STUDY ON THE CHEMICAL CONSTITUENTS AND ANTIBACTERIAL ACTIVITY OF ESSENTIAL OIL OF ACORUS CALAMUS L . RHIZOMES OF RUPENDEHI DISTRICT ( NEPAL )

The essential oil from the air dried powder of wild Acorus calamus L. rhizomes was isolated using Clevenger-type hydrodistillation apparatus and its chemical constituents were analyzed by gas chromatography-mass spectrometric (GCMS) analysis. A total numbers of fourteen chemical constituents were identified and quantified occupying 100 % of total oil composition. The major chemical constituents are reported to be β-asarone (68.96 %), 4-hydroxy-4-methyl-2pentanone (8.73 %) shyobunone (8.65 %), p-methoxyphenyl butanone (4.16 %) and the minor chemical constituents are δ-cadinene (0.84 %), isoelemicine (2.68 %), muurolene (1.92 %) and cis-ocimene (1.44 %). The antibacterial activity of the essential oil isolated from the wild Acorus calamus L. rhizomes was carried out using paper disc method against one Gram positive bacteria of Staphylococcus aureus and three Gram negative bacteria of Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris. The isolated essential oil from the dry powder of Acorus calamus L. rhizome showed a good potentiality of the antibacterial effect against tested bacteria.


INTRODUCTION
Acorus calamus L. of the genus Acorus belong to the family Acoraceae.It is known as BHOJO in Nepal and commonly known as sweet flag.It is reported that about 40 species of Acorus is distributed from temperate to sub tropical wetland and native of central Asia and Eastern European countries (Gilani et al. 2006, Agarwal et al.1956).This plant species grow along swamp, rivers, lakes and marshy places up to 2000 meters altitude in different parts of Nepal and the Himalayas of India (Raina et al. 2003, Kim et al. 2009).It is about two meters of height with stout and knobby shallow branching rhizomes.Tufts of basal leave occur at intervals along these rhizomes while coarse fibrous roots develop below and the plant multiplies by its rhizomes.The rhizome is long indefinite branched, smooth, pinkish or pale green and it is internally whitish pink in color with pleasant aroma and slightly bitter in taste (Balakumbahan et al. 2010).
Acorus calamus L. has been used as traditional folk medicine in Nepal (Satyal et al. 2013).In America and Indonesia, it is used for cure of gastrointestinal disorder such as colic pain, diarrhea and in the therapy of diabetics (Gilani et al. 2006, Si et al. 2010).It has been an integral part of Indian and Chinese medicinal systems for hundreds of years (Wu et al. 2009, Lee et al. 2011).People of Baitadi and Darchula districts of Nepal utilize the juice of the Acorus calamus L. rhizome as an anthelmintic and it is chewed to treat coughs, cold and sore throat (Satya et al. 2013, Uprety et al. 2011).This plant contains bioactive chemical constituents such as essential oils, alkaloids, steroids, phenols, coumarins, flavonoids etc which are valuable for treating various human ailments (Balakumbahan et al.2010).Despite an insufficient research works on the chemical components and bioactivities of Acorus calamus L., there is a dearth of information about its secondary metabolites and biological activities.Therefore, the present study is aimed to investigate the essential oil composition of the rhizome of air dried wild Acorus calamus L. from Rupandehi district of Nepal using GC-MS analysis and to evaluate their antibacterial activities.

Plant material
The air dried wild Acorus calamus L. rhizome was collected in the month of April 2011 from marshy places of Devdaha-9, Rupandehi district, Nepal which is located in 27° 40 ' 48" N and 83° 34' 12" E at about 250 meters above the sea level.The plant was identified at Central Department of Botany, Tribhuvan University, Kirtipur, Nepal.The rhizomes were washed with cold water to remove the contaminant soil and allowed to air dry in shade at room temperature for about two months and crushed to get powder using electrical grinder.

Extraction of essential oil
The essential oil from the powdered Acorus calamus L. rhizome was extracted using Clevenger-type hydrodistillation apparatus.Amount of 100 grams of the powdered sample was kept in a round bottom flask along with distilled water and fitted with Clevenger-type distillation apparatus.The content of the flask was heated Study on the chemical constituents and antibacterial activity of essential oil ….
in heating mantle at boiling temperature for 4 hours followed by standing for one hour at room temperature.The isolated essential oil was dried using anhydrous sodium sulphate and stored in airtight reagent bottle at 4 °C for further analysis.

GC-MS analysis
The essential oil obtained from the wild Acorus calamus L. rhizomes was analyzed by GC-mass spectrometer (QP-2010, Shimadzu Co. of Japan) operated in the electron impact (EI) mode with the electron energy of 70 eV and ion source temperature of 200 °C under the following conditions: injection volume of 1 μL with split ratio of 1:90; Helium as carrier gas at 1 ml/min constant flow mode, injector temperature of 280 °C, oven temperature of 40 to 250 °C at the rate of 7 °C/min.Mass spectra were recorded in the scan range between 35-500 atomic mass units.Identification of the chemical constituents was based on their retention indices determined by reference and comparison of their mass spectral fragmentation patterns using previously reported references, and NIST 05, WILEY and SZTERP libraries.

Antibacterial screening test
Antibacterial evaluation of the essential oil was performed using the paper disc diffusion method elaborated by Bauer et al. (1960).Bacterial strains were collected from Center Department of Microbiology, Tribhuvan University, Kirtipur.The studied strains include Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris).For the positive control, tetracycline paper disc was taken and petroleum ether disc was taken for negative control.The zone of inhibition was obtained by measurement of the radius from the centre of the disc to the edge of the inhibition growth (Oliver et al. 1959) and the obtained values were converted in percentage of zone of inhibition.

RESULTS AND DISCUSSION
The amount of isolated essential oil obtained from the powder form of the air dried wild Acorus calamus L. rhizome was quantified and was found to be 0.8 % v/w on dry weight basis.The extracted oil was pale yellow color and slightly viscous with sweet herbal aroma.Different chemical constituents of the essential oil isolated from the air dried wild Acorus calamus L. rhizomes are tabulated in Table 1.The GC-mass spectrometric analysis of the isolated essential oil revealed the presence of all together fourteen chemical components in the gas chromatogram (Fig. 1) and representing 100 % of total isolated oil compositions.The chemical types of secondary metabolites were monoterpene hydrocarbons, alcohol derivative of monoterpene, sesquiterpenes, carbonyl compound and ester derivatives.The major chemical constituents identified were of β-asarone (68.96 %), 4hydroxy-4-methyl-2-pentanone (8.73 %) shyobunone (8.65 %), p-methoxyphenyl butanone (4.16 %), isoelemicine (2.68 %) and the minor chemical constituents of δ-cadinene (0.84 %) and cis-ocimene (1.44 %) with a little amount of cuparene (0.29 %) and other secondary metabolites.α-asaron was not detected in the isolated oil.Some of the chemical constituents having same chemical composition are on different retention time (Rt).

Acorus calamus L .rhizome collected from Rupandehi district of Nepal
The present study revealed that the chemical constituents of the different essential oil and their percentages are different from previously published works of Nepal and Indian origin (Uttarakhand, India).The major component of the essential oils from the plant of Eastern Nepal (Biratnagar) origin and Indian (Uttarakhand) origin was reported β-asarone of 84.0-86.9% (Satyal et al. 2013, Gyawali & Kim 2009) and of 81.1-92.4% (Bist et al. 2011) with other minor chemical constituents.Similarly, a very high amount of β-asarone was reported from Acorus calamus L. rhizomes found all over India and other places of world (Raina et al. 2003).
The result of the antibacterial screening test of essential oil isolated from Acorus calamus L. rhizomes at different concentration (0.25, 0.50, 0.75 and 1.00 mg/disc) is depicted in Fig. 2. The essential oil was found to be equally active against Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli and Proteus vulgaris in 0.25 mg/disc concentration.Among these four bacteria, good potential antibacterial effect was shown against Klebsiella pneumoniae at 1.0 mg/disc concentration.The potential antibacterial activity of this essential oil attributed to β-asarone as well as synergistic effect of other complex chemical compositions present in natural product either in major or minor amounts.
β-asarone revealed beneficial in cognitive impairment associated disorders such as Alzeimer disease(McGraw et  al. 2002).Quantitative differences in the amount of βasarone in the air dried wild Acorus calamus L. rhizomes essential oil have been considered to have taxonomic importance within the genus(Rost et al. 1979).Differences in the chemical compositions and yield of the essential oil are mostly due to different environmental condition, geographical location and time of collection of plant materials.

Fig. 2 .
Fig. 2. Antibacterial activities of Acorus calamus L. rhizomes essential oil from Rupandehi district of Nepal

Table 1 . Chemical constituents of the isolated essential oil of Acorus calamus L. rhizome collected from Rupandehi district of Nepal
Chemical constituents are presented based on retention time (Rt), molecular weight (MW) and mass spectrum (MS)