Devendra Sir Cover Page copy.jpg BIBECHANA 17 (2020) 104-109 104 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 Phytoconstituents and biological analysis of Acorus calamus rhizome of Sindhupalchowk District, Nepal Shanta Pokhrel * , Kalpana Chaulagain Department of Chemistry, Tri-Chandra Multiple Campus, Tribhuvan University, Nepal ∗E-mail: shantabhattarai2014@gmail.com Article Information Received: October 25, 2019 Accepted: December 2, 2019 Keywords: Acoruscalamus rhizome, Antibacterial activity, Antioxidant activity ABSTRACT Phytochemical and biological activities of methanolic and hexane extract of Acorus calamus rhizome were carried out. Phytochemical analysis showed the presence of flavonoid, glycoside, saponin, resin and carbohydrates which are responsible for the antibacterial activities. The antibacterial potential was studied against Staphylococcus aureus (gram positive bacteria) and Escherichia coli, Salmonella typhi (gram negative bacteria) using Agar Well Diffusion Method. The activity was shown by both methanolic and hexane extract of Acorus calamus rhizome against both Gram positive and Gram negative bacteria. Antioxidant activity of methanolic extract was evaluated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity showed potent antioxidant activities with IC50 value 3.74 μg/mL slightly higher than standard ascorbic acid (IC50 = 3.56 μg/mL). 1. Introduction Acorus calamus Linn. (familyAraceae) commonly referred to as calamus or sweet flag, Bojho in Nepali, is a well-known medicinal plant.The rhizomes were utilized extensively by the Chinese, Indians and American Indians as well as by other cultures, and many of these uses continue to this day [1] including in Nepali traditional medicine [2]. People of Far Western Region (Baitadi and Darchula) 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 [3,5]. Medicinal plants, possessing secondary metabolites, are potential sources of curative drugs with the very long list of chemicals and its curative nature. Plant products are used as main source of medicine throughout the world for treating various human ailments [6]. The interest to study the plants is because of their medicinally and pharmacologically important active ingredients. Plant produces a plethora of natural products, such as alkaloids, flavonoid, glycoside, steroid, saponin and resin which has often been correlated with medicinal and pharmacological properties of the plants. The 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.v17i0.24674 http://nepjol.info/index.php/BIBECHANA mailto:shantabhattarai2014@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ https://doi.org/10.3126/bibechana.v17i0.24674 Shanta Pokhrel, Kalpana Chaulagain / BIBECHANA 17 (2020) 104-109 105 rhizome, roots and essential oil distilled from have been reported to possess several important biological activities including antimicrobial [7-10], anticellular and immunosuppressive [11]. The aim of present study was to investigate the phytochemical constituents of extracts with methanol and hexane solvents, their antioxidant activity and antibacterial activities of extract Acorus calamus rhizomes against different strains of bacteria. 2. Experimental Collection and preparation of plants extracts The Acorus calamus rhizome was collected from the Sindhupalchowk, Nepal. The collected rhizomes were washed by fresh water to remove other contamination like dust, soil, insect larvae, eggs etc. then clean plant were air dried in shade for about a month, then obtained dried leaves were grinded to power and store in clean plastic bag. The powdered form (60 g) of plants rhizomes was extracted by Soxhlet method in n-Hexane, and methanol solvents (250 mL) at room temperature. The solvent used for Soxhlet extraction, which has undergone several cycle for about 8-10 hours was collected and obtained solvents were individually concentrated by rotary evaporator under reduced pressure, maintaining temperature lower than the boiling point of respective solvents used. These extracts were stored in the refrigerator until use in vile tubes. Phytochemical screening Analysis of crude methanolic and hexane extracts of Acorus calamus rhizome for various phytochemical constituents were carried out using standard protocols [12]. The analysis of the presence of main group of natural constituents present in the different plant extract was done by the color reaction using different specific reagents. Antibacterial activity Antibacterial activity of the plant extract was performed by agar well diffusion method. Effectiveness of antimicrobial substance was evaluated by determination of zone of inhibition (ZOI) [13]. The microbial strains staphylococcus aureus cocci ATCC 25923 (gram-positive) and Escherichia coli ATCC 25922 and Salmonella typhi (gram-negative) bacteria were obtained from MED- MICRO Nepal Lab, Kathmandu. The 50 µL of the working solution of the plant extract, DMSO as negative control (NC) and 25 µL of Ofloxacin (antibiotic- ear and eye drop) as positive control (PC) at the same time in separated well (6 mm) were loaded into the respective wells with the help of micropipette. The plates were then left for half an hour with the lid closed so that the extract diffused into media. The plates were incubated overnight at 37 ℃. After 24 hours of incubation, the plates were observed for the presence of inhibition of bacterial growth that indicated by a clear zone around the wells. The size of the zone of inhibition was measured and the antibacterial activity expressed in term of the average diameter of zone of inhibition in millimeters. The ZOI were measured with the help of millimeter ruler and mean was recorded. Antioxidant activity The free radical scavenging activity of samples and standard ascorbic acid solution in methanol was determined based on their ability to react with stable 1, 1-diphenyl-2-picrylhyrazyl (DPPH) free radical [14]. The IC50 (50% inhibitor concentration) value is indicated as the effective concentration of the sample that is required to scavenge 50% of the DPPH free radicals. IC50 values were calculated using the inhibition curve by plotting extract concentration vs the corresponding scavenging effect. Different concentrations of test samples of 20, 40, 60, 80 and 100 µ/mL were made from stock solutions. Then 2 mL of all the concentration of test solution were mixed 2 mL of 2 mM DPPH solution. The test tubes were shaken vigorously for the uniform mixing then the solutions was kept for 30 minutes in dark place at room temperature. The control was prepared as above but without the plant extracts (methanol + DPPH). After 30 minutes absorbance of the entire sample was measured at 517 nm using a UV-visible spectrophotometer. The antioxidant activity of the samples was expressed as IC50 (inhibitory concentration), which was defined as the concentration (in μg/mL) of sample required to inhibit the formation of DPPH radicals by 50%. Ascorbic acid was used as positive control. Free Shanta Pokhrel, Kalpana Chaulagain / BIBECHANA 17 (2020) 104-109 106 radical scavenging activity was calculated by using following equation (1): Radical Scavenging (%) = A0 − As A0 ∗ 100 (1) where, Ao = Absorbance of the control (DPPH solution + methanol) As = Absorbance of test sample The % scavenging was then plotted against concentrations used and from the graph IC50 was calculated. 3. Results and Discussion Phytochemical screening The different phytochemicals in the crude methanol and n-hexane extracts were identified by the color reaction with different reagents. The results obtained for methanolic and n-hexane extract of the plant is tabulated below (Table 1). A preliminary study has reported the rhizome extracts contains large number of bioactive secondary molecules like alkaloids, tannins, carbohydrates, flavonoids, glycoside (Table-1). The presence of these components in this species is an indication that it may have some medicinal potential. The rhizome of Acorus calamus are used traditionally for treatment of emetic, stomachic, dyspepsia, colic, remittent fevers, nerve tonic, in bronchitis, dysentery in children, insectifying and in snake bite [6]. These results are consistent with reported in the literature [6] with slight difference. This is due to the variation in altitude of plants and different environmental conditions. Table 1: Phytochemical screening of various extracts of Acorus calamus. S.N. Phytochemicals n-Hexane Extracts Methanol Extracts 1. Alkaloids - + 2. Flavonoids + + 3. Steroids - + 4. Glycosides - + 5. Tannins + + 6. Carbohydrates - - 7. Phenol - - (+) sign indicated the presence of phytochemicals whereas (-) sign indicated the absence of phytochemicals Antibacterial assay The diameter of zone of inhibition (ZOI) produced by the plant extracts on particular bacteria was measured for the estimation of their antibacterial activity. The Methanol and hexane extracts of rhizome of Acorus calamus were studied. Results obtained from the antibacterial analysis of different extracts are tabulated (Table 2). Figure 1 (a, b, c) show the results of antibacterial activity of methanolic and hexane extract of Acorus calamus rhizome. It is clear that the extract shows higher antibacterial activity against S. aureus than the Salmonella typhi and E. coli. Hexane extracts shows higher antibacterial activity against S. aureus than the Salmonella typhi and E. coli gives ZOI value 15 mm, 11 mm and 11 mm respectively in 50% concentration. But in crude hexane extract of Acorus calamus only show antibacterial activity against S. aureus gives ZOI value 14 mm and others do not show antibacterial activity. Table 2: Antibacterial analysis of methanol and hexane extracts of Acorus calamus rhizome. S.N. Plant extract (Solid) Bacteria ZOI (mm) of Extracts at Concentration 100 mg/ mL ZOI (mm) of Description Ofloxacin as Control at 100 mg/mL 1. Hexane 50% Crude E. coli 11 0 30 S. aureus 15 14 28 S. typhi 11 0 26 2. Methanol 10% 1% E. coli 10 0 30 S. aureus 12 10 28 S. typhi 0 12 26 Methanol extracts of Acorus calamus against Staphylococcus aureus and Salmonella typhi gives ZOI value 10 mm and 12 mm respectively in 1% concentration. But in 10% concentration of methanol extracts against Staphylococcus aureus and Shanta Pokhrel, Kalpana Chaulagain / BIBECHANA 17 (2020) 104-109 107 Escherichia coli gives ZOI value 10 mm and 12 mm and other are inactive. Hexane extract shows highest zone of inhibition against Staphylococcus aureus 15 mm in 50% concentration than that in methanol (12 mm in 10% concentration). According to Chhatopadhyay et al. the rhizome of A. calamus exhibited 16 mm zone of inhibition, which support the present finding [16]. Fig. 1: Antibacterial screening of methanol and hexane extract of Acorus calamus rhizome against (a) Escherichia coli, (b) Staphylococcus aureus, (c) Salmonella typhi. Antioxidant activity The antioxidant activity of the methanolic solution of different samples were explored by using 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. DPPH assay Antioxidants are the compounds which terminate the attack of reactive species and reduce the risk of disease [16]. Most of the plants have antioxidant property. In this study, the antioxidant activity of each plant extract was measured by using 1,1- diphenyl-2-picrylhydrazyl radical (DPPH), is a very stable free radical with purple color. It is scavenging by antioxidants through the donation of proton forming the reduced DPPH. The DPPH radical assay was performed for methanol extract of Acorus calamus rhizome by using ascorbic acid as standard. In this assay the mixture of DPPH with different concentration of extract solution and ascorbic acid were separately incubated at room temperature and absorbance was recorded at 517 nm by spectrophotometer. Calibration curve was constructed by measuring the absorbance of ascorbic acid in order to calculate IC50 value. The value obtained from plant extract was compared with ascorbic acid. The observed absorbance with the different concentration of ascorbic acid was plotted in the graph as shown below. The comparison of percentage radical scavenging at different concentration between plant extract and ascorbic acid as standard was shown in Table (3) and Figure (2). The comparison of percentage radical scavenging at different concentration between plant extract and ascorbic acid as standard was shown in graph. The antioxidant potential is in an inverse relation with IC50value, lower value of IC50 indicates high antioxidant potential. The IC50 values of the plant extracts along with the standard ascorbic acid is tabulated below. It is found that the IC50 value of Acorus calamus rhizome in methanol extract is 3.74 μg/mL and that of standard ascorbic acid is 3.56 μg/mL. By comparing the reference standard ascorbic acid, the methanol extract showed less a b c Shanta Pokhrel, Kalpana Chaulagain / BIBECHANA 17 (2020) 104-109 108 antioxidant activity. Kumar reported that the chloroform extract of Acorus calamus showed more antioxidant property and is potent DPPH free radical scavenger [17]. World Health Organization (WHO) mentioned that medicinal plants would be the best source to a variety of drugs. About 80% of individuals from developed countries use natural medicine. Alkaloids and tannin have antioxidant capacities because they have been found to possess antioxidant and free radical scavenging effect [17]. Fig. 2: Graph of absorbance vs concentration of ascorbic Acid. Table 3: Percentage of radical scavenging with different concentrations Radical scavenging (%) Sample Concentration (µg/mL) 20 40 60 80 100 MR 13.21 19.38 27.50 58.66 72.59 Ascorbic Acid 13.12 23.75 31.87 42.58 52.05 where, MR =Methanol Rhizome Extract Fig. 3: A plot of percentage radical scavenging activity vs concentration of methanol extract and ascorbic acid Table 4: Comparison of IC50 values of methanol extract and ascorbic acid. S. No Sample IC50(µg/mL) 1. Standard Ascorbic acid 3.56 2. Methanol rhizome extract 3.74 0 0.2 0.4 0.6 0.8 1 1.2 0 20 40 60 80 100 120 A b so rb an ce Concentration Absorbance Absorbance y = 15.804x - 9.144 y = 9.669x + 3.667 0 20 40 60 80 20 40 60 80 100% R ad ic al s ca v en g in g Concentration (µg/mL) methanol ascorbic acid Shanta Pokhrel, Kalpana Chaulagain / BIBECHANA 17 (2020) 104-109 109 4. Conclusion Phytochemical screening of different rhizome extract of Acorus calamus showed the flavonoids and tannins were present in both the extracts while alkaloids, glycosides and steroids were only in methanol extract. The phytochemical screening and antibacterial assay of the plants indicated positive application of these plant extracts for medicinal use wherein their antioxidant property is an added advantage. More studies on this plants help to study the use of the plant. Acknowledgements Authors are thankful to Department of Chemistry, Tri-Chandra Multiple Campus, Tribhuvan University, Kathmandu, Nepal for providing laboratory facilities to conduct experimental work. We are thankful to Med-Micro Nepal Lab, Thapathali, Kathmandu, Nepal for antibacterial tests. References [1] T. J. Motley, The ethnobotany of sweet flag, Acorus calamus (Araceae), Econ. Bot. 48 (1994) 397-412. 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