterial activity.

RESULTS

The components of the oil, the percentage of each constituent and their RI values are summarized in Table 1.

Table 1. Percentage composition of the volatile oil of the leaves of C. tamala.
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The constituents are arranged in order of GLC and GC-MS elution on silicon DB-1 and ULBON HR-1 fused silica column, respectively. The oil was characterized by high amount of aromatic components (97.4 %). The predominant constituents were eugenol (74.4 %) and isoeugenol (21.1 %). The other aromatic components present in the volatile oil were characterized as acetyl eugenol (1.2 %) and ethyl cinnamate (0.7 %). There were two monoterpene hydrocarbons, α-thujene and α-pinene, present in trace amounts. Among five sesquiterpenes (2.1 %), β-elemene (1.1 %) and β-caryophyllene (0.2 %) were the hydrocarbons. There were two sesquiterpene alcohols and one sesquiterpene oxide, all occurring in trace amounts. All the components were positively identified. The oil was devoid of aliphatic constituents.
The Tejpat leaf oil was examined for antibacterial activity against E. coli, S. aureus and B. subtilis and antifungal activity against Aspergillus niger, A. fumigatus, A. flavus and Candida albicans. The oil showed significant antimicrobial and antifungal activity in comparison to standard, tetracycline and fluconazole. The observations are recorded in the Tables 2 and 3.

Table 2. Antibacterial activity of the volatile oil of C. tamala leaves..
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Table 3. Antifungal activity of the volatile oil of C. tamala leaves.
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DISCUSSION

The essential oil of C. tamala leaves from Nainital (Uttarakhand) contained mainly (E)- cinnamaldehyde, linalool and (E)-cinnamyl acetate as the major components (Lohani et al. 2012). The prominent components of the essential oil of the tejpat leaves from north Cacher hills (Assam) were α-linalool (60.73 %), α-pinene (10.54%) and β-pinene (10.42 %). Eugenol and cinnamaldehyde were detected only in trace amounts (Nath et al., 1994). The volatile oil of C. tamala collected from Imphal, Manipur contained eugenol (41.8%), eugenyl acetate (47.1 %) and phellandrene (2.5%) as the main chemical constituents (Rana et al., 2012). The important components of the volatile oil of the leaves of C. tamala grown in Pakistan were β-caryophyllene (25.3%), linalool (13.4%) and caryophyllene oxide (10.3%) (Ahmed et al., 2000). Eugenol (66.1%) was the main constituent of the volatile oil of C. tamala leaves of Gorakhpur region followed by spathulenol (4.8%), viridiflorene (2.4%), methyl eugenol (1, 9%) and aromodendrene (1.5%) (Kapoor et al. 2009). The essential oil of tejpat leaves from Nepal possessed linalool (54.66%), α-pinene (9.6%) and p-cymene (6.4%) as the predominant constituents (Upadhya et al.,1994). The C. tamala leaf oils of both wild and cultivated sources occur in various chemotypes as eugenol, cinnamaldehyde, cinnamaldehyde-linalool and linalool rich types. C. tamala from Rajouri and Billawar forests in Jammu and Kashmir and Tehri Garhwal in Uttarakhand are of eugenol rich type. The chemical composition of C. tamala oils of Jeolikot and Chamoli forests are of cinnamdehyde rich types (Rema et al., 2005). The C. tamala leaves available in Delhi market are of eugenol chemotype grown in Tehri Garhwal region, India.

CONCLUSION

The leaf volatile oil of Cinnamomum tamala was characterized by high amount of aromatic components (97.4 %) including eugenol (74.4 %), isoeugenol (21.1 %), acetyl eugenol (1.2 %) and ethyl cinnamate (0.7 %). The oil was devoid of aliphatic constituents. It showed significant antibacterial activity against Escherichia coli (NCTC-6571), Staphylococcus aureus (NCTC-10418) and Bacillus subtilis and antifungal effect against Aspergillus flavus, As. niger, As. fumigatus and Candida albicans. This establishes the essential oil composition of C. tamala and its suitability as an antimicrobial to control diseases.

ACKNOWLEDGEMENTS

The authors are thankful to the Head, SAIF, Central Drug Research Institute (CSIR) for recording GC-MS data of the volatile oil.

CONFLICT OF INTEREST
None declared.

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