Comparative Study of Chemical Composition and Antioxidant Activity of Essential Oil Extracted from Acorus calamus L . Leaves

Acorus calamus L. (Araceae) is perennial, semi aquatic and aromatic medicinal herb exist in both temperate and sub temperate region with sharp pointed, long erect leaves. Acorus calamus is a well known herb with useful bioactive properties in indigenous medicine for centuries in India (Mehrotra et al., 2003). This plant is an evergreen herb up to 6 feet tall and is cosmopolitan in distribution ranging from Europe, East Asia, and North America. It has been reported that in America and Indonesia Acorus calamus is used as herbal medicines to treat gastrointestinal disorders like colic pain, diarrhea and diabetes (Gilani et al., 2006; Si et al., 2006). Its uses for the treatment of fever, cough, bronchitis, inflammation, depression, tumors, hemorrhoids, numbness, skin diseases, general debility and as antidotes for several poisoning have been reported (Vaidyaratnam, 1994). Terpenoids, flavonoid and Journal of Herbal Drug

quinines as major constituents have been reported in A. calamus (Patra and Mitra, 1979).

Plant material
The plants were collected from the twenty different ecological niches of Uttarakhand in India (Table 1).The herb was taxonomically authenticated by Dr. D. S. Rawat, Department of Biological Science, College of Basic Science and Humanities.

Isolation of essential oil
The essential oils from fresh crushed and chopped leaves were isolated by hydrodistillation (Clevenger, 1928).The hydrodistillation was continued for about 7-8 hr.The oils were extracted with DCM and desiccated over anhydrous Na 2 SO 4 .

GC-MS Analysis
The GC-MS analysis was carried out using GCMS-QP 2010 with mass selective detector having capillary column (DB-5, 30m × 0.25mm, 0.25µm).The column initial oven temperature was 600C and then programmed at 30C /min to final oven temperature 240oC, isothemal for 30 min .Injection temperature was 260°C.Helium was carrier gas with flow rate of 3.0 ml/min and split ratio 40:1.For mass detection electron ionisation (70 eV) technique was used.The constituents ofessential oils were identified on the basis of their mass spectra obtained then matching them with those in NIST-MS, FFNSC Wiley Library and authenticating by comparing with literature reports and GC retention indices (Adams, 2007).

Antioxidant activity
In present investigation, the in vitro antioxidant property of leaf essential oils from Acorus calamus was studied by DPPH radical scavenging method compared to standard antioxidant.DPPH scavenging activity was evaluated according to the method developed earlier and recently being used by (Prakash et al.,2011).The assay mixture contained 5 ml of 0.004% methanol solution of DPPH and different amount of test sample solution of different concentrations.The percent DPPH free radical inhibition (IC 50 %) was calculated by using the equation.IC 50 %=[( control-sample)/control]×100.Standard curve was drawn by plotting percent inhibition against concentrations.Finally using this curve IC50 values for standard and essential oil were calculated.The lower IC 50 value indicated more radical scavenging activity.

Statistical analysis
The experimental data were obtained by executing the experiments in triplicates.The data was analysed by Tukey's test in conjunction with an ANOVA (post-hoc) analysis with the help of SPSS 16 version.

Essential oil analysis
Depending upon environmental factors, seasonal factors, growth stage, climatic conditions and locality development the yield of essential oils ranged from 0.2-1.4% (v/w) among the accessions.A total of 37 constituents, contributing 80-86.8% of the essential oils were identified.Persual of Table 2 indicates the names and respective percentage of identified constituents while figure 1 represents the major compounds.All the oils were different in their qualitative and quantitative make-up of major and minor constituents and showing vast molecular diversity In term of class composition the components of essential oil were mainly composed of phenylpropanoids (49.7-72.9%),monoterpenoids (2.0-12.8%)and sesquiterpenoids (7.8-24.4%)(Figure 2 and Table 3).A positive correlation between major constituents and essential oil was observed (Table 4).The results vary from the previous reports.A report by Raal et al. (2016) on Acoruscalamus essential oil revealed the dominating presence of oxygenated sesquiterpenes in his report.In present communication the dominating constituents in the leaf essential oils from Acorus calamus were βasarone (43.4-60.7%),α-asarone (2.6-7.9%),shyobunone (3.4-6.3%), and α-humulene (0.3-1.6%) in twenty different accessions.The constituents linalool (0.4-7.1%), caryophyllene (1.1-3.2%) and α-elemol (0.5-1.0%), were identified in all the accession except 1L and 2L.It was also observed that germacrene D-4ol (4.7%) was identified in 6L while palmitic acid (3.6%) was in 13L whereas in other accession these compounds were totally missing.Similarly Z-methyl isoeugenol (0.8-3.1%) and Z-isoelemicin (3.2-5.4%)showed variable concentrations which were totally absent in samples collected from 2L and 6L respectively.
In our previous study by Kumar et al. (2009) and Joshi et al. (2012) we have published a report on antibacterial and anthelmentic activity in rhizome essential oils of Acorus calamus from different locations.

Antioxidant activity
Natural antioxidants are responsible for preventing the oxidative stress and much safer to use due to its less toxicity and side effects.Antioxidants play a major role for prevention of tissue damage that stimulates the wound healing process (Barku et al., 2013).DPPH antioxidant activity is based on the capability of DPPH a stable free radical which is discolored in the presence of antioxidants (Subathraa and Poonguzhali, 2012).The results of the antioxidant activity are demonstrated in Figure 3 and Table 5.
Strong to good DPPH radical scavenging property in Acorus calamus leaf essential oils was observed as a function of concentrations at selected dose levels.The maximum scavenging activity (IC 50 10.79±0.042bμg/ml) was observed in the sample 2L.The essential oil of this collection contained highest amount (7.9%) of α-asarone.The maximum radical scavenging activity of this sample might be possibly due to the presence of αasarone.The results are in total agreements with the previous reports by Manikandan and Devi (2005); Devi et al. (2014) which revealed that the antioxidant property of Acorus calamus extract is partly expected due to the active constituents, α-asarone.
Eessential oil of Acorus calamus is known for its medicinal and insecticidal properties.β-asarone the major chemical constituents reported in Phytochemical investigation has been reported to possess many biological activities along with a potential therapeutic agents for Alzheimer's disease (Geng et al., 2010), However, the other isomeric phenylpropanoid αasarone have been reported to posses neuroprotective, anticonvulsive, anti-oxidative, cognitive enhancing action and to improve various central nervous system disorders along with prevention of the noise stress induced memory impairment (Manikandan and Devi 2005;Shin et al., 2014;Kim et al., 2015).
The compounds shyobunone and isoshyobunone isolated from essential oil have been reported to possess insecticidal and repellant activity against Lasioderma serricorne and Tribolium castaneum (Chen et al., 2015).
The essential oil of Pakistanian Acorus calamus have been reported to exhibit potential pesticidal activity (Tariq et al., 2010).It has been reported that the essential oils containing linalool and the corresponding acetate play a major role in the anti-inflammatory activity (Peana et al., 2002).

Conclusion
Fromthe above result it is infered that the herb Acorus calamus is a rich source of phenylpropanoids with major compounds like β-asarone, α-asarone and Z-isoelemicin.The present study revealed that βasarone is positively correlated with the total amount of essential oil.These results confirmed that essential oil composition of herb can be different in quality and quantities in different geographical and environmental condition.Hence, the chemical diversity of the herb Acorus calamus essential oil can be a good source for herbal, neutraceuticals and phenylpropanoids, the biologically important class of terpenoids.

Acknowledgements
Authors are thankful to Jawahar Lal University, New Delhi, India for providing GC/MS facility.DST, New Delhi is thankfully acknowledged for financial assistance.

Figure 1 .
Figure 1.Structure of major chemical constituents of Acorus calamus leaf essential oils.

Figure 2 .
Figure 2.Comparative class composition of terpenoids of A. calamus leaves from 20 different altitudes.

Figure 3 .
Figure 3. DPPH radical scavenging activity of leaf essential oils from Acorus calamus

Table 1 .
Collection sites of plant.

Table 2 .
Comparative GC/MS analysis of A. Calamus leaves essential oil from 20 different altitudes of Uttarakhand.

Table 3 .
Comparative class composition of terpenoids of ACLEO from 20 different altitudes.

Table 4 .
7%) in leaf and rhizome essential oils of Acorus calamus.This variability in the leaf essential oils of Acorus calamus might be possibly due to their ploidy natures, seasonal factors or geographical conditions.Correlation between the total EOs content and main constituents of essential oil of A. calamus Leaves.

Table 5 .
IC 50 of A. calamus leaf essential oil of different altitudes. SN.

Sample Name IC 50 values (μg/ml) in triplicate
= Standard antioxidants, Values are means of three replicates ±SD.Within a column, mean values followed by the same letter are not significantly different according to Tukey's test (p<0.05). *