Composition, antioxidant and anti-inflammatory activities of different polarity extracts of Artemisia nilagirica collected from hilly areas in the Himalayan terrain of Uttarakhand European Journal of Chemistry 14 (3) (2023) 323-329 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2023 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.14.3.323-329.2449 European Journal of Chemistry View Journal Online View Article Online Composition, antioxidant and anti-inflammatory activities of different polarity extracts of Artemisia nilagirica collected from hilly areas in the Himalayan terrain of Uttarakhand Vaishali Garia 1, Shiv Kumar Dubey 1,*, Ananya Bahuguna 1, Ravendra Kumar 2, Om Prakash 2 and Dharmendra Singh Rawat 3 1 Department of Biochemistry, College of Basic Sciences and Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar-263145, Uttarakhand, India 2 Department of Chemistry, College of Basic Sciences and Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagr-263145, Uttarakhand, India 3 Department of Biological Sciences, College of Basic Sciences and Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar-263145, Uttarakhand, India * Corresponding author at: Department of Biochemistry, College of Basic Sciences and Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar-263145, Uttarakhand, India. e-mail: sk.dubey@gbpuat-cbsh.ac.in (S.K. Dubey). 10.5155/eurjchem.14.3.323-329.2449 Received: 17 May 2023 Received in revised form: 05 July 2023 Accepted: 11 July 2023 Published online: 30 September 2023 Printed: 30 September 2023 The plant Artemesia nilagirica, collected from the terrain of the Himalayan region in Uttarakhand, India, was evaluated for its phytochemical composition, antioxidant and anti- inflammatory activities in vitro. The different polarity extracts of the plant were prepared and subjected to GC-MS analysis for their phytochemical composition. Twenty-six compounds were identified in the hexane extract of Artemisia nilagirica that represents 73.30% of the total area. The main compounds were tetracontane (15.21%), heneicosane (6.52%), and phytyl tetradecanoate (5.11%). The methanol extract yielded 26 compounds, accounting for 83.78% of the total compounds detected. The main compounds were palmitic acid (13.25%), alpha-linolenic acid (10.32%), oleamide (9.41%), phytol (8.58%), muco- inositol (7.27%), and neophytadiene (5.05%). The hexane and methanol extracts showed significant metal chelating activity having IC50 values of 2.23±0.01 and 2.41±0.02 µg/mL, respectively, with standard EDTA having an IC50 value of 2.22±0.01 µg/mL. The methanol extract showed a better anti-inflammatory property having an IC50 value of 2.97±0.03 µg/mL compared to the standard diclofenac potassium having an IC50 value of 3.79±0.01 µg/mL. The hexane extract showed better antioxidant activity in terms of metal chelating activity and reducing power activity than the methanol extract. The anti-inflammatory activity of both hexane and methanol extracts showed better results than the standard marketed drug diclofenac potassium. The current study reveals that Artemisia nilagirica plant extracts have potent antioxidant and anti-inflammatory activities. The effective biological compounds of plant extracts, such as phenols and flavonoids, can be potential alternatives to standard pharmaceuticals. Biological activity Artemisia nilagirica Mass spectrometry Antioxidant activity Gas chromatography Anti-inflammatory activity Cite this: Eur. J. Chem. 2023, 14(3), 323-329 Journal website: www.eurjchem.com 1. Introduction Plants have a long history of usage as traditional remedies to cure a variety of disorders. There are still many plants that need to be investigated for the existence of biologically active substances [1]. The phytochemical components of medicinal plants are responsible for their therapeutic quality. Medicinal plants can be found primarily in the eastern Himalayas, Western Ghats, and Andaman and Nicobar Islands. India is dubbed as the 'World’s Botanical Garden' because it produces the most therapeutic herbs. Ayurveda and naturopathy as health-protecting and curing disease practices are popular and prevailing in India since time immemorial. Ayurveda emerged in India around 600 BC and uses medicinal plants for the treatment of various ailments [2]. Numerous plants having restorative properties exhibit extraordinary antioxidant potential as well as anti- inflammatory activities. When oxygen molecules break apart into single unpaired electron oxygen atoms, they become unstable free radicals that seek other atoms or molecules to attach to. Free radicals can negatively impact a number of significant types of biological components, including lipids, proteins, and nucleic acids, changing the normal redox state and increasing oxidative stress [3]. The antioxidants present in plants reduce oxidative stress in cells and are then helpful in the therapy of numerous human diseases, including malignancy, cardiovascular diseases, and incendiary infections [4]. The anti- inflammatory properties of plants are attributed to their potential to inhibit or down-regulate the secretion of pro- inflammatory stimuli such as cytokines, etc. ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.14.3.323-329.2449 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.14.3.323-329.2449 mailto:sk.dubey@gbpuat-cbsh.ac.in http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.14.3.323-329.2449&domain=pdf&date_stamp=2023-09-30 324 Garia et al. / European Journal of Chemistry 14 (3) (2023) 323-329 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.3.323-329.2449 For thousands of years, the selective use of crude plant extracts has been the earliest ritual in the ancient Indian medicinal system, 'Ayurveda,' as well as in traditional Chinese medicine. To produce high particle medications from plant components, modern methodologies have combined multidisciplinary technologies and specific chemical substances extracted and identified. Plants that produce a significant amount of polysaccharides, steroids, terpenoids, flavonoids, alkaloids, and antibiotics are useful for developing medications for a variety of diseases and disorders, including cancer therapies [5]. One of the most common groups of plants used in traditional medicine is the Asteraceae family [6]. Artemisia nilagirica (C.B. Clarke) Pamp is widely found in the hilly areas of India. It is known as 'Nagdona' in Hindi. It is an aromatic, herbaceous perennial plant that belongs to the Asteraceae family. It is also commonly found in Europe, Asia, northern Africa, Alaska, and North America. It grows well in the nitrogenous soils of hilly regions of India, such as the Uttarakhand Himalayan region. The chemical composition of most members of the family is similar. All species, for example, are good sources of inulin, a natural polysaccharide with strong prebiotic characteristics [7]. Artemisia nilagirica has been effective against asthma, caused by exposure to common allergens present in the environment. The plant shows anticancer and antioxidant activity which may be due to the free radical quenching properties of sesquiterpene lactones and flavonoids present in the ethanolic extract of the plant. Essential oils of Artemisia, generally extracted from aromatic plants, contain a variety of volatile components, for example, terpenoids, phenylpropanoids, and aliphatic mixtures [8]. The purpose of the current study was to evaluate the antioxidant and anti-inflammatory properties of Artemisia plant in relation to its phytochemical composition from the Himalayan region. Plant extracts were prepared using two different solvents, hexane and methanol. The extracts were then subjected to a GC-MS analysis to determine their phyto- chemical composition. Following phytochemical analysis, the biological activities of the extracts, such as their antioxidant and anti-inflammatory properties, were evaluated using standard protocols. Twenty-six compounds were identified in the hexane extract as well as the methanol extract of A. nilagirica. The results of the present study indicate that Artemisia nilagirica plant extracts have potent antioxidant and anti-inflammatory activities. The hexane extract showed better antioxidant activity in terms of metal chelating activity and reducing power activity than the methanol extract. The effective biological compounds of plant extracts, such as phenols and flavonoids, can be potential alternatives to traditional medications. The study reveals that the plant exhibits potent antioxidant and anti-inflammatory activities in vitro. 2. Experimental 2.1. Plant collection and authentication Artemisia nilagirica was collected from the hilly region of Uttarakhand near Jeolikote at an elevation of 1320 m in September 2020. The plant sample was submitted to Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, India. The identification of the plant sample was carried out by Dr. Dharmendra Singh Rawat (Assistant Professor and Plant Taxonomist), Department of Biological Sciences, College of Basic Sciences and Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, India. 2.2. Extract preparation The whole plant of A. nilagirica was shade dried for four days and was coarsely powdered and successively extracted using the cold percolation method [9]. The extraction was done by using different polarity solvents based on the order of their increasing polarity. The yield obtained in the case of the methanol extract was 3.257 g from 240.57 g of the whole plant having a 1.35% yield while in the case of the hexane extract it was 2.255 g from 243.57 g of the plant having a yield of 0.92%. 2.3. Analysis and identification of compounds To identify the phytochemical composition of plant extracts, an analytical technique, gas chromatography mass spectrometry (GC-MS), was carried out using Shimadzu GCMS- QP2010 Plus equipment with helium as the carrier gas having a pressure of 73.3 kPa and a velocity of 40.1 cm/sec. The column flow rate was maintained at 1.21 mL/min. The carrier gas saver, high pressure injection, and splitter hold were turned off, and the oven temperature was set to 60 °C RAMP@ 3 °C/min up to 210 °C (isotherm for 2 min), then 6 °C/min up to 280 ° C (isotherm for 2 min), then hold for 11 min, flame thermionic detector (FTD) [10]. The Kovats indices (KI) of the peaks in the DB-5 column were compared to the values in the literature and compared to the standard library spectra, which were constructed using pure substances and components of known essential oils. The fragmentation pattern of the mass spectra produced by GC-MS analysis was compared with those contained in the WILEY 8 library [11] and the published literature for further identi- fication [12]. The peak areas obtained without FID response factor correction were used to calculate the relative amounts of identical components. 2.4. Antioxidant activities 2.4.1. 2,2’-Diphenylpicrylhydrazyl (DPPH) free radical scavenging activity The free radical scavenging activity of methanol and hexane extracts was determined with the help of the previously reported method [13]. The dark-colored crystalline powder DPPH is made up of stable free radical molecules. The DPPH solution in methanol was freshly prepared and stored in an amber-colored bottle in the dark to prevent the UV rays to enter inside. In methanol, a stock solution of 0.1 mM DPPH was prepared. The free radical scavenging activity of various polarity solvent extracts, referred to as test samples, was inves- tigated. Varied polarity extracts were prepared at different concentrations of 5, 10, 15, 20, and 25 µg/mL. Then 0.1 mL of each concentration of both extracts was mixed with 2.9 mL of 0.1 mM DPPH solution and kept in the dark for 30 min incubation. Using a UV spectrophotometer (Thermo Scientific Genesys 10S UV-VIS) the absorbance was determined at 517 nm. Ascorbic acid was utilized as the standard. The percentage of scavenging activity of the DPPH free radical was calculated with the help of the following formula [13]. DPPH radical scavenging activity (%) = (𝐴𝐴𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐−𝐴𝐴𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑐𝑐𝑠𝑠) 𝐴𝐴𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 × 100 (1) where, Acontrol is the absorbance of the DPPH radical solution and Asample is the absorbance of the test sample. The IC50 value was derived by plotting the percentage scavenging of the DPPH free radical versus the concentration of the test sample. The ability of a sample to inhibit a reaction is measured by the half-maximum inhibitory concentration or IC50. Here, the concentration of the test sample that scavenges Garia et al. / European Journal of Chemistry 14 (3) (2023) 323-329 325 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.3.323-329.2449 50% of the DPPH free radical is called IC50. Low IC50 means high antioxidant activity and vice versa. 2.4.2. Reducing power activity The extracts were prepared at different concentrations of 50, 100, 150, 200, and 250 µg/mL in their respective solvents. A volume of 2.5 mL from each concentration was added to 2.5 mL of phosphate buffer (200 mM) having a pH of 6.6 and 2.5 mL of potassium ferricyanide (1% w/v). After that, the samples were incubated in a water bath for 20 minutes at 50 °C. After incubation, 2.5 mL of trichloroacetic acid (10% v/v in distilled water) was added. The mixture was then centrifuged at 50.54 g (650 rpm) for 10 minutes. Then, 5 mL of supernatant was taken and mixed with 5 mL of distilled water. The UV spectro- photometer (Thermo Scientific Genesys 10S UV-VIS) was used to measure the absorbance of the resulting solution at 700 nm after 1 mL of ferric chloride was added to the mixture. Readings were taken in triplicate. The gallic acid standard was also subjected to the same process, with measurements collected in triplicate. The same technique was used on the control, with the exception that no samples were added [14]. The percentage of reducing power activity of the extracts and the gallic acid standard was calculated with the following formula [14], Reducing power activity (%) = (𝐴𝐴𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐−𝐴𝐴𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑐𝑐𝑠𝑠) 𝐴𝐴𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 × 100 (2) where Acontrol = Absorbance of the control and Asample = Absorbance of the test sample. The percentage activity graph of reducing power against concentration was plotted to calculate the IC50 value of the extracts and standard. The low value of IC50 indicates high reducing activity. 2.4.3. Metal chelating activity The metal chelating activity is based on the chelation of ferrous ions by ferrozine. Ferrozine forms a complex with ferrous ions and forms a red color [15]. Different concent- rations of 5, 10, 15, 20, and 25 µg/mL of plant extracts were prepared in their respective solvents and added to 0.05 mL of 2 mM FeCl2.4H2O. To it, 0.2 mL of 5 mM ferrozine was added and the volume was made up to 5 mL by adding methanol. The mixture was incubated for 10 min at room temperature and the absorbance was measured at 562 nm. The standard for this activity was EDTA, which was subjected to the technique described above. The following formula [15] was used to calculate the % inhibition metal chelating activity of the extracts and the EDTA standard % Metal chelating effect = (𝐴𝐴𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐−𝐴𝐴𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑐𝑐𝑠𝑠) 𝐴𝐴𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 × 100 (3) where Acontrol = Absorbance of control and Asample = Absorbance of the test samples. The graph of % chelating activity against concentration was plotted to calculate the IC50 value of the extracts and the standard. A low value of IC50 indicates high metal chelating activity. 2.5. In vitro anti-inflammatory activity In vitro anti-inflammatory activity was investigated according to the protocol developed by Kar et al. [16]. Various concentrations i.e., 5, 10, 15, 20, and 25 µg/mL of methanol and hexane extracts, were made in their respective solvents. The volume of 2 mL of samples at different strengths was mixed with 2.8 mL of freshly produced phosphate buffer at pH = 6.4. The mixture was then spiked with 0.2 mL of egg albumin. As a result, the total volume was 5 mL. The mixture was incubated for 15 min at 37 °C and for 5 min at 70 °C. The absorbance was finally measured at 660 nm. The standard was chosen as diclofenac potassium. The following formula [16] was used to calculate the anti-inflammatory activity of the extracts and diclofenac potassium standard, Anti − inflammatory activity (%) = (𝐴𝐴𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐−𝐴𝐴𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑐𝑐𝑠𝑠) 𝐴𝐴𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 × 100 (4) where Asample = Test sample absorbance and Acontrol = Control absorbance. 2.6. Statistical analysis The mean and standard deviation of triplicates of plant extracts were calculated using Microsoft Excel. To evaluate their significance (p < 0.05), all results were subjected to a 5%- point Duncan test for one-way analysis (ANOVA) [17]. SPSS software was used to determine the significance and association of various extracts [17]. 3. Results and discussion 3.1. GC-MS analysis of the hexane extract of the whole A. nilagirica plant Twenty-six compounds were identified in the hexane extract of A. nilagirica which comprised a total of 73.30% of the area. The main compounds were tetracontane (15.21%), hene- icosane (6.52%), phytyltetradecanoate (5.11%), tetratetra- contane (4.96%), squalene (2.93%), alpha-linolenic acid (2.58%), and 24-norursa-3,12-diene (2.10%). The compounds present in lesser amounts were isononacosane (0.50%), phytol (0.51%), lyratyl acetate (0.55%), beta-amyrin (0.58%), cis-9- hexadecenal (0.65%) and many more. Alkanes were the class of compounds that were found most abundantly in the hexane extract of the plant, which occupied 46.99% area followed by saturated fatty acids, which occupied 7.05%, triterpenes occupying 3.51% of the area followed by unsaturated fatty acids, which occupied 2.58% area. Sterols comprised 2.55% of the class of chemical compounds. Other minor compounds comprised 10.62% area of a total of 73.30% area of chemical compound classes. Table 1 shows the chemical composition of A. nilagirica hexane extract and Figure 1 shows the gas chromatogram of the hexane extract of the whole plant of A. nilagirica. 3.2. GC-MS analysis of the methanol extract of the whole plant of A. nilagirica Twenty-six compounds were identified in the methanol extract of A. nilagirica, contributing to 83.78% of the total compounds identified. The main compounds were palmitic acid (13.25%), alpha-linolenic acid (10.32%), oleamide (9.41%), phytol (8.58%), muco-inositol (7.27%), and neophytadiene (5.05%). Minor compounds include oleic acid (0.84%), 2- (dimethylamino) ethyl 3-cyclopentylpropanoate (0.78%), triacontane (0.63%), linoleoyl chloride (1.73%), octadecyl trifluoroacetate (0.57%) and urs-12-ene (0.93%). Unsaturated fatty acids were the major class of compounds comprising 14.54% area followed by saturated fatty acids comprising 13.25% area of the total composition of the methanol extract. The fatty acid amide comprised 9.41% area and oxygenated diterpenes comprised 8.58%. Esters and triterpenes were also present in large amounts. Other minor compounds had a percentage area of 41.31%. The precise information about the compounds is provided in Table 2 and Figure 2. 326 Garia et al. / European Journal of Chemistry 14 (3) (2023) 323-329 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.3.323-329.2449 Table 1. Chemical composition of A. nilagirica hexane extract. No Compound Chemical formula Kovat’s index %Area Class of compounds 1 1-Isopropenyl-3-propenylcyclopentane C11H18 1095 1.09 Monocyclic monoterpenoid 2 Lyratyl acetate C12H18O2 1387 0.55 Ester 3 (-)-Oplopanone C15H26O2 1734 0.76 Sesquiterpenoid 4 Phytyl tetradecanoate C34H66O2 1762 5.11 Saturated fatty acid 5 cis-9-Hexadecenal C16H30O 1800 0.65 Aldehyde 6 4-tert-Butylcalix[4]arene C44H56O4 1859 2.05 Macrocyclic phenols 7 24-Norursa-3,12-diene C29H46 1897 2.10 Alkene 8 trans-trans-10,11-Epoxy farnesenic acid methyl ester C16H26O3 1921 0.70 Ester 9 Phytol C20H40O 1949 0.51 Acyclic diterpene alcohol 10 Palmitic acid C16H32O2 1983 1.94 Saturated fatty acid 11 Eicosane C20H42 2000 1.47 Alkane 12 Methyl linolenate C19H32O2 2067 1.24 Ester 13 alpha-Linolenic acid C18H32O2 2078 2.58 Polyunsaturated fatty acid 14 Heneicosane C21H44 2109 6.52 Alkane 15 Docosane C22H46 2200 5.73 Alkane 16 Heneicosanal C21H42O 2307 0.97 Aldehyde 17 Pentacosane C25H52 2500 6.26 Alkane 18 Squalene C30H50 2663 2.93 Triterpene 19 Isononacosane C29H60 2862 0.50 Acyclic branched alkane 20 Celidoniol deoxy C29H60 2900 3.97 Alkane 21 Brassicasterol C29H48O 3063 1.88 Sterol 22 Campesterol C28H48O 3305 0.67 Sterol 23 beta-Amyrin C30H50O 3337 0.58 Pentacyclic triterpenoid 24 Hexatriacontane C36H74 3600 2.37 Alkane 25 Tetracontane C40H82 4000 15.21 Alkane 26 Tetratetracontane C44H90 4395 4.96 Alkane Total 73.30 Figure 1. Gas chromatogram of the hexane extract of the whole plant of A. nilagirica. 3.3. In vitro antioxidant activity Three methods were used to evaluate the antioxidant activity in vitro. These were free radical scavenging activity, metal chelating activity, and reducing power activity. All of these activities were evaluated by comparing them with the activity shown by the appropriate standard. Table 3 lists the mean IC50 values with SD of the DPPH free radical scavenging activity, the metal chelating activity, and the reducing power activity of the extracts of hexane and methanol from the whole plant of A. nilagirica. 3.3.1. DPPH free radical scavenging activity By monitoring the change in violet color induced by electron transfer, the DPPH free radical activity is used to determine the antioxidant activity. At room temperature, DPPH is a stable free radical that creates a diamagnetic molecule by accepting electrons or other free radicals. Due to the existence of an odd electron, DPPH has the highest absorbance at 517 nm. As antioxidant molecules quench DPPH free radicals, lowering absorbance, the freshly generated DPPH solution fades and vanishes from deep blue to colorless [18,19]. The methanol and hexane extracts have IC50 values of 35.26±0.99 and 23.68±0.20 µg/mL, respectively, with standard ascorbic acid having an IC50 value of 2.54±0.03 µg/mL. Here, IC50 is the concentration of the test sample (antioxidant) that is required to inhibit DPPH by 50%. Lower IC50 implies higher antioxidant activity. The DPPH free radical scavenging activity of the extracts might be due to the presence of phenolic and flavonoid content in the extracts [20]. The stronger antioxidant activity of the methanol extract of A. nilagirica may be attributed to its higher content of α- linolenic acid occupying 10.32% which can be correlated with the previous report on the plant Portulaca oleracea L. [21]. Heneicosane (6.52%), which is present in good amounts in the hexane extract of A. nilagirica, has also been reported to have antioxidant activity in the plant Ceropegia bulbosa [22]. Garia et al. / European Journal of Chemistry 14 (3) (2023) 323-329 327 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.3.323-329.2449 Table 2. Chemical composition of the methanol extract of A. nilagirica. No Compound Chemical formula Kovat’s index %Area Class of compounds 1 Myrtanal C10H16O 1180 0.60 Monoterpene 2 3-Cyclopentylpropionoate, 2-dimethylaminoethyl ester C12H23NO2 1474 0.98 Ester 3 Linoleoyl chloride C18H31ClO 1566 1.73 Fatty acid ester 4 Farnesal C15H24O 1715 0.82 Sesquiterpenoid 5 Cryptomeridiol C15H28O2 1780 0.88 Sesquiterpenoid 6 (7Z,10Z,13Z)-Hexadeca-7,10,13- trienal C16H26O 1824 0.82 Polyunsaturated fatty acid 7 Neophytadiene C20H38 1827 5.05 Diterpene 8 Phytol C20H40O 1949 8.58 Acyclic diterpene alcohol 9 Methyl palmitate C17H34O2 1950 1.42 Fatty acid methyl ester 10 Muco-Inositol C7H14O6 1974 7.27 Carbohydrate 11 Palmitic acid C16H32O2 1984 13.25 Saturated fatty acid 12 alpha-Linolenic acid C18H32O2 2078 10.32 Polyunsaturated fatty acid 13 Octadecanol C20H37F3O2 2121 0.57 Saturated fatty alcohol 14 Oleic acid C18H34O2 2141 0.84 Unsaturated fatty acid 15 9-Hexadecenal C16H30O 2147 2.29 Mono-unsaturated fatty aldehyde 16 Linolenic acid C19H32O2 2199 2.56 Polyunsaturated fatty acid 17 Oleamide C18H35NO 2375 9.41 Fatty acid amide 18 9-Acetyl-S-octahydrophenanthrene C16H20O 2455 4.40 Ketone 19 Calix[4]arene C28H24O4 2676 0.53 Macrocyclic phenols 20 beta-Monolinolein C21H38O4 2706 3.64 Monoglycerides 21 Triacontane C30H62 3000 0.63 Alkane 22 6-Nitro cholesteryl acetate C29H47NO4 3135 2.21 Ester 23 Campesterol C28H48O 3305 1.32 Sterol 24 Friedoolean-6-ene C30H50 3343 0.57 Triterpene 25 alpha-Amyrin C30H50O 3382 2.16 Pentacyclic triterpenoid 26 Urs-12-ene C30H50 5002 0.93 Triterpene Total 83.78 Table 3. Mean IC50 values with SD of DPPH free radical scavenging activity, metal chelating activity, and reducing power activity of the hexane and methanol extracts of the whole A. nilagirica plant. Sample Mean IC50 values with SD (µg/mL) DPPH free radical scavenging activity Metal chelating activity Reducing power activity Artemisia nilagirica methanol extract 35.26±0.99 2.41±0.02 2.03±0.02 Artemisia nilagirica hexane extract 23.68±0.20 2.23±0.01 1.86±0.01 Standard 2.54±0.03 (Ascorbic acid) 2.22±0.01 (EDTA) 4.29±0.01 (Gallic acid) Figure 2. Gas chromatogram of the methanol extract of the whole A. nilagirica plant. 3.3.2. Metal chelating activity The hexane and methanol extracts showed comparable metal chelating activity having IC50 values of 2.23±0.01 and 2.41±0.02 µg/mL, respectively, compared to standard EDTA that had an IC50 value of 2.22±0.01 µg/mL. Squalene (2.93%), which has been reported to have antioxidant properties, might have also contributed to the metal chelating activity, as it is present in plant extracts [23]. Tetracontane (15.21%) present in the hexane extract of A. nilagirica has been reported to exhibit antioxidant activity, as also observed in a previous study conducted on the plant Plectranthus amboinicus [24]. Oleamide (9.41%) present in the methanol extract of A. nilagirica has been reported to have antioxidant activity in endophytic fungi [25]. 3.3.3. Reducing power activity of Fe3+ The value of IC50 is the amount of antioxidant required to reduce ferric ions (Fe3+) to ferrous ions (Fe2+) by 50%. The IC50 values of the hexane extract, the methanol extract, and the standard (gallic acid) are 1.86±0.01, 2.03±0.02, and 4.29±0.01 µg/mL, respectively. The hexane extract showed high anti- oxidant activity in terms of reducing power activity followed by the methanol extract compared to the standard. A major compound present in the hexane extract is squalene (2.93%) which has been reported to have antioxidant properties. Therefore, the reducing power activity of the hexane extract could be due to the presence of squalene [23]. 328 Garia et al. / European Journal of Chemistry 14 (3) (2023) 323-329 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.3.323-329.2449 Table 4. IC50 with SD of the anti-inflammatory activity of standard and hexane and methanol extracts of the whole plant of A. nilagirica. Samples Mean IC50 values with SD (µg/mL) Artemisia nilagirica hexane extract 3.67±0.01 Artemisia nilagirica methanol extract 2.97±0.03 Diclofenac potassium 3.79±0.01 3.4. In-vitro anti-inflammatory activity The IC50 values of the plant extracts were calculated with the help of a scattered curve diagram. The IC50 values of the extracts of hexane and methanol were 3.67±0.01 and 2.97±0.03 µg/mL, respectively. Diclofenac potassium showed an IC50 value of 3.79±0.01 µg/mL. Table 4 shows the IC50 of the anti- inflammatory activity of plant extracts from the whole plant of A. nilagirica. The methanol extract showed high anti-inflam- matory activity compared to the standard diclofenac potas- sium; however, the hexane extract showed values comparable to the standards. The anti-inflammatory effect of the methanol extract may be due to the presence of phytol (8.58%) and neophytadiene (5.05%) which are present in large amounts and have been reported to have anti-inflammatory properties [26]. Palmitic acid (13.25%) which is present in large amounts in methanol extract, is also known to have anti-inflammatory activity, as reported in fenugreek (Trigonella foenum - graecum) [27]. 4. Conclusion The biological characteristics of plant extracts continue to be intriguing, inspiring the creation of novel pharmaceuticals. Ethnobotany, phytochemistry, medicinal chemistry, and pharmacology must all be used together to find plant-derived compounds that can be beneficial for a variety of therapeutic purposes. The results of the present study indicate that Artemisia nilagirica plant extracts have strong antioxidant and anti-inflammatory activities. The plant extracts exert potent anti-inflammatory activity in vitro; therefore, their anti- inflammatory characteristics can be exploited further in detail to utilize them for pharmaceutical industries on a larger scale. The current study reveals that the hexane extract showed better antioxidant activity in terms of metal chelating activity and reducing power activity than the methanol extract. The anti-inflammatory activity of both the hexane and methanol extracts showed better results than the standard marketed drug diclofenac potassium. The results are still preliminary, but if different polarity extracts extracted from the plant are further investigated for their therapeutic potential, they could be used as cost-effective alternatives to synthetic drugs in the pharmaceutical industry for anti-inflammatory and antioxidant properties. Acknowledgements We thank the Advanced Instrumentation Research Facility of Jawaharlal Nehru University (JNU), New Delhi, India, for its support in their study of the GC-MS analysis of plant extracts and the Department of Biochemistry, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, India, for their infrastructure. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been followed. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Shiv Dubey; Methodology: Vaishali Garia; Validation: Vaishali Garia, Ananya Bahuguna; Formal analysis: Vaishali Garia, Shiv Dubey, Ananya Bahuguna; Investigation: Shiv Dubey, Om Prakash, Ravendra Kumar, Dharmendra Rawat; Resources: Shiv Dubey, Om Prakash, Ravendra Kumar, Dharmendra Rawat; Data curation: Shiv Dubey, Om Prakash, Ravendra Kumar, Dharmendra Rawat; Writing original draft: Vaishali Garia, Shiv Dubey; Writing review and editing: Vaishali Garia, Shiv Dubey; Visualization: Ravendra Kumar, Dharmendra Rawat; Supervision: Shiv Dubey, Om Prakash ORCID and Email Vaishali Garia garianitisha8@gmail.com https://orcid.org/0000-0001-7201-7037 Shiv Kumar Dubey shivdub@gmail.com sk.dubey@gbpuat-cbsh.ac.in https://orcid.org/0000-0001-5841-2807 Ananya Bahuguna ananyabahuguna95@gmail.com https://orcid.org/0000-0003-1142-1575 Ravendra Kumar ravichemistry.kumar@gmail.com https://orcid.org/0000-0002-0296-5231 Om Prakash oporgchem@gmail.com https://orcid.org/0000-0002-2767-6997 Dharmendra Singh Rawat ds.rawat@gbpuat-cbsh.ac.in https://orcid.org/0000-0003-2613-2709 References [1]. 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Extraction, characterization and antioxidant activity of fenugreek (Trigonella-Foenum Graecum) seed oil. Mater. Sci. Energy Technol. 2019, 2, 349–355. Copyright © 2023 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution, or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Plant collection and authentication 2.2. Extract preparation 2.3. Analysis and identification of compounds 2.4. Antioxidant activities 2.4.1. 2,2’-Diphenylpicrylhydrazyl (DPPH) free radical scavenging activity 2.4.2. Reducing power activity 2.4.3. Metal chelating activity 2.5. In vitro anti-inflammatory activity 2.6. Statistical analysis 3. Results and discussion 3.1. GC-MS analysis of the hexane extract of the whole A. nilagirica plant 3.2. GC-MS analysis of the methanol extract of the whole plant of A. nilagirica 3.3. In vitro antioxidant activity 3.3.1. DPPH free radical scavenging activity 3.3.2. Metal chelating activity 3.3.3. Reducing power activity of Fe3+ 3.4. In-vitro anti-inflammatory activity 4. Conclusion Acknowledgements Disclosure statement CRediT authorship contribution statement ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: