Composition, antioxidant and anti-inflammatory activities of different polarity extracts of Anaphalis busua from the Himalayan terrain of Uttarakhand European Journal of Chemistry 14 (1) (2023) 114-120 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.14.1.114-120.2395 European Journal of Chemistry View Journal Online View Article Online Composition, antioxidant and anti-inflammatory activities of different polarity extracts of Anaphalis busua from the Himalayan terrain of Uttarakhand Ananya Bahuguna 1, Shiv Kumar Dubey 1,*, Vaishali Garia 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, Pantnagar-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.14.1.114-120.2395 Received: 23 December 2022 Received in revised form: 18 January 2023 Accepted: 25 January 2023 Published online: 31 March 2023 Printed: 31 March 2023 The current study describes the analysis of the phytochemical composition and biological activities of various polarity extracts of the Anaphalis busua plant that was collected at an altitude of 1654 m in the Himalayan terrain of Uttarakhand, India. The extracts were prepared by the cold percolation method, which was then subjected to GC-MS for phytochemical analysis. A total of 31 compounds were identified that constituted 94.95% of the total methanolic extract. Mome inositol (31.03%) was identified as the main compound in the methanolic extract. Twenty-two compounds that comprise 68.24% of the total hexane extract were identified. Tetracontane (19.33%) was present in a significant proportion. The methanolic extract demonstrated potent antioxidant activity in terms of DPPH radical scavenging and metal chelating activity that have IC50 values of 81.71±1.334 and 11.26±0.005 µg/mL, respectively, compared to standards ascorbic acid and EDTA that have IC50 values at 12.71±0.02 and 11.36±0.06 µg/mL, respectively. The methanolic extract showed potent anti-inflammatory activity with an IC50 value of 24.10±0.09 µg/mL in comparison to standard diclofenac potassium with an IC50 value of 18.95±0.03 µg/mL. In vitro studies reveal that A. busua has a strong therapeutic potential and, if further explored, may prove to be a powerful antioxidant, anti-inflammatory, and cost-effective agent compared to synthetically derived agents from pharmaceutical industries. Anaphalis busua Biological activity Mass spectrometry Antioxidant activity Gas chromatography Anti-inflammatory activity Cite this: Eur. J. Chem. 2023, 14(1), 114-120 Journal website: www.eurjchem.com 1. Introduction The diverse and variable pharmacological effects of medicinal plants are primarily dependent on their phyto- chemical constituents [1]. One of the largest families of Asteraceae has several edible plants and medicinal values. Anaphalis busua (Buch.-Ham.) DC. or Tall Pearly Everlasting belonging to this family has various medicinal properties, i.e., antioxidant activity, antibacterial, antifungal, anti-inflam- matory, and many more [2]. Medicinal plants play an important role in the livelihood worldwide and are used as raw materials for the extraction of active constituents in pure form (eg., alkaloids such as quinine and quinidine from cinchona bark, emetine from ipecacuanha root, glycosides from digitalis leaves, sennosides from senna leaves), as precursors for synthetic vitamins or steroids and as preparations for indigenous and herbal medicines [3]. Medici- nal plants are the localized and global heritage of natural antioxidants in addition to other significant bioactive leads that are implemented in the prevention and treatment of diseases such as atherosclerosis, heart stroke, diabetes, cancer, and the ageing process that is prevalent in both rural and urban areas [4,5]. India’s forests are the fundamental repository for a plethora of fragrant plants. In India, plants are generally known to possess some medicinal properties [6,7]. Another report recorded that a total of 2500 plants are traditional medicine, of which 100 plants are used regularly. The Himalayan range has a splendid history of knowledge of plant-based therapy. Uttarakhand, near the Himalayas, which is home to many medicinally important plant families, is the centre of medicinal plant species due to its prolific biodiversity [8]. Anaphalis is the largest genus belonging to the Asteraceae family and has more than 110 distinct species of herbaceous plants spread pre- dominantly in central and southern Asia. It is well variegated in the Himalayas and the Qinghai-Tibet Plateau region. The fresh leaves of this plant and some other species of Anaphalis are ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.14.1.114-120.2395 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.14.1.114-120.2395 mailto:sk.dubey@gbpuat-cbsh.ac.in http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.14.1.114-120.2395&domain=pdf&date_stamp=2023-03-31 Bahuguna et al. / European Journal of Chemistry 14 (1) (2023) 114-120 115 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.114-120.2395 macerated and applied to the cut wounds under a ragged bandage. When taken before meals, the leaves induce appetite; in addition, it is administered to recovering patients for its sedative and tonic properties. It exhibits antiasthmatic, anticoughing, expectorant, and antiphlogistic activity [9]. A. busua, an upright perennial herb, is called ‘Buki Phool’ in Nepal [2]. The plant is also recognized by another name, Bugla. The cough, cold, and sore throat are treated with its flowers and leaves [10]. Discrete parts from various Anaphalis species are used as remedies for various diseases and wounds and may also be used as an antiseptic. The roots of A. busua are used as juice, which is then applied externally to the affected areas. The Himalayan terrain has many medicinal plants with pleotropic medicinal properties because of its phytogeo- graphical location, which are still unexplored for their medicinal properties. Therefore, the present study aims to collect the A. busua plant from the Himalayan terrain. The plant of study was further taken for extraction in two different polarity solvents. The phytochemical composition of the extracts was then analyzed using GC-MS. After chemical analysis, biological activities such as antioxidant and anti- inflammatory activities of the extracts were evaluated using standard protocols. A. busua possesses a variety of phyto- chemicals that make it beneficial as an antinociceptive, antioxidant, antibacterial, antifungal, and anti-inflammatory agent. The review of the literature shows that not much work has been done on A. busua from hilly terrain of Uttarakhand, India, for its phytochemical and medicinal properties. There- fore, it can be studied for consumption as a drug of plant origin with little or no side effects compared to synthetic drugs available on the market. 2. Experimental 2.1. Plant collection and authentication The plant A. busua was collected from the Gethia and Bhowali hilly regions of Uttarakhand at an elevation of 1654 m in September 2020. Dr. Dharmendra Singh Rawat (Plant taxonomist), Department of Biological Science, College of Basic Sciences and Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, India, established the botanical identification of the plant. 2.2. Extract preparation The whole plant of A. busua was shade dried and then grinded to powdered form which was subsequently dissolved in hexane and methanol. The resulting mixture was kept at room temperature for a week with intermittent shaking for successive extraction using the cold percolation method [11]. The resulting extract was filtered twice using Whatman filter paper to remove any undissolved moieties. The filtered solvent of the hexane and methanol extract was evaporated using a rotatory evaporator to obtain the dried form of the extract. The respective extracts were weighed to calculate the yield. The plant extract obtained was resinous and sticky in nature. Nonpolar compounds constituted the major part of the extract compared to the polar ones. The hexane extract had a yield of 3.33%, while the methanolic extract had a yield of 2.37%. 2.3. Analysis and identification of compounds with the help of gas chromatography mass spectrometry (GC-MS) GC-MS was used to analyze and identify the phytochemical content of essential oil using GCMS-QP 2010 Plus equipment with helium as the carrier gas at a pressure of 73.3 kPa and a split ratio of 10:0. The overall flow rate was 16.3 mL/min during the study, with a column flow rate of 1.21 mL/min, a linear velocity of 40.1 cm/sec, and a purge flow of 3 mL/min. The carrier gas saver, high pressure injection, and splitter hold were all 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 minutes, flame thermionic detector (FTD). 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 NBS 54 K L spectrometer database, the WILEY 8 library, and the published literature for further identification [12]. Peak areas obtained without FID response factor correction were used to calculate the relative amounts of identical components (Tables 1 and 2). 2.4. Antioxidant activities 2.4.1. 2,2’-Diphenylpicrylhydrazyl (DPPH) free radical scavenging activity The antioxidant activity of the methanol and hexane extracts was determined using a previously published method [13]. DPPH is a free and stable radical that can receive a hydro- gen atom and an electron, transforming it into a stable diamagnetic molecule. The freshly produced DPPH solution in methanol was stored at 4 ° C in an amber bottle in the dark. A stock solution of 0.1 mM DPPH was produced in methanol. The test sample comprised varied polarity solvent extracts that were subjected to free radical scavenging activities. The test samples were developed in methanol and hexane at varying concentrations, that is, 5, 10, 15, 20, and 25 µg/mL. The absorbance was estimated at 517 nm using a UV spectro- photometer after 30 min of incubation [13]. Using the same approach, ascorbic acid was used as a reference for determining the free radical scavenging activity. The formula used to compute the percentage scavenging activity of the DPPH free radical was as follows: Percentage (%) scavenging = (1-At/Ao) × 100% (1) where Ao is the absorbance of the DPPH solution and At is the absorbance of the test sample. The IC50 value was derived by plotting the percentage of DPPH free radical scavenging versus the concentration of the test sample. The IC50 concentration of a sample is the concent- ration at which 50% of the DPPH free radical is scavenged. A sample with a lower IC50 value has higher antioxidant activity. The IC50 values of the samples were compared with standard ascorbic acid. 2.4.2. Metal chelating activity Metal chelating activity is based on the ability of ferrozine to chelate metals. Ferrozine is a powerful metal chelator that forms a ferrozine-ferrous complex with ferrous ions and gives it a red color having maximum absorbance at 562 nm [14]. Different concentrations of methanol and hexane extracts (5, 10, 15, 20, and 25 µg/mL) were prepared. The volume was increased to 5 mL by adding 0.2 mL of 5 mM ferrozine and 0.2 mL of methanol. The mixture was incubated for 10 min at room temperature and the absorbance was measured at 562 nm. The standard was EDTA, which was subjected to the same process as the plant extract sample. The following formula was used to calculate the percent inhibition of metal chelation in the extract and standard. C%= (A0-At)/Ao× 100 (2) where Ao = absorbance of the control, At = absorbance of the test sample. 116 Bahuguna et al. / European Journal of Chemistry 14 (1) (2023) 114-120 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.114-120.2395 Table 1. Chemical composition of the methanolic extract of the whole plant of A. busua. No Compounds Chemical formula Kovats index % Area Class of compound 1 Trans beta caryophyllene C15H24 1494 1.95 Sesquiterpene hydrocarbon 2 1(10),4-Cadinadiene C15H24 1523 0.91 Sesquiterpene hydrocarbon 3 Cubebol C15H26O 1580 0.68 Sesquiterpene oxygenated 4 Mome Inositol C7H14O6 1647 31.03 Cyclitol 5 Phytone C18H36O 1754 1.45 Sesquiterpene oxygenated 6 Humulane-1,6-dien-3-ol C15H26O 1757 0.69 Sesquiterpene oxygenated 7 1-Methyl-4-methylene-2-(2-methyl-1-propenyl)-1-vinylcycloheptane C15H24 1796 0.53 Sesquiterpene hydrocarbon 8. Acetylcholine C7H17NO3 1808 0.50 Ester 9 Methyl 14-methylpentadecanoate C17H34O2 1814 1.49 Fatty acid methyl ester 10 7-Hexadecenoic acid, methyl ester C17H32O2 1886 0.64 Fatty acid methyl ester 11 1,3,14,16-Nonadecatetraene C19H32 1924 0.73 Alkene 12 Palmitic acid C16H32O2 1968 9.82 Fatty acid 13 Palmitic acid glycidyl ester C19H36O3 1984 0.53 Fatty acid ester 14 cis-3,14-Clerodadien-13-ol C20H34O 2010 1.85 Diterpene oxygenated 15 Neophytadiene C20H38 2045 1.25 Diterpene hydrocarbon 16 Phytol C20H40O 2045 0.59 Acyclic diterpene alcohol 17 Methyl linoleate C19H34O2 2093 0.93 Polyunsaturated fatty acid ester 18 Stearic acid C18H36O2 2167 1.29 Long chain fatty acid 19 α-Linoleic acid C18H32O2 2173 5.63 Polyunsaturated fatty acid 20 Oleic acid C18H34O2 2175 10.80 Long-chain monounsaturated fatty acid 21 Propyleneglycol monooleate C21H40O3 2527 1.81 Fatty acid ester 22 Campesterol C28H48O 2632 4.98 Phytosterol 23 2-Linoleoylglycerol C21H38O4 2713 3.76 Unsaturated fatty acid monoglyceride 24 Cycloeucalenol acetate C30H50O 2816 0.52 Sterol ester 25 Lanosterol C30H50O 2882 0.87 Tetracyclic triterpenoid 26 Phytyl myristate C34H66O2 2890 0.74 Phytyl fatty acid ester 27 Eicosyl benzoate C27H46O2 2949 0.82 Benzoic acid ester 28 3,5-Dihydroxy-6,7,8-trimethoxyflavone C18H16O7 2957 0.75 Flavonoids 29 16-Keto-26-hydroxycholesterol C27H44O3 3010 0.92 Cholesterol 30 6-Nitrocholest-5-en-3-ol acetate C29H47NO4 3108 5.08 Ester 31 Lanosteryl tosylate C37H56O3S 4024 0.70 Sterol Total 94.95 To calculate the IC50 value of the extract and standard, the percentage of chelating activity against concentration was drawn. The higher metal chelating activity is indicated by a decreased IC50 value. 