Evaluation of Antioxidant and Anti-Inflammatory Activities, and Metabolite Profiling of Selected Medicinal Plants of Nepal

This study aimed to evaluate the antioxidant, antiarthritic, and anti-inflammatory properties of extracts from the leaves of twelve different medicinal plants in Nepal. We then evaluated the total phenolic, flavonoid, and tannin contents of the extract using in-vitro assays and characterized it using GC-MS analysis. Results revealed that most of the leaf extracts contained phenolic compounds, flavonoids, tannins, alkaloids, and saponins. Few plants also showed the presence of glycosides, phytate, and vitamin C. Among the studied plants, Neolamarckia cadamba exhibited the highest total phenolic and tannin contents, as 241.53 ± 0.20 µg of gallic acid equivalent/mg and 74.48 ± 1.081 µg of tannic acid equivalent/mg, respectively. Ipomoea batatas exhibited the highest total flavonoid content, as 53.051 ± 1.11 µg of quercetin equivalent/mg. Moreover, Raphanus sativus demonstrated significant ferrous ion chelating, 2,2-diphenyl-1-picrylhydrazyl, hydrogen peroxide scavenging, and total antioxidant activities with IC50 value of 4.76 ± 0.68 µg/mL, 5.84 ± 0.14 µg/mL, 6.89 ± 0.16 µg/mL, and 8.99 ± 0.20 µg/mL, respectively. Similarly, Colocasia esculenta and Cicer arietinum exhibited the highest hydroxyl radical and nitric oxide scavenging activities, measuring IC50 value of 7.22 ± 0.56 µg/mL and 9.06 ± 0.10 µg/mL, respectively. Among all the extracts, Amorphophallus paeoniifolius displayed significant human red blood cell (HRBC) membrane stabilization activity (IC50 = 6.22 ± 0.78 µg/mL). Furthermore, Raphanus sativus, Chenopodium album, Cicer arietinum, and Murraya koenigii exhibited the highest inhibitory activities against protein denaturation with bovine serum albumin, antiarthritic, lipoxygenase inhibitory, and proteinase inhibitory, measuring IC50 of 7.48 ± 0.48 µg/mL, 9.44 ± 1.62 µg/mL, 14.67 ± 1.94 µg/mL, and 28.57 ± 2.39 µg/mL, respectively. In conclusion, this study demonstrated the twelve leaf extracts' significant antioxidant, antiarthritic, and anti-inflammatory activities.


Introduction
Natural products have been utilized as herbal/natural medicine from ancient times and are a major option for healing for many of the population until today [1].Te knowledge that humans gathered from using herbal medicine from ancient times can be a good resource that increases the likelihood of discovering bioactive compounds and formulations [1].Tis reason has made phytochemical screening from several natural products with an ethnomedicinal value to increase rapidly in these days [2].Also, the relatively lower adverse efects and toxicity of herbal medicines compared to chemically synthesized drugs are the reason for the inclination towards herbal research [3].Various active phytoconstituents were extracted from herbs and plants that prevent and cure chronic illnesses caused by oxidative stress.Many phytoconstituents were already reported to have antiarthritic, anti-infammatory, and antioxidant activities [4].
As such, andrographolide is the biologically active diterpenoid lactone extracted from Andrographis paniculata (an ancient herb found in Asian countries) used to treat fu, upper respiratory tract, and sore throat [5].Tey are multitarget medications with anti-infammatory and antioxidant in various cell types [6].Using multiple doses of 25, 50, and 100 mg/kg in rats, they found andrographolide to decrease the level of articular elastase, TNF-α, IL-6, CXC chemokine ligand 2, and myeloperoxidase and increase the concentration of catalase, superoxide dismutase, and glutathione (antioxidant enzymes) [7].
Infammation is a defensive mechanism triggered by harmful foreign stimuli, such as pathogens, viruses, dust particles, irritants, and damaged cells, to initiate healing [8,9].It comprises various steps, starting with an induction phase, continuing with a peak of infammation, and ending with the resolution phase [10].Induction phase is needed for efective host defense.It is caused due to external and endogenous noxious stimuli resulting from mechanical, chemical, or biological cell destruction [11].Te resolution phase is necessary for reducing infammation and restoring cell homeostasis after removing the noxious stimuli.Exaggerated immune responses such as human leukocyte antigen-B27 in arthritis, nucleotide-binding oligomerization domain-2, and interleukin-10-R mutations are hereditary factors that can override suitable resolution mechanisms [12].
Tis study aims to evaluate antioxidant, anti-infammatory, and antiarthritic activities of twelve native medicinal plants found in Nepal and characterize their extract using the LC-MS technique.Tis study helps to understand the biological activities of these plants and identify bioactive compounds responsible for those activities.1.

Plant Extraction Process.
First, the fresh leaves of all twelve plants were washed with fresh water to remove the dust; after that, the fresh leaves of individual plants were introduced with liquid nitrogen separately to perform the freeze-drying process.5 mL of ethanol (70% v/v) was mixed with one gram of freeze-dried sample for about fve minutes in a magnetic stirrer at high speed, followed by centrifugation at 4500 rpm for 10 minutes.After that, the supernatant was stored and fltered with the help of flter paper.Te remaining residue was re-extracted following the same procedures, and the collected supernatants were combined.Ten, the combined supernatant was dried in a rotatory evaporator at 40 °C.Te dried extract was accurately weighed and stored at − 20 °C in an airtight screw-capped glass bottle and was used for assay by dissolving the extract in ethanol in a required concentration [20].

Determination of Phytochemical Constituents
2.3.1.Determination of Moisture Content.Te sample was exactly weighed before and after oven drying to measure the loss on drying.Moisture content (MC) was calculated as follows [21]: Moisture content on a wet basis (% moisture) � wt of wet sample (g) -wt of dry sample (g) wt of wet sample (g) × 100%. (2)

Estimation of Chlorophyll
Content.Some fresh leaves of selected plants were taken and cut individually.Te chopped leaves of those plants were accurately weighed (1 g) and grounded using a mortar pestle separately.Ten, 20 mL of 80 percent acetone and 0.5 g of magnesium carbonate powder were added.Te leaves were again appropriately ground for 4 minutes at 40 °C, and the samples were put into the refrigerator for the next 24 hours.After 24 hours, the samples were centrifuged at 500 rpm for 5 minutes.Te supernatants were transferred to 100 mL of volumetric fask, and 80% acetone was added to the prepared solution to maintain the fnal volume.Te absorbance was measured using a spectrophotometer at 645 and 663 nm [20].

Preliminary Phytochemical Screening of Diferent
Plant Extracts.Te preliminary phytochemical screening of various plant extracts was carried out using the standard procedure mentioned in previous studies.Test for alkaloids are as follows:: Test with Dandruf reagent [22], test with Mayer's reagent, test with Wagner's reagent, and test with Hager's reagent [23].Estimation of saponins are as follows: Foam test [24] and determination of phytate content [25].

Determination of Total Phenolic Content (TPC).
Te method explained by Hazra et al. was used for the determination of total phenolic content by using the Folin-Ciocalteu phenol reagent [24].A total of 2 mL of plant extract (100-2000 μg/mL) and 2 mL of Folin-Ciocalteu phenol reagent were mixed, and the solution was made 9 mL each with distilled water.Te solution of sodium carbonate was added [1 mL of 10% (w/v)] after 5 minutes and mixed thoroughly.Te total 10 mL of the reaction mixtures were kept for 1 hour at room temperature, and the absorbance was measured using a UV spectrophotometer at 765 nm.Using an equation derived from the gallic acid standard calibration curve, the total phenolic content for each extract was expressed in milligrams of gallic acid equivalent per milligram of extract (a µg of GAE/mg of extract).Te entire experiment was carried out in triplicate [26].Ten, it was centrifuged at 5000 rpm for 5 minutes, and 20 µL supernatant phenolic content was taken (free from tannins due to precipitation formation with PVP).Absorbance was determined at 725 nm and expressed in terms of µg tannic acid equivalent per milligram (µg TAE/mg extract) using a standard curve [28].

Gas Chromatography and Mass Spectroscopy (GC-MS).
In this study, we performed GC-MS spectroscopic analysis of ethanolic leaf extracts to determine their phytoconstituents.GC-MS extract analysis was conducted at the Nepal Academy of Science and Technology (NAST), Lalitpur, Kathmandu, Nepal.Te analysis was carried out by using Agilent 7890A spectrometer system with fused silica column Agilent 19091s-433 (5% phenyl methyl siloxane 30 m × 250 µm, flm thickness × 0.25 µm), interfaced with GC-Agilent 5975C inert MSD with triple axis detector.In this spectroscopy, helium gas was used as carrier gas with a fow speed of 1 mL/minute.Te spectrophotometer ion source temperature was 230 °C, with an interface temperature was 320 °C, pressure 6.6018 psi, and the injector volume was 2 µL in split mode with a ratio of 75 : 1 (75 mL/minutes) at a temperature of 320 °C.Initially, the spectrophotometer column temperature was started at 32 °C for 5 minutes.After that, it was changed to 70 eV and ion source temperature 280 °C in the scan range of 50-100 amu for 5 minutes.Te total run time of individual samples was 59.6 minutes.After the analysis, the relative percent amount of each component was calculated by comparing the average peak % area [29].

In-VitroAnti-Infammatory Activity and Antiarthritic Activity
2.6.1.Protein Denaturation Assay.We used the method explained by Palit and his colleagues to determine the antiarthritic activity, with slight modifcation [36].0.10 mL of diferent plant leaf extract dissolved in DMSO was used separately at 100 − 2000 μg/mL concentration.Te solution mixture was again mixed with 2.40 mL of 3.5% bovine serum albumin (BSA) solution.Te 1N of hydrochloric acid at pH 6.3 was prepared and added to the extract solution.After that, the solution was incubated at 37 °C for 20 minutes.Te reaction mixture was then heated at 71 °C for 1 minute.After cooling solution, 2.5 mL of phosphate-bufered saline (PBS) with pH 6.3 was added to individual samples.Te absorbance of the fnal reaction mixture was measured at 660 nm by using a UV spectrophotometer.Product control was prepared with BSA, and the percentage of protein denaturation inhibition will be estimated by using the following formula: where test is mean absorbance of plant extract and reagents and control is mean absorbance of solvents and reagents without inhibitor.

Membrane Stabilization
(1) Erythrocyte Suspension.With few modifcations, the erythrocyte suspension was prepared using the technique outlined by Pieroni et al. [37].Healthy human volunteers provided the blood sample, which was centrifuged for 10 minutes at 1500 rpm.Te red blood cell (RBC) pallet was washed three times with PBS (pH 7.4) following centrifugation.Te bufy coat and supernatant liquid were aspirated out following each wash.Te RBCs were resuspended in PBS at a concentration of 20% (v/v).
(2) Heat-Induced Hemolysis.Te method explained by Gunathilake et al. was used to determine the heat-induced hemolysis with some modifcations.A 0.05 mL at varying concentrations of 100 − 2000 μg/mL of hydroethanolic extract of diferent plant leaves was taken separately, and a 0.05 mL of blood erythrocyte suspension was mixed with 2.95 mL of PBS at pH value 7.4.Ten, the mixture was incubated for 20 minutes at 54 °C in the water bath with continuous shaking.Te mixture was incubated and centrifuged at 2500 rpm for 3 minutes.By using a UV spectrophotometer, the absorbance of supernatant was measured at 540 nm.In the experiment, PBS will be used as blank [38].Te hemolysis level will be calculated using the following equation: (3) Proteinase Inhibitory Activity.Te method explained by Truong et al. was used to evaluate the proteinase inhibitory activity of plant extract with slight modifcation [38].A 0.06 mg trypsin and 1 mL of 20 mM Tris-HCl bufer with pH 7.4 were mixed with 1 mL of individual plant extract at 100 − 2000 μg/mL concentration.Te mixture was incubated at 37 °C for 5 minutes, and then, 1 mL of 0.7% (w/v) casein protein was added.Again, the reaction mixture was incubated for 20 minutes.Ten, 1 mL of 70% perchloric acid was added to end the reaction.Te reaction mixture was centrifuged at 3000 rpm at 4 °C for 10 minutes.Te absorbance of the supernatant will be measured at 210 nm against the bufer solution as blank by using a spectrophotometer.
% inhibition � 1 − Abs of test − Abs of control Abs of control   × 100. (5) 2.6.3.Lipoxygenase Inhibition Assay.Te method explained by Wu et al. was used to evaluate the lipoxygenase inhibition activity of plant extract with some modifcations [39,40].Te linoleic acid was used as substrate, and the 5-lipoxidase enzyme was used.Te solution mixture containing 1 mL of sodium borate bufer (0.1M) with pH 8.8 and 10 µL lipoxygenase (fnal concentration 8000 U/mL) was incubated with 1 mL plant extract at a concentration of 100 − 2000 μg/mL.Te reaction mixture was incubated at room temperature at 30 ± 2 °C for 5 minutes.Te 10 µL linoleic acid (10 mmol) was added and initiated the reaction.By using a UV spectrophotometer, the absorbance was calculated at 234 nm.Te % inhibition of lipoxygenase will be calculated by using the following formula:

Statistical Analysis.
Te resultswere expressed as mean ± standard error mean (SEM).Data analysis was performed using GraphPad Prism Version 5 and 8.0.Te mean was compared by one-way analysis of variance followed by Tukey's multiple comparison test.Te values were statically signifcant at three levels, * * * p < 0.001, * * p < 0.01, and * p < 0.05 but nonsignifcant (NS) if p > 0.05.

Percentage
Yield.An extraction technique is considered ideal when it produces high yields of extracts while requiring little modifcation to the extract's functional qualities [37].Te diference in the efcacy of biological activities was observed in extracts generated using various extraction procedures [41].As a result, it is important to choose the best extraction technique and solvent based on the sample matrix's characteristics, the analytes' chemical properties, the interaction of the matrix and the analytes, and the efectiveness and desired attributes [37].Extraction yields were determined by evaporating the diferent ethanolic extracts obtained in the ratio 1 : 5 of extract and solvent at 37 °C in an incubator to dryness and measuring the remaining solid residue.Te extractive yield of each ethanolic leaf extract was determined and stated in Table  1 shows the data of the combined chlorophyll and moisture content.All plant leaves were incubated at 37 °C, and it was found that Amaranthus viridis exhibited the highest moisture content (2.82 ± 0.28 mg/mL), while Chenopodium album exhibited the lowest (0.24 ± 0.001 mg/mL).Amorphophallus paeoniifolius had the highest chlorophyll content, measuring 13.59 ± 0.003, while Cicer arietinum had the lowest, measuring 0.10 ± 0.035, respectively.

Total Phenolic Content (TPC).
Te phenolics are the important phytoconstituents having redox properties responsible for scavenging free radicals [42].In present studies, the total phenolic content of diferent ethanolic plant extracts weredetermined using the Folin-Ciocalteu assay by creating a standard calibration curve with gallic acid into the relationship between absorbance and concentration (y = 0.0098x + 0.0058 and R 2 = 0.995).Using the equation derived from the calibration curve of gallic acid, the difference in total phenolic content was large among all extracts, ranging from 25.    Whereas increasing the extract concentration, the total tannin content was also increased (supplementary Table 3).

GC-MS Analysis.
Te present study used GC-MS analysis to determine the phytoconstituents present in ethanolic extract of twelve medicinal plants.In the GC-MS analysis, the compounds were predicted by calculating the spectrum's average peak percentage area and retention time.
All the phytoconstituents present in several plant leaf extracts are listed in Table 3.Also, the mass spectrophotometer determines the compounds eluted at diferent time frames and helps to identify the compounds' nature and structure (supplementary Figure 1).Te GC-MS analysis helps predict the phytoconstituent present in diferent leaves plant extract.It may lead to the isolation of those phytoconstituents present in high concentration into the specifc plant extract.
Similarly, determining their pharmacological activity will be helpful for the discovery of new molecules.
3.8.In-Vitro Antioxidant Activity.Te antioxidant activity of ethanolic extract (100 − 2000 μg/mL) from twelve different plants was carried out by taking ascorbic acid, BHT, and BHA, as standard compounds.Te plant extracts were tested against the oxidant compound for showing signifcant antioxidant properties.Te plant extracts were tested by taking diferent concentrations and found that the activity was concentration-dependent, i.e., increment in the concentration of plant showed better antioxidant activity and vice versa.Almost all plant extracts showed signifcant antioxidant activity.Te ferric ion-reducing antioxidant power assay of twelve plant extracts was carried out.Te activity was confrmed when the color of the solution was changed from yellow to green and Prussian blue based on the reduction property of extracts and standard, which was measured at 700 nm wavelength using a UV spectrophotometer.Te absorbance value was increased with the increasing concentration of plant extract (Table 4).So, the activity was found to be concentration-dependent.

In-Vitro Anti-arthritic and Anti-Infammatory Activities.
Te antiarthritic and anti-infammatory properties of ethanolic extract (100 − 2000 μg/mL) of the plants A. viridis, I. batatas, A. paeoniifolius, N. cadamba, R. sativus, C. arietinum, C. esculenta, M. koenigii, B. nigra, C. tamala, and C. album were compared with the standard compound indomethacin, aceclofenac, etoricoxib, and aspirin.Te values in the table are the IC 50 values, indicating that the compound with less IC 50 results in good inhibitory activity.Among the standard compounds, indomethacin responds better to all the protein denaturation activity with BSA, human red blood cells (HRBC) membrane stabilization, lipoxygenase (LOX), proteinase inhibitory and antiarthritic activity, and etoricoxib for protein denaturation with BSA and human red blood cells (HRBC) membrane stabilization, and aceclofenac for protein denaturation with BSA and lipoxygenase (LOX) inhibitory activity and aspirin for protein denaturation with BSA.
Among the plant extract, the best response of human red blood cells (HRBC) membrane stabilization was confrmed by A. paeoniifolius (IC 50 5).

Discussion
Te phenolic content of purple-feshed, orange-feshed Beauregard, and white-feshed Bonita sweet potato leaves was estimated by Su et al. 2019 using the Folin-Ciocalteu method.Te leaves were extracted using 70% acetone and were diluted to 12.5-200 μg/mL.Also, the result at the highest concentration was found to be in 36.8 ± 4.8 mg GAE/ gm in the leaves of purple-feshed, 41.2 ± 5.0 mg GAE/gm in Beauregard, and 46.7 ± 2.1 mg GAE/gm in Bonita.Comparing the result with the present study of ethanolic leaf extract of I. batatas shows less phenolic content i.e. 30.30± 0.27 µg GAE/mg than the previous study [43].Also, Jyothi et al. 2019 estimated that the total phenolic in ethanolic leaves extract of C. esculenta was 26.5 mg GAE/g at 1000 μg/mL concentration.Te present study result revealed that total phenolic content was highest, i.e., 51.73 ± 0.14 µg GAE/mg compared to previous studies at the same concentration [44].Te total favonoid content of methanolic extract of A. viridis leaves was estimated by Salvamani et al. and Sarker and Oba, which was found to be 152.12mg rutin equivalent/ gm and 182.46 ± 0.26 mg rutin equivalent/gm, respectively.In the present study of ethanolic extract of leaves of A. viridis, the total favonoid content is 23.708 ± 0.81 µg GAE/mg.Tis shows that the favonoid content is lower than the previous studies [45,46].
Ooko Abong et al. researched phytochemical screening in leaves and roots of various sweet potato species.Tey determined the total tannin content for which 0.15 gm and 0.25 gm of the freeze-dried powdered leaves and roots were taken, and values were represented as mean±standard deviation.Also, the species of sweet potato (K/KA/2004/205) had the highest tannin content of 5.05 gm/100 gm in leaves (40 times) as compared to roots.Te tannin content estimated in the present study in the leaf extract of I. batatas is 48.50 ± 1.045 µg TAE/mg, which is lesser than the previous study [47].Also, the presence of tannin content in the tuberous extract of A. paeoniifolius with 50% ethanol was determined using the standard protocol described by Majumder et al. 2020.Te diferent concentrations of the samples extracts were prepared as 25-400 µg/mL, and at the highest concentration, 400 µg/mL (0.4 mg/mL) of plant extract was found to be 938.95± 90.5 mg TAE/gm.In the present study of ethanolic extract of leaves of A. paeoniifolius at a concentration of 400 μg/mL, the tannin content is 54.35 ± 1.217 µg TAE/mg.Tis clarifes that in the species of A. paeoniifolius, the presence of tannin content is more in the tuberous extract than in the leaf extract [48].
Oxidative stress is a condition of imbalanced free radicals in the biological system.It is generated as a by-product of oxygen metabolism.Te excessive production of these free radicals plