HR-LCMS Analysis for the identification of bioactive compound of Justicia adhatoda L. leaf extract and the potential Antioxidant and Anti-lipid peroxidation Assay In vitro

*Corresponding author: Elareen Belljoy Donshiew Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. HR-LCMS Analysis for the identification of bioactive compound of Justicia adhatoda L. leaf extract and the potential Antioxidant and Anti-lipid peroxidation Assay In vitro Elareen Belljoy Donshiew *, Ridashisha Rymbai and Surya Bhan Department of Biochemistry, North Eastern Hill University, Shillong, Meghalaya, India-793022 GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 326-343 Publication history: Received on 18 September 2025; revised on 24 October 2025; accepted on 28 October 2025 Article DOI: https://doi.org/10.30574/gscbps.2025.33.1.0414 Abstract Throughout human history, medicinal plants have always been used as medicine to treat various diseases, extracts and active compounds of the medicinal plant most use of the therapies and play a major role in medication since the beginning of human civilization and also contribute to the manufacturing of drugs these days. The purpose of this study was to assess the Justicia adhatoda L. butanol (JABE) and methanol (JAME) leaves extracts for potential against lipid peroxidation activity, as well as for several antioxidant assays. A number of characteristics were examined, such as antilipid peroxidation, scavenging of free radicals (ferric reducing power potential, iron chelating activity, hydroxyl, hydrogen peroxide, superoxide and nitric oxide radicals). The JABE and JAME extracts was also subjected to HR-LCMS analysis. The HR-LCMS analysis revealed the makeup of a variety of bioactive compounds, such as Andrographolide, Vasicinone, vincamine, Curcumin, Kaempferitrin, 6-Gingerol, Sitagliptin, Kynurenic acid, Oleoyl ethanolamide, Luotonin A and many more, which exhibit numerous pharmacological activities such as antioxidant, anti-inflammatory, anticancer, and antidiabetic, antihypertensive, antimicrobial, antiaging, hepatoprotective, lipid lowering potential. These fractions also demonstrated a strong correlation with the IC50 values for the scavenging of hydrogen peroxide radicals, superoxide radicals, anti-lipid peroxidation, nitric oxide, iron chelating and hydroxyl radical efficacy. Additionally, the maximum scavenging activity for the above mention assays and for the reduction of Fe3+ to Fe2+ was demonstrated by both the fractions. The current findings indicated that Justicia adhatoda L. leaves, specifically the JABE and JAME fractions, have therapeutic potential as agent to prevent damage caused by free radicals. Keywords: Medicinal plant; Free radical; HR-LCMS; Hepatoprotective; Antioxidant; Lipid peroxidation 1. Introduction The use of plants as medicines is as old as human civilization itself, and more than 10% of the approximately 258,650 species of higher plants known to exist worldwide are used to treat sick population [1]. Plants have been essential to preserving human health and improving the quality of human life for thousands of years. Numerous naturally occurring antioxidants have been identified and extracted from therapeutic plants. Moreover, phytochemicals' antimicrobial, antithrombotic, and vasodilator potential have raised interest in them as complementary and alternative therapies [2]. Reactive oxygen species, including nitric oxide, peroxide, hydroxyl, and superoxide radicals, apart from the dangers oxidative stress poses to biological systems, it is also the cause of chronic illnesses like cancer, heart disease, aging, diabetes, and cataracts [3]. Numerous studies have proposed a link between consuming these phytochemicals through food and preventing a range of abnormalities brought on by stress [4–8]. Given that the majority of people in underdeveloped nations use traditional medicines, it is imperative that medicinal plants be described according to their pharmacological characteristics [8–10].Many medicinal plants and herbs contain high concentrations of antioxidants, such as polyphenols, vitamin C, vitamin E, selenium, β-carotene, lycopene, lutein, and other carotenoids, which act as antioxidants by neutralizing, quenching, reducing, or breaking down peroxides [11]. In order to prevent the possible harm of synthetic antioxidants, recent research is concentrating on substituting naturally occurring antioxidants for GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 326-343 327 synthetic ones [12–15]. A synergic effect of phytochemicals from medicinal plants exists to exhibit the observed pharmacological properties [16]. The increasing use of phytochemicals in traditional medicinal systems is because of the current trend toward applying green products [17]. Reactive oxygen species [ROS], are different types of activated oxygen that include non-free radical species like hydrogen peroxide (H2O2) and free radicals like superoxide ions (O2-) and hydroxyl radicals (OH-) [18]. Degenerative or pathological processes like aging, cancer, coronary heart disease, Alzheimer's disease, neurological disorders, atherosclerosis, cataracts, and inflammation are all significantly impacted by these ROS [19]. Numerous ROSs are produced in living organisms by a variety of mechanisms, such as peroxisomes, macrophages, activated polymorphonuclear leukocytes, and regular aerobic respiration. These seem to be the primary endogenous sources of the majority of oxidants that cells generate. Tobacco smoke, ionizing radiation, certain pollutants, organic solvents, and pesticides are examples of exogenous sources of free radicals [18]. The action of many vitamins, minerals, and other phytochemicals to prevent damage from ROS is referred to as "antioxidant" [20]. Although they are utilized in processed foods, synthetic antioxidants like butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have negative side effects. Due to safety concerns, plant-based antioxidants are now preferred over synthetic ones [21]. As a result, studies on the antioxidant potential of plants are crucial [22]. Currently interest in antioxidant compounds has increased because they play key function in health and diseases and also have nutritional value. The antioxidant activities of plants are different when different prooxidants are used. HR-LCMS is an important analytical technique e.g. metabolomics experiments [23]. HR-LCMS-based approaches are expected to be of particular importance in plants, owing to the highly rich biochemistry of plants, which covers many semi-polar compounds, including key secondary metabolite groups, which can best be separated and detected by HRLCMS approaches [24]. According to estimates from the World Health Organization (WHO), 80% of the world's population rely on traditional medicine for their primary medical need entailed applying plant extract or active ingredients [25]. The selected plant Justicia adhatoda L. found in Shillong Meghalaya, India is a member of Acanthaceae locally known as ‘dieng kthang’ in Khasi Hills and ‘devlamach’ in Garo Hills. Justicia adhatoda L. is a shrub widespread throughout the tropical region of Southeast Asia. It is a perennial evergreen and highly branches shrubs, 1-1.5 m height, with bitter taste. The tender leaves of this plant are used as vegetables after cooking by the Khasi and Garo tribes. It is also been used traditionally for years as herbal medicine for treating cold, cough, bleeding, skin disease, wounds, headache, fever and for lowering blood pressure. The terms J. adhatoda L. is frequently referred to as Malabar nut or Vasaka [26]. It has been widely used in Chinese and Indian medicines. Extracts from various part of the plant has been used for the treatment of a variety of diseases such as leprosy, skin diseases, piles, asthma etc. Vasicine is one of the compounds from the plant which belongs to quinazoline alkaloid class. Vasicine has been demonstrated to be active against tuberculosis [27]. Vasicinone is another compound found which also is classified as quinazoline alkaloid. Both vasicine and vasicinone contribute to the antioxidant activities of the plant extract [28]. In addition, vasicol and vasicinolone are also found in the plant extract [29], extract has been shown to possess anti-inflammatory, antioxidant properties [30-32]. Hence, this study was aimed to determine the composition of bioactive compound by HR-LCMS and in vitro antioxidant activities of butanol and methanol extract using different assays. 2. Materials and Methods 2.1. Plant material collection During the month of SeptemberOctober 2021, aerial plant parts of Justicia adhatoda L. were gathered from LabanBarik area Shillong, East Khasi Hills District Meghalaya, India, leaves were collected, identified, and after being cleaned with distilled water, the renewed foliage was dried for 14 days at room temperature (25-30 °C), out of direct sunlight, and ground into a powder using an electronic grinder. The crushed sample leaves were kept at room temperature in dry, airtight plastic bags 2.2. Preparation of Butanolic and Methanolic Extract To aid in the extraction of phytoconstituents, a 25g powdered sample was steeped in 250 ml of butanol (JABE), methanol (JAME) solvents in a conical flask for 48 hours, with continuous stirring. Whatman (No. 1) filter paper were then used to filter the entire mixture. Using a rotary evaporator set to lower pressure and temperature, the filtrate was further evaporated and freeze-dry, the pure crude extracts were then collected and stored under -20°C for further analysis. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 326-343 328 2.3. Chemicals Hydrogen peroxide, Ferric Chloride, Di-Sodium hydrogen Phosphate obtained from Merck Specialties Pvt. Ltd., Mumbai, India, butanol and methanol, gallic acid, ascorbic acid, Potassium persulphate, Potassium ferricyanide and Trichloroacetic acid, ferric chloride, ferrozine, ethylenediaminetetraacetic acid EDTA, N-1-naphthyl ethylene diamine dihydrochloride [NED], sulfanilamide, H3PO4, riboflavin, nitro blue tetrazolium (NBT) were was purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Sodium phosphate,1,1,3,3-tetraethoxypropane (TEP), thiobarbituric acid (TBA) were procured from Sigma-Aldrich (St. Louis, USA). Other solvent used were of analytical grade. 2.4. In vitro antioxidant study 2.4.1. Reducing power assay: Ferric reducing antioxidant power (FRAP) assay This method is based on the conversion of the Fe3+/ferricyanide complex to its ferrous form to formed a violet-colored solution, whose intensity is proportional to the sample concentration. A higher absorbance of the reaction mixture is indicative of a greater reducing power of the extract [33]. The ferric ion reducing power test was used to measure the reducing power of the JABE and JAME extract of Justicia adhatoda L. 2 ml of phosphate buffer (0.2 M) with a pH of 6.6 was mixed with 2 ml of various concentration of plant extracts (µg/ml), and 2 ml of potassium ferricyanide were added. For 20 minutes, the mixture was incubated at 50°C. 2 ml of 10% trichloroacetic acid (w/v) was added to the mixture, it was centrifuge for 10 minutes at 3000 rpm, 4 °C. Then equal volume of the supernatant with distilled water and 0.1 ml of 0.1% ferric chloride was mixed, incubated at room temperature for 10 minutes, absorbance of chromogen formed was read at 700 nm, increased absorbance value indicated high reduction capacity [34]. At each concentration, experiment was carried out in triplicate. 2.4.2. Iron chelating assay The ability of antioxidants to stop electron transport by creating a coordination complex with metal ions, which stops oxidation reactions and the production of free radicals. Consequently, the red color of the iron (II)–ferrozine complex is decolorized when other chelating agents are present as they compete with ferrozine for the ferrous ions. The decreased in absorbance at 562 nm indicates the chelating activity of ferrous ions [35]. A common spectrophotometric technique, was used to measure the chelating activity of ferrous ions. 1 ml of 0.125 Mm ferrous sulfate solution, 1 ml of JABE and JAME extracts at different concentrations (μg/ml) and 1 ml of ferrozine (0.3125 mM), was added to start the reaction. Vortexed, and allowed to stand at room temperature for 10 minutes. EDTA was used as a positive control. The absorbance was read at 562 nm using UV-Vis spectrophotometer. At each concentration, tests were performed in triplicates. The tendency of the sample to chelate the ferrous ion was calculated using the following formula: % Iron II chelating activity = [(A0A1)/A0] × 100 Where, A0 = control absorbance A1 = extract or EDTA absorbance 2.4.3. Hydroxyl Radical (OH-) Scavenging assay This assay relies on the extract's capacity to prevent the deoxyribose degradation caused by hydroxyl radicals through the Fenton's reaction, which uses a reaction combination of Fe3+-EDTA-ascorbic acid and H2O2. [36-37]. Reagent solution was prepared by sequential addition of 50 mM KH2PO4-KOH buffer (pH 7.4), 200 mM FeCl3 (1:1 v/v), EDTA (1.04 mM), H2O2 (2.0 mM) 200μl, ascorbic acid (1.0 mM), 2-deoxy-2-ribose (28 mM) 100μl, and 100μl the extract at different concentration (μg/ml) were all added to a reaction mixture. For 1 h, the mixture was incubated at 37°C. Following incubation, 1ml of 2.8% trichloroacetic acid (TCA) and 1% thiobarbituric acid (TBA) were added, and the mixture was then incubated for an additional 20 minutes at 100 °C to formed a pink coloured complex. Once cooled, the absorbance at 532 nm was measured. All of the reagents, excluding the extract, were present in the blank solution. Gallic acid was used as positive control. Each experiment was carried out in triplicates. The hydroxyl radical scavenging activity percentage was calculated using the formula below: % OHscavenging activity = [(A0 - A1) / A0] × 100 Where, A0 = control absorbance A1 = extract or standard. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 326-343 329 2.4.4. Nitric oxide (NO) scavenging assay Sodium nitroprusside (SNP) generated nitric oxide on its own. This nitric oxide then combines with molecular oxygen to form nitrite ions, which may be measured with the Griess reagent. The extract's nitric oxide scavengers compete with molecular oxygen, which lowers the amount of nitrite ions produced. [38]. Nitric oxide radical scavenging activity of JABE and JAME was determined, Nitric oxide generated from SNP in aqueous solution at physiological pH interacted with oxygen to produce nitrite ions which were measured using Griess reaction method. 3 ml of 10mM SNP in phosphate buffer 0.2M, pH 7.4 was added to 1 ml of different concentrations of extracts preparation (µg/ml). A similar procedure was repeated with methanol as a blank, were incubated at room temperature for 30 minutes. Following incubation, 1.5 ml of sample solution incubated and 1.5 ml of Griess reagent (0.1% N-1-naphthyl ethylene diamine dihydrochloride [NED], 1% sulfanilamide, and 2% H3PO4) was added (The extracts mixed with an equal volume of freshly prepared Griess reagent). Nitrite ion diazotization with sulfanilamide and pink chromophore was produced by a subsequent interaction with NED, whose Spectrophotometric measurements of absorbance were made at 546 nm against reagent blank [39]. Three duplicates of each test were performed. Ascorbic acid was used as the positive control. The percentage nitrite radical scavenging activity of the extracts and ascorbic acid were calculated using the following formula: % NO scavenging activity = [(𝐴0 – A1)/A0] × 100 Where 𝐴0 = absorbance of control 𝐴1 = absorbance in the presence of extracts / standard. 2.4.5. Hydrogen peroxide (H2O2) scavenging assay The decrease in absorbance that occurs when the antioxidant chemical reduces H2O2, is basis of this scavenging assay. The scavenging capacity of extracts for H2O2 was established with minor adjustments. H2O2 (100 mM) was made into a solution with 40 mM phosphate buffer saline at pH 7.4. [40]. 2 ml of hydrogen peroxide solution with 1 ml of varying concentration of the extracts, were added vortexed, then incubated. After 10 mins, the absorbance of hydrogen peroxide at 230 nm was measured against blank solution that contained phosphate buffer but no hydrogen peroxide. A different blank sample was utilized for background subtraction at every concentration. Gallic acid was employed as positive control. Each test was conducted in triplicate. Percent inhibitory activity was calculated using the following formula: % H2O2 scavenging activity = [(A0 – A1)/A0] × 100 Where, A0 = is the absorbance of the control A1 = is the absorbance of the extract/standard, 2.4.6. Superoxide (O2-) scavenging assay The assay relies on the extract's capacity to scavenge the superoxide radicals produced in the riboflavin-light-nitro blue tetrazolium (NBT) system, thereby preventing the formation of formazan [41], 50 mM sodium phosphate buffer (pH 7.6), 20µg riboflavin, 12 mM ethylenediaminetetraacetic acid (EDTA), 1.22 mM NBT were added. The total volume of reaction mixture was 3 ml which was prepared by sequential addition of 1 ml of plant extracts at varying concentration. The samples of different concentrations were prepared in 50 mM sodium phosphate buffer (pH 7.6.) The photo-induced reactions were initiated by illuminating the reaction mixtures. Illuminating the reaction mixture with varying concentrations of plant extracts for 90 seconds initiated the reaction at room temperature. To ascertain the amount of formazan produced, the absorbance of the reaction mixture at 562 nm was measured right after illumination is completed. Ascorbic acid was used as the positive control, and each test was run in triplicates, the percentage inhibition was determined using the following formula: % O2scavenging activity = [(A0 – A1) / A0] × 100 Where, A0 = absorbance of control A1 = absorbance of test/ standard 2.4.7. In vitro anti-lipid peroxidation assay A pink substance with an absorption maximum at 532 nm is formed when malondialdehyde (MDA), a byproduct of lipid peroxidation (the breakdown of polyunsaturated fatty acids), combines with thiobarbituric acid (TBA). MDA was GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 326-343 330 measured using a slightly modified version as described by Smail Aazza et al., (2011) [42]. 2.0 ml of the TCA HCl reagent 15% (w/v) TCA, 0.375% (w/v) TBA, and 0.25 N HCl and the plant extract at different concentration were present in the reaction mixture with a final volume of 1.0 ml. To remove the TCA precipitate that formed the light pink supernatant (MDA), the reaction mixture was incubated in a water bath at 90 °C for 10 minutes, cooled, and centrifuged at 10,000 rpm for 10 minutes. Ascorbic acid was used as a standard. Absorbance of clear supernatant of MDA generated was measured in each sample at 532 nm against the reagent blank. All tests were performed in triplicate. The percentage of lipid peroxidation inhibition was calculated using the following equation: % Lipid peroxidation= [ (A0 – A1)/A0] × 100 Where, A0 = control absorbance A1 = extract / ascorbic acid absorbance 2.4.8. High Resolution Liquid Chromatography and Mass Spectrometry (HR-LCMS) Analysis After being prepared in their respective solvent, the JABE and JAME was analyzed using HR-LCMS. Using the Agilent high resolution liquid chromatography and mass spectrometry model-G6550A with 0.01% mass resolution, chemical fingerprints of a chosen medicinal plant extract were created. MS was used as the acquisition method, with a scanning rate of one spectrum per second with a minimum range of 50 (M/Z) and a maximum of 1000 Dalton (M/Z). each spectrum with scanning rate per second, at 250 °C the gas chromatography was kept with a flow of 13 psi/minute. With an auxiliary speed of 100μl/minute, an ejection speed of 100μl/minute, a flush out factor of 5μl and 8μl injection volume used for HR-LCMS, Hip sampler with modelG4226A was used. Within half an hour during the first two minutes of the acquisition time, the solvent composition A:B flow was 95:5. HR-LCMS used a solvent i.e., water 100%, Acetonitrile at 100%. 2.4.9. Statistical analysis In vitro and other parametric assays were performed and shown as mean ± standard error of mean of the results of three replicates per sample used to express the values of the JABE and JAME extract of Justicia adhatoda L. leaves for iron chelating activity, hydroxyl activity, hydrogen peroxide activity, superoxide scavenging activity, nitric oxide scavenging activity, ferric reducing power activity and anti-lipid peroxidation assay. Antioxidant potential of different assays was determined as IC50 values by applying Graph Pad Prism 5 and Microsoft Excel 2021 used to examine the results. 3. Results 3.1. In vitro antioxidant study 3.1.1. Ferric reducing antioxidant power (FRAP) Assay Depending on each compound's reducing capability, the test solution's yellow color in this assay changes to a variety of green and blue hues. The presence of radicals (i.e. antioxidants) causes the conversion of the Fe3+ / ferricyanide complex utilized in this approach to the ferrous form indicated by the production of brilliant Prussian blue. Figure 1 displays the findings of the FRAP assay of the JABE and JAME at 700 nm with reference to the standard ascorbic acid. The standard curve was plotted using OD value against various concentrations of both the extracts and ascorbic acid standard. Additionally, it has been shown that when the concentration of study plant extracts in the test sample increases, the absorbance values continue to increase significantly. The ferric reductive activity increased in proportion to the five tested concentrations of the JABE and JAME. In terms of their ability to reduced, all of the concentrations were statistically comparable to the ascorbic acid standard concentration. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 326-343 331 Figure 1 In vitro ferric-reducing antioxidant power of JABE and JAME. Results are expressed as means ± SD for replicate measurements, n = 3; JABE: Justicia adhatoda L. butanolic extract; JAME: Justicia adhatoda L. methanolic extract; ASA: Ascorbic acid 3.1.2. Hydrogen peroxide (H2O2) radical scavenging activity Table 1 displays the findings of the hydrogen peroxide scavenging activity of the JABE and JAME at 230 nm with reference to the standard. The standard curve used to determine each sample's IC50 (µg/ml) was plotted using the percentage of inhibition against various concentrations of both extracts and gallic acid. The results showed that the IC50s for the JABE extract, JAME extract, and gallic acid were 170.59 ± 0.20, 187.58 ± 0.18, and 26.32 ± 0.25, respectively. Hydrogen peroxide, although it is not very reactive by itself, it can occasionally be harmful to cell because it can produce hydroxyl radical inside the cells. Consequently, elimination of H2O2 is crucial for safeguarding the systems. 3.1.3. Nitric oxide (NO) scavenging activity Mammalian cells produce the free radical nitric oxide (NO), which regulates a number of physiological functions. However, a number of disorders may be initiated and developed as a result of excessive NO generation [43]. Antioxidant potential in JABE, JAME may compete with oxygen to react with nitric oxide [44], so inhibiting the production of nitrite. This might explain the nitric oxide scavenging activity that was reported to be concentration dependent. The quantity of nitrous acid drops when a scavenger, is present. In comparison to the standard ascorbic acid, which had respective IC50 values of 34.59 ± 0.36 µg/m, the percentage inhibition of JABE and JAME of triplicate readings revealed IC50 values of 250.31 ± 0.25 and 602.33 ± 0.20 µg/ml respectively. 3.1.4. Superoxide (O2-) scavenging activity The first reduction product of oxygen that is quantified by the suppression of O2 production is superoxide anion [45]. In aerobic organisms, the superoxide radical is ubiquitous in nature. Despite being slightly reactive with biological molecules, the superoxide radical might change into an extremely harmful and toxic hydroxyl radical. The dismutation of reactive superoxide anion to oxygen and hydrogen peroxide is catalyzed by superoxide dismutase. Plant extract's ability to scavenged anions may be attributed to its suppression of superoxide production. The JABE and JAME was shown to have this effect, the findings indicate that the possibilities for scavenging potential of JABE and JAME were discovered to be comparable to the ascorbic acid that was used as favorable control. The superoxide radical scavenging activity of JA extracts shown in Table 1. The scavenging activities was comparable to the standard in concentration dependent manner. The IC50 value was found out to be 31.38 ± 0.64 for ascorbic acid, 81.12 ± 0.29 for JABE, 63.82 ± 0.25 for JAME respectively. 3.1.5. Hydroxyl (OH-)radical scavenging activity At all extract concentrations, the JABE and JAME showed strong hydroxyl radical scavenging action. Justicia adhatoda L. leaf extract's capacity to scavenge hydroxyl radicals was observed to be in a concentration dependent manner. The hydroxyl radical scavenging activity of JABE and JAME was statistically comparable to that of a standard reference gallic acid. The hydroxyl radical scavenging activity varied the extract's ability to scavenge hydroxyl radicals elevated drastically with increasing concentrations, with the highest extract concentration showing the highest activity. Findings of the present study also revealed that the JABE and JAME IC50 value of 275.56 ± 6.38 and 344.71 ± 7.85 µg/mL comparable to that of the standard, gallic acid, whose IC50 value was 234.78 ± 8.54 µg/mL as illustrates in table 1. GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 326-343 332 3.1.6. Iron-Chelating activity The investigation indicates that there was a concentration related increase in iron chelating activity of the JABE and JAME. The potential to inhibit the formation of iron (II)–ferrozine complex was significantly different among all the concentrations. The highest extract concentration showed significantly higher activity than those of the lower extract concentrations. The iron-chelating activities exhibited by JABE and JAME were statistically comparable to that of the standard reference, EDTA. Furthermore, it was observed that the IC50 value of the JABE and JAME showed 280.51 ± 3.45 and 380.39 ± 1.89 µg/mL, whereas the IC50 value for the standard, EDTA, was 194.55 ± 2.54 respectively (table 1). 3.1.7. Lipid Peroxidation Inhibition activity Lipid peroxidation inhibition increased with concentration in the JABE and JAME leaf extract. The Justicia adhatoda L. leaf's ability to inhibit lipid peroxidation extract varied considerably, the increase in extract concentration demonstrated substantially higher inhibition compared to the inhibition at the lowest extract concentration. JABE and JAME suppression lipid peroxidation at various concentrations were statistically comparable to the standard ascorbic acid. The JABE and JAME displayed an IC50 value of 110.78 ± 2.58 ,238.37 ± 8.25 which is comparable to standard ascorbic acid 105.78 ± 5.23 respectively. Table 1 Results of all the % Inhibition and IC50 studies. Values are presented as M ± SEM (n = 3) M: Mean; SEM: Standard error of mean; JABE: Butanolic extract; JAME: Methanolic leaves extract; IC50, half maximal inhibitory concentration; MDA: malondialdehyde; EDTA :ethylenediaminete traacetic acid. Result of Radical Generated Samples IC50 (µg/ml) from standard curve Hydroxyl (OH-) Gallic acid JABE JAME 234.78 ± 8.54 275.56 ± 6.38 344.71 ± 7.85 Iron (II)-ferrozine complex Hydrogen peroxide (H2O2) Nitric oxide (NO) Superoxide (O2-) EDTA JABE JAME Gallic acid JABE JAME Ascorbic acid JABE JAME Ascorbic acid JABE JAME 194.55 ± 2.54 280.51 ± 3.45 380.39 ± 1.89 26.32 ± 0.25 170.59 ± 0.20 187.58 ± 0.18 34.59 ± 0.36 250.31 ± 0.25 605.33 ± 0.20 31.38 ± 0.64 81.12 ± 0.29 63.82 ± 0.25 GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 326-343 333 MDA Ascorbic acid JABE JAME 105.78 ± 5.23 110.78 ± 2.58 238.37 ± 8.25 3.1.8. HR-LCMS Analysis Major phytoconstituents were chromatographically identified from the Justicia adhatoda L. butanolic and methanolic extracts producing spectra with more than 500 compounds and their corresponding retention times in both positive and negative ionization modes. The reference library was used to identify and compare these peaks. Every peak in Figure 2 (a) and (b) chromatograms represented a distinct compound found in the JABE and JAME. The medicinal significance and distinctive details of the bioactive chemicals isolated by HR-LCMS analysis were shown in Table 2 (a) and (b), were among the main bioactive compounds identified. These compounds have strong antioxidants with bactericidal, anti-inflammatory, anti-aging, anti-diabetic, anti-cancer, neuroprotective, and hepatoprotective properties. Furthermore, phenolics and flavonoids serve as protective substances [46]. Due to their antimicrobial qualities, plant metabolites act as chemical barriers against infections and are utilized in a variety of food sectors as flavouring, colouring, and texturizing [47]. 2 (a) GSC Biological and Pharmaceutical Sciences, 2025, 33(01), 326-343 334 2 (b) Figure 2 HR-LCMS chromatograms of (a) JABE, (b) JAME based on retention time plotted against relative abundance. Peaks are the fingerprints of bioactive components in both positive and negative modes Table 2 (a) Major bioactive compounds identified by HR-LCMS Analysis: JABE positive and negative ionization mode Sr No. Bioactive compound Molecular Mass (g/mol) RT min) Pharmacological Significance 1. Andrographolide 350.2093 11.90 Antioxidant, anticancer, antihypertensive, anti-inflammatory [48]. 2. Luotonin A 285.0902 12.231 Antioxidant, antihyperlipidemic, antidiabetic, anticancer, antimicrobial [49]. 3. Oleoyl ethanolamide 325.2981 19.40 Anti-inflammatory, antiantioxidant [50]. 4. vasicinone 202.0742 8.366 Anti-inflammatory, antioxidant, anticancer, antiaging, antimicrobial, antidiabetic, hepatoprotective [51]. 5. Kynurenic acid 189.042 8.726 Anti-inflammatory, antidiabetic, antihyperlipidemic, Antioxidant [52] 6. Vincamine 354.1943 Antioxidant, antidiabetic, antihyperlipidemic [53] 7. Betaine 117.0791 1.215 Antioxidant, anti-inflammatory [54] 8. Sitagliptin 407.1181 10.221 Antioxidants, anti-inflammatory [55] 9. 6-Gingerol 294.3859 13.49 Antioxidant, lipid-lowering, antihyperglycemic [56] 10 Curcumin 368.1260 13.889 Antioxidative, anti-inflammatory, hypolipidemic effect [57]. 11. Benzoic acid [Negative] 122.0368 6.20 Antioxidants, antidiabetic, anti-inflammatory [58]. 12. (±)9-HpODE 312.2301 14.91 Antioxidant, antidiabetic [59]. 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