BIBECHANA 17 (2020) 89-95 BIBECHANA ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher: Department of Physics, Mahendra Morang A.M. Campus, TU, Biratnagar, Nepal Chemical profiling and antioxidant activities of essential oil from the rhizomes of Acorus calamus L. Sumnath Khanal 1 , Devi Prasad Bhandari 2 , Laxman Bhandari 2 , Sabita Dangol 1 , Achyut Adhikari 1 1 Central Department of Chemistry, Tribhuvan University, Nepal 2 Natural Products Research Laboratory, Thapathali, Kathmandu, Nepal * E-mail: achyutraj05@gmail.com Article Information Received: August 10, 2019 Accepted: December 01, 2019 Keywords: Acorus calamus L. DPPH assay essential oil IC50 ABSTRACT Acorus calamus L. is an indigenous herb in Nepal. It belongs to family Acoraceae and grows in wetland with scented rhizomes. It is also known as Sweet flag in English and commonly as Bojho in Nepal. The present investigation reveals the chemical compositions and antioxidant activity of rhizome essential oil of A. calamus. The essential oil of rhizomes of Acorus calamus L. from Kaski district, Nepal was extracted by hydrodistillation method and volatile constituents were analyzed using Gas chromatography-Mass spectrometry technique. The antioxidant potential of essential oil was analyzed by 1,1-Diphenyl-2- Picrylhydrazyl (DPPH) scavenging assay. A GC-MS analysis revealed the presence of β-asarone (22.38%), α-asarone (14.97%), 1-(4,6-dimethoxy-2,3- dimethylphenylethanone (14.24%), Isoelemicin (5.68%), cis-methylisoeugenol (4.26%), α-calacorene (4.16%), and other 20 minor components. From DPPH assay, half-maximal inhibitory concentration (IC50) value of essential oil was found to be 109.83 µg/mL. These findings have strengthened the A. calamus is good source of compounds like β-asarone, α-asarone and can be used as potential antioxidants. 1. Introduction Acorus calamus L. belongs to the Acoraceae family, is an important medicinal plant of South Asia commonly recognized as Bojho (in Nepali), Sweet flag (in English). It is a well-known herbal drug usually used in conventional medicines [1]. It grows as an herb with thick rhizomes in wetlands, mainly marshes [2]. It is widely distributed in South Asia along with some countries of Europe. The calamus species are used in the treatment of mental ailments, dysentery, chronic diarrhea, bronchial catarrh and abdominal tumors [3]. More specifically, A. calamus is an indigenous herb used in the treatment of fever, cough, bronchitis, inflammation, tumor, skin diseases and some other bacterial and fungal infections [4,5]. As an analgesic, it is used for relief of toothache, This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons.org/licenses/by-nc/4.0/ DOI: https://doi.org/10.3126/bibechana.v17i0.25201 http://nepjol.info/index.php/BIBECHANA mailto:achyutraj05@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ https://doi.org/10.3126/bibechana.v17i0.25201 BIBECHANA 17 (2020) 89-95 headache, cough, asthma, and bronchitis and as a sedative [6]. Additionally, it has significant antibacterial and antifungal properties [7]. The essential oil from the rhizome of A. calamus has been reported to have several biological activities such as antifungal, antibacterial [8] and its ethanolic extract has anti-cellular immunosuppressive properties [9]. Monoterpene hydrocarbons, sequestrine ketones, α- asarone (2,4,5-trimethoxy-1-propenyl benzene) and β-asarone are the major compounds of rhizome essential oil from A. calamus [10]. β-asarone (42.4% - 60.7%) as the major constituents followed by α-asarone (2.6% - 7.9%) have been reported from India [1]. However, β-asarone (40.59% - 76.33%) as the major constituent, α-asarone (1.29% - 10.48%) and some other minor compounds have been reported from Nepal [10]. The chemical constituents, yield, and activities are influenced by weather, soil conditions, time of harvest and some other minor factors [11]. Indian origin A. calamus has IC50 value 475.48 ± 0.08 µg/mL whereas 11.72 ± 0.03 µg/mL for standard [1]. The essential oil of A. calamus from different markets of Nepal exhibited the IC50value 312.64±1.14 µg/mL [12]. Antioxidants are the compounds that can slow or retard the oxidation of an oxidizable material, even when used in a small amount (commonly 1−1000 mg/mL) as compared to the amount of material they must protect [13]. The DPPH (2,2-diphenyl-1- picrylhydrazyl) is a well-known radical and by DPPH assay antioxidant potential can be determined. Because of a strong absorption band centered at about 517 nm, the DPPH radical has a deep violet color in solution, and it becomes colorless or pale yellow when neutralized. This property allows visual monitoring of the reaction. To the best of our knowledge, the chemical profile and antioxidant potential of rhizome essential oil of A. calamus from Kaski, Nepal has not been investigated yet. So, the objectives of this research are to find its volatile constituents by GC-MS and its role as an antioxidant. DPPH, purple Antioxidants DPPH-H yellow Fig. 1: Mechanism of antioxidants with DPPH radical. 2. Materials and Methods Plant materials Rhizomes of A. calamus L. were collected from the Togi village of Kaski district, Nepal in September 2018. The plant species was authenticated at National Herbarium and Plant Laboratories, Godavari, Nepal. Extraction of essential oils The rhizomes of A. calamus L. are washed with clean water and 200 g of it was subjected to .Sumnath Khanal et al./ hydrodistillation using the Clevenger apparatus with 600 mL of distilled water. The extraction of essential oil was continued for 6 hours. The vapors of water along with volatile oils were condensed into a liquid by a condenser which was fitted with Clevenger. The essential oil was collected in the BClevenger.oftubemeasuring ofecause immiscible nature, essential oil was separated from water and its volume was noted from which the percentage yield could be calculated. The oil was collected on a small glass bottle and a pinch of anhydrous sodium sulfate was added to remove moisture. Finally, the bottle was sealed, labeled and stored in the refrigerator until further analysis. Gas chromatography-Mass spectrometry (GC- MS) analysis The chemical composition of essential oil was analyzed by Gas Chromatography (Shimadzu GC 2010) having an RTX-5 MS column (60 m×0.32 mm×25µm) and using Helium as the carrier gas. The sample (100 µL) diluted with spectroscopic grade hexane in a ratio 1:10 was injected into the GC inlet maintaining constant flow rate of 0.68 mL min -1 and purge flow 3 mL min -1 in split mode. The initial column temperature was set at 40 o C. The qualitative analysis of oil was further continued in a Shimadzu GCMS-QP 2010 Plus. The MS Library used for comparison was FFNSC 1.3, NIST 2017. Oil components were identified based on their retention indices (RI) and by comparison of their mass spectral fragmentation patterns. DPPH assay This is a quick and easy method to analyze the scavenging potential of antioxidants. Free radical scavenging activity of essential oil was measured by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical, as previously described by Jamuna et al. [14]. For this, 100 mL solution of 0.1 mM concentration of DPPH was prepared and a stock solution of essential oil of 1 mg/mL.From the sample stock solution 50, 100, 150, 200, 250 μg/mL solutions were prepared. Then, to the sample solutions of different concentrations, 1mL DPPH solution was added. Finally, the absorbance at 517 nm was taken after incubation at room temperature for 30 minutes. Ascorbic acid was used as the standard for antioxidants. The percentage of inhibition was calculated by using formula, % I = AC−AO AC × 100% where, AC = absorbance value of the control (1 mL methanol+1 mL DPPH solution), AO = absorbance value of the sample solution, and I % = percentage of inhibition The radical scavenging activities of essential oil were expressed in terms of their IC50 values. 3. Results and Discussion Chemical profile The yellow-colored essential oil was obtained and the percentage yield of oil was 0.9 %. The percentage yield of essential oil in this study somewhat different from the previously published reports from India and Nepal. The Gas Chromatogram of oil showed a total of 29 peaks corresponding to total 29 compounds. The oil was further continued in a Shimadzu GCMS-QP 2010 Plus for the qualitative analysis. The MS Library used for comparison was FFNSC 1.3, NIST 2017, and from it, the 26 oil components were identified based on their retention indices (RI) and by comparison of their mass spectral fragmentation patterns. β-asarone (22.38%), α-asarone (14.97%), 1-(4,6-dimethoxy-2,3-dimethylphenylethanone (14.24%), Isoelemicin(5.68%), cis- Methylisoeugenol (4.26%), α-calacorene (4.16%), are major components and other are minor constituents (less than 3%), shown in table 1. The major components of oil of Indian origin were reported to be β- asarone (43.4-60.7%), α-asarone (2.6-7.9%), shyobunone (3.4-6.3%) and β- isoelemicin (3.2-5.4%) [1]. In the same way, Nepalese origin A. calamus has major constituents β- asarone and α-asarone are found 40.59% and 1.29% respectively from Banke, 76.33% and 10.41% respectively from Salyan [10]. This deviation in percentage yield and chemical constituents can be explained in terms of Sumnath Khanal et al./ BIBECHANA 17 (2020) 89-95 contribution from several factors including age, vegetative cycle stage, climate, seasons, soil composition, altitude, etc. Fig. 2: Gas chromatogram of rhizome essential oil of A. calamus. The abundant constituents β-asarone (1,2,4- Trimethoxy-5-[(Z)-prop-1-enyl] benzene) and α- asarone(1,2,4-Trimethoxy-5-[(E)-prop-1-enyl] benzene) are used in killing pests and bacteria [15]. Asarone is not metabolized to trimethoxyamphetamine, which has been reported by online vendors [16]. The Council of Europe Committee of Experts on Flavoring Substances concluded that β-asarone is clearly carcinogenic and has proposed limits for its concentration in flavorings such as bitters made from A. calamus [17]. β-Asarone exhibits anti- fungal activity by inhibiting ergosterol biosynthesis in Aspergillus niger [18]. Antioxidant activity DPPH assay is fundamentally based on the capability of DPPH free radical which is discolored in the presence of antioxidants present its sample. The results of the antioxidant activity of this study are demonstrated below. The IC50 values of ascorbic acid and essential oil of A. calamus was calculated and shown below in table 2. The half inhibitory concentration (IC50) of A. calamus oil was found to be 109.83 µg/mL whereas that of standard ascorbic acid is 25.38 µg/mL. This implies that A. calamus oil has 0.231 times antioxidant potential than that of ascorbic acid. These results are in close agreement with the previous report suggested by Bhandari et. al., [12] which revealed that the antioxidant property of A. calamus oil is 0.277 times than that of ascorbic acid. In the same way, this result also correlated with the report suggested by Parki et al. [1]. Abundance Retention time (Minutes) Sumnath Khanal et al./BIBECHANA 17 (2020) 89-95 https://en.wikipedia.org/wiki/Pest_(organism) https://en.wikipedia.org/wiki/Carcinogen https://en.wikipedia.org/wiki/Bitters https://en.wikipedia.org/wiki/Acorus_calamus Table 1. The chemical composition of rhizome essential oil of A. calamus. Peak Retention time Retention index Area % Name of the Compounds Identification methods 1 9.397 831 0.36 Furfural RI, MS 2 18.02 1046 0.41 β-Ocimene RI, MS 3 34.158 1375 0.54 1,3-Dimethyl-8-propan-2-yltricyclo [4.4.0.02,7] dec-3-ene RI, MS 4 34.794 1398 0.38 1-Ethenyl-1-methyl-2,4-bis(prop-1-en-2-yl) cyclohexane RI, MS 5 36.138 1419 0.52 Isoledene RI, MS 6 36.707 1434 0.68 Calarene RI, MS 7 37.42 1455 4.26 cis-Methylisoeugenol RI, MS 8 37.12 1463 1.59 2,3-5,6-Bis(1,5-octanediyl)-2,5-dibora-1,4- dioxane RI, MS 9 39.282 1491 0.73 Viridiflorene RI, MS 10 39.389 1497 0.40 α-Muurolene RI, MS 11 40.323 1543 2.91 Cedrol RI, MS 12 40.617 - 2.01 δ-Cadinene MS 13 40.323 1537 2.21 9- Cedranone RI, MS 14 40.617 1538 1.18 (2S,3S,6S)-6-Isopropyl-3-methyl-2-(prop-1-en- 2-yl)-3-vinylcyclohexanone RI, MS 15 40.676 1544 4.16 α-Calacorene RI, MS 16 41.203 1550 0.97 Elemicin RI, MS 17 41.409 1565 5.68 Isoelemicin RI, MS 18 42.144 1614 2.30 Acorenone B RI, MS 19 43.755 1617 22.38 β-Asarone RI, MS 20 44.101 - 8.19 - 21 45.182 1634 14.24 1-(4,6-dimethoxy-2,3-dimethylphenyl) ethanone RI, MS 22 44.284 1635 2.48 Isolongifolol RI, MS 23 44.747 1659 0.76 - 24 45.097 1659 0.59 Cadin-4-en-10-ol RI, MS 25 45.933 1663 1.1 6-Methyl-2-(4-methylcyclohex-3-en-1-yl) hepta-1,5-dien-4-ol RI, MS 26 46.301 1679 14.97 α-Asarone RI, MS 27 46.442 1683 0.91 (5-Methyl-8-propan-2-yl-3,4,4a,7,8,8a- hexahydronaphthalen-2-yl) methanol RI, MS 28 47.723 - 0.9 - 29 48.981 1771 2.18 4α-hydroperoxy-2,5,5,8α-tetramethyl-4- methylidene-7,8-dihydro-6H-chromene RI, MS Sumnath Khanal et al./BIBECHANA 17 (2020) 89-95 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 % In h ib it io n Concentration(µg/mL) Ascorbic acid A. calamus Fig. 3: Percentage Inhibition vs Concentration plot of Ascorbic acid and A. calamus esssential oil Table 2: IC50 values of standard ascorbic acid and A. calamus essential oil. S.N Name IC50 (µg/mL) 1. Standard Ascorbic acid 25.38 2. The essential oil of A. calamus 109.83 4. Conclusions The rhizome essential oil of A. calamus from Kaski, Nepal was analyzed by GC and GC-MS and was found to be rich of β-asarone, α-asarone, asarone, α-calacorene and cis-Methylisoeugenol. Furthermore, essential oil of rhizome of A. calamus is a good source of antioxidants. Acknowledgment We would like to express our sincere gratitude to Department of plant resources for providing lab facilities to conduct this research work. Financial support (Award no. MRS/74_75/S&T-24) to Mr. Sumnath Khanal from University Grant Commission (UGC) is heartily acknowledged. References [1] A. Parki, P. Chaubey, O. Prakash, R. Kumar, and A. K. pant, Seasonal Variation in Essential Oil Compositions and Antioxidant Properties of Acorus calamus L. Accession, Medicines 4(2017) 81.doi:10.3390/medicines4040081 [2] P. 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