2.4.3. Reducing power activity The reducing power activity of the extracts was investi- gated using a method described previously [15]. Different concentrations of methanol and hexane extract (50, 100, 150, 200, and 250 µg/mL) were prepared. Around 2.5 mL of each conc. of plant extract was mixed with 2.5 mL of phosphate buffer (200 mM, pH = 6.6) and 2.5 mL of potassium ferricyanide (1% w/v). The mixture was then incubated in a 50 °C water bath for 20 min. After incubation, 2.5 mL of trichloroacetic acid (10% v/v in distilled water) was added to the mixture, which was further centrifuged for 10 min at 50.54 g (1000 rpm). Subsequently, 5 mL of the supernatant was combined with 5 ml of distilled water. The absorbance of the resulting solution was measured at 700 nm using a UV spectrophotometer after adding 1 ml of ferric chloride to the mixture. The experiment was carried out in triplicate. Gallic acid was used as a control and was tested in the same way. The control was subjected to the identical procedure, except that distilled water was used instead of the sample. The following formula was used to compute the extract and standard’s reducing power percent activity. % Reducing power activity = (Ao-At)×100/Ao (3) where Ao = absorption of the control, At = absorption of the test sample. The RP50 value of the extract and standard was calculated by plotting a graph between the reducing power activity against the concentration. The value of RP50 is the significant amount of total antioxidants required to convert ferric ions into ferrous ions by 50%. A lower value of RP50 indicates a higher reduction property. 2.5. In vitro anti-inflammatory activity The approach described by Kar et al. was used to investigate in vitro anti-inflammatory activity [16]. Various concentrations, that is, 5, 10, 15, 20, and 25 µg/mL of methanol and hexane extracts, were produced in their respective solvents. A volume of 2 mL of a specified quantity of plant extract was mixed with 2.8 mL of freshly prepared phosphate buffer of pH = 6.4. Subsequently, 0.2 mL of egg albumin was added to the mixture, bringing the total amount to 5 mL. After 15 min of incubation at 37 °C, the mixture was incubated for 5 min at 70 °C. At 660 nm, the absorbance was measured. The standard was subjected to the same procedure as the plant extract. In this study, diclofenac potassium was used as a standard. % Inhibition =100 × (1 − 𝑉𝑉𝑉𝑉 𝑉𝑉𝑉𝑉 ) (4) where Vt = test sample absorbance and Vc = control absorbance. 3. Statistical analysis To determine the mean and standard deviation of triplicates of plant extracts, statistical analysis was performed using the SPSS16.00 program. To evaluate their significance (p < 0.05), all results were subjected to a 5% point Ducane test for one-way analysis (ANOVA). SPSS software was used to determine the significance and association of various extracts. 4. Results and discussion 4.1. GC-MS analysis of a methanolic extract of the whole plant of A. busua In the methanolic extract, 31 compounds were identified using GC-MS, representing 94.95% of the total composition of the methanol extract. Some of the compounds that are present in the highest quantity are mome inositol (31.03%), oleic acid (10.80%), palmitic acid (9.82%), α-linoleic acid (5.63%) and, 6- nitrocholest-5-en-3-ol acetate. (5.08%). Bahuguna et al. / European Journal of Chemistry 14 (1) (2023) 114-120 117 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.114-120.2395 Table 2. Chemical composition of the hexane extract of the whole plant of A. busua. No Compounds Chemical formula Kovats index % Area Class of compound 1 Pogostol C15H26O 1530 0.97 Sesquiterpene oxygenated 2 Hexahydrofarnesyl acetone C18H36O 1754 0.90 Sesquiterpene oxygenated 3 Humulene-1,6-dien-3-ol C15H26O 1757 0.50 Sesquiterpene oxygenated 4 Palmitic acid C16H32O2 1968 1.20 Saturated Fatty acid 5 cis-3,14-Clerodadien-13-ol C20H34O 1984 1.04 Diterpene oxygenated 6 Docosane C22H46 2105 0.66 Hydrocarbon 7 Celidoniol deoxy C29H60 2109 1.81 Hydrocarbon 8 Eicosane C20H42 2113 2.88 Hydrocarbon 9 Pentacosane C25H52 2175 3.76 Hydrocarbon 10 Heneicosane C21H44 2208 5.10 Hydrocarbon 11 Campesterol C28H48O 2632 0.64 Phytosterol 12 Clionasterol C29H50O 2731 0.52 Phytosterol 13 Stigmasterin C29H48O 2739 1.87 Phytosterol 14 2,6,10-Trimethyl,14-ethylene-14-pentadecane C20H38 3030 1.21 Sesquiterpene oxygenated 15 2-Dotriacontanone C32H64O 3338 0.55 Ketone 16 Decanoic acid C30H58O2 3400 1.89 Saturated fatty acid 17 Tetratriacontane C34H70 3401 14.14 Hydrocarbon 18 4-tert-Butylcalix[4]arene C44H56O4 3412 2.38 Macrocyclic phenols 19 Pentatriacontane C35H72 3500 2.64 Hydrocarbon 20 Hexatriacontane C36H74 3600 3.20 Hydrocarbon 21 Octadecyl benzoate C25H42O2 3717 1.05 Ester 22 Tetracontane C40H82 3997 19.33 Hydrocarbon Total 68.24 Figure 1. Gas chromatogram of the whole plant part of the A. busua methanolic extract (ABME). The remaining compounds that occupy a % area less than 5% are mentioned in Table 1. Other constituents with a contribution of less than 1% were thought to be present in tiny amounts. Table 1 and Figure 1 provide detailed information on the compounds. 4.2. GC-MS analysis of the hexane extract of the whole plant of A. busua Twenty-two compounds were identified with the help of GC-MS representing 68.24% of the total hexane extract composition. The compounds that are present in a prominent amount are tetracontane (19.33%), tetratriacontane (14.14%), heneicosane (5.10%), pentacosane (3.76%) and hexatriacon- tane (3.20%). The compounds occupying less than 3% of the area are given in Table 2. While the other compounds that contribute less than 1% were present in small amounts. The precise information on the compounds has been conferred in Table 2 and Figure 2. 4.3. In vitro antioxidant activity The antioxidant activity of the hexane and methanolic extracts of the whole plant of A. busua was achieved using three separate methods: radical scavenging activity of 2,2-diphenyl- 1-picrylhydrazyl (DPPH), metal chelating activity of Fe2+, and reducing power activity of Fe3+. The antioxidant activity of the hexane and methanolic extracts was compared to that of a standard reference antioxidant to assess all activities. 4.3.1. DPPH radical scavenging activity DPPH free radical activity is used to assess antioxidant activity by observing the change in violet color caused by electron transfer. DPPH is a stable free radical that forms a diamagnetic molecule by receiving electrons or other free radicals at ambient temperature. The maximum absorbance of DPPH is at 517 nm due to the presence of its odd electron. The freshly prepared DPPH solution fades and disappears from deep blue to colorless or bleached, as antioxidant molecules quench the DPPH free radicals, decreasing the absorbance. The faster absorbance decreases, the more potent the antioxidant becomes in terms of hydrogen-ion-donating capacity [17,18]. In this study, the DPPH free radical scavenging activity of plant extracts was assessed and their ability to operate as a powerful antioxidant was examined in a dose-dependent manner. Diffe- rent concentrations of plant extract (5-25 µg/mL) were used to evaluate the inhibitory activity on the DPPH free radical. The IC50 value is defined as the amount of total antioxidants required to inhibit the DPPH free radical by 50%. The IC50 value of the DPPH free radical scavenging activity of A. busua is in order of ABME (81.71±1.33 µg/mL) > ABHE (177.83±15.31 µg/mL) with the standard ascorbic acid having an IC50 value at 12.71±0.02 µg/mL (Table 3). 118 Bahuguna et al. / European Journal of Chemistry 14 (1) (2023) 114-120 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.114-120.2395 Table 3. Mean IC50 values with SD of in vitro antioxidant activity of the whole plant of A. busua *. Sample Mean IC50 values with SD (µg/mL) DPPH radical scavenging activity Metal chelating activity of Fe Reducing power activity ABME 81.71±1.334 11.26±0.005 10.76±0.01 ABHE 177.83±15.311 11.64±0.03 10.04±0.01 Standard Ascorbic acid: 12.71±0.020 EDTA: 11.36±0.06 Gallic acid: 2.146±0.05 * ABME: Anaphalis busua methanolic extract; ABHE: Anaphalis busua hexane extract. Figure 2. Gas chromatogram of the whole plant part of A. busua hexane extract (ABHE). 4.3.2. Metal chelating activity of Fe2+ Ferrozine, the standard chelating agent, forms a quanti- tative complex with Fe2+, leading to a red color. Formation of the ferrozine-Fe complex is hindered in the presence of other chelating agents, resulting in a reduction in the intensity of the red color. Calculating the color reduction can be used to estimate the chelating activity of coexisting chelators. The extract with the highest activity is inferred with the ferrous ferrozine complex, which implies that it has chelating activity and captures ferrous ions before ferrozine [19]. In this study, the metal chelating capacity of plant extracts as well as their potential to act as a strong antioxidant was evaluated in a dose- dependent manner. The amount of plant extract present (5-25 µg/mL), that is considered for evaluation has good metal chelating activity. The IC50 value is the value that is defined as the significant amount of total antioxidant required to chelate metal ions by 50%. The methanolic extract, as well as the hexane extract, showed potent antioxidant properties com- pared to standard EDTA. The IC50 value of the metal chelating activity of A. busua is in the order of ABME (11.26±0.005 µg/mL) > ABHE (11.64±0.03 µg/mL) with standard EDTA having its IC50 value at 11.36±0.06 µg/mL as shown in Table 3. Methanol and hexane extract of A. busua show comparable metal chelating activity with an IC50 value of 11.26±0.005 and 11.64±0.03 µg/mL, respectively. The primary methanolic extract compound is mome-inositol, which has been docu- mented to exhibit antioxidant properties, perhaps the cause of this effect [20]. Tetracontane, which occupies the highest percentage (19.33%) of constituents followed by tetratriacon- tane (14.14%) in the hexane extract, has been reported to exhibit antioxidant activity [21]. 4.3.3. Reducing power activity of Fe3+ Reducing power is an evaluation of a bioactive compound’s ability to donate electrons and is related to its antioxidant action. When Fe3+ is reduced, it produces a prussian blue color with maximum absorption at 700 nm. The higher reducing capacity is shown by an increase in absorbance. Depending on the capacity of the extract to reduce Fe3+ to Fe2+, the yellow color of the test solution changes to green or blue. The decreasing power is proportional to the absorbance [22]. The reducing power capacity of plant extracts was investigated in this study and their ability to be a potent reducing power agent was evaluated in a dose-dependent way. The plant extract concentrations selected for the study (50-250 µg/mL) have good reducing power activity. The RP50 value is the significant amount of total antioxidant necessary to convert ferric ions into ferrous ions by 50%. Bahuguna et al. / European Journal of Chemistry 14 (1) (2023) 114-120 119 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.114-120.2395 Table 4. IB50 of anti-inflammatory activity of plant extracts of the whole plant of A. busua. No Sample name Mean IC50 values with SD (µg/mL) 1 ABME 24.10±0.09 2 ABHE 25.97±0.61 3 Diclofenac 18.95±0.03 The reducing power activity of methanolic and hexane extract is almost comparable; however, it has less reducing power activity compared to standard gallic acid. The RP50 value of reducing power activity of A. busua is in order of ABHE (10.04±0.01 µg/mL) > ABME (10.76±0.01 µg/mL) with the standard gallic acid having its RP50 value at 2.146±0.05 µg/mL (Table 3). The methanol and hexane extract of A. busua show comparable reducing power activity with an IC50 value of 10.76±0.01 µg/mL and 10.04±0.01 µg/mL, respectively. In addition to mome-inositol, several additional substances in the methanolic extract have been linked to antioxidant activity, including campesterol, lanosterol, campesterol methyl ester, 16-keto-26 hydroxycholesterol, cholest-5-en-3-ol and 7-hexa- decenoic acid [23]. The antioxidant potential of the methanolic extract of Anaphalis busua is completely consistent with this statement. The heneicosane present in good amounts in the hexane extract exhibited antioxidant activity [24]. 4.4. In vitro anti-inflammatory activity Inflammation is said to be attributable to protein denaturation [25]. Inflammation and its associated diseases are becoming a major health problem for most people [26]. Denaturation converts the tertiary structure of a protein to a secondary structure. In the presence of phosphate buffered saline salt, albumin protein denatures at physiological pH. The potential of plant extracts to suppress denaturation was investigated for anti-inflammatory effects. The effect of heat on albumin denaturation was evaluated [25]. In this study, the anti-inflammatory capacity of plant extracts was evaluated and their ability to be a potent anti-inflammatory was examined dose-dependently. The plant extract concentration selected for the study (5-25 µg/mL) has good anti-inflammation activity. Compared to hexane extract, the methanolic extract was found to possess better anti-inflammatory properties. IB50 is the 50% inhibition of protein denaturation. Table 4 shows the IB50 values of the anti-inflammatory activity of A. busua, which are in the order of ABME (24.10±0.09 µg/mL) > ABHE (25.97±0.61 µg/mL) with the standard diclofenac having its IB50 value at 18.95±0.03 µg/mL. The methanolic extract with an IC50 value of 24.10±0.09 µg/mL shows potent anti-inflammatory activity compared to the standard diclofenac with an IC50 value of 18.95±0.03 µg/mL. Mome inositol has also been known to have anti-inflammatory properties [20]. A similar anti-inflammatory activity of A. busua methanolic extract could be possible due to the presence of mome inositol or the synergistic effect of other major/minor compounds present in the extracts. Oleic acid is another compound present in a significant amount that has shown anti- inflammatory activity [24]. The hexadecanoic acid identified in the methanolic extract is known to exhibit strong antimicrobial and anti-inflammatory activities [27]. 5. Conclusions The plant A. busua thrives as a weed in hilly areas of Uttarakhand and is found in abundance, however, it has not been explored much for its medicinal and therapeutic properties. Preliminary results show that A. busua hexane and methanol extracts were found to be effective against metal chelating activity compared to EDTA, which could be further exploited for advanced studies to be used for their therapeutic properties. Additionally, locally grown weed can also be used for its anti-inflammatory properties, as it has demonstrated effective anti-inflammatory activity compared to standard diclofenac, which is a marketed drug. Plant extracts could be used as powerful antioxidants that could be environmentally friendly and economically viable for the pharmaceutical and cosmetic sectors, as the plant is found in abundance as a weed in hilly areas. Acknowledgements Support is acknowledged from the Advanced Instrumentation Research Facility of Jawaharlal Nehru University (JNU), New Delhi for facilitating GC- MS analysis of the plant extracts and from the Department of Biochemistry, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, India, for providing infrastructure to complete the research work. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered to. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Shiv Dubey; Methodology: Ananya Bahuguna; Validation: Ananya Bahuguna, Vaishali Garia; Formal analysis: Ananya Bahuguna, Shiv Dubey, Vaishali Garia; 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: Ananya Bahuguna, Shiv Dubey; Writing review and editing: Ananya Bahuguna, Shiv Dubey; Visualization: Ravendra Kumar, Dharmendra Rawat; Supervision: Shiv Dubey, Om Prakash Funding Govind Ballabh Pant University of Agriculture and Technology http://dx.doi.org/10.13039/501100003408 ORCID and Email Ananya Bahuguna ananyabahuguna95@gmail.com https://orcid.org/0000-0003-1142-1575 Shiv Kumar Dubey shivdub@gmail.com sk.dubey@gbpuat-cbsh.ac.in https://orcid.org/0000-0001-5841-2807 Vaishali Garia garianitisha8@gmail.com https://orcid.org/0000-0001-7201-7037 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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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). https://www.intechopen.com/chapters/61866 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 with the help of gas chromatography mass spectrometry (GC-MS) 2.4. Antioxidant activities 2.4.1. 2,2’-Diphenylpicrylhydrazyl (DPPH) free radical scavenging activity 2.4.2. Metal chelating activity 2.4.3. Reducing power activity 2.5. In vitro anti-inflammatory activity 3. Statistical analysis 4. Results and discussion 4.1. GC-MS analysis of a methanolic extract of the whole plant of A. busua 4.2. GC-MS analysis of the hexane extract of the whole plant of A. busua 4.3. In vitro antioxidant activity 4.3.1. DPPH radical scavenging activity 4.3.2. Metal chelating activity of Fe2+ 4.3.3. Reducing power activity of Fe3+ 4.4. In vitro anti-inflammatory activity 5. Conclusions Acknowledgements Disclosure statement CRediT authorship contribution statement Funding ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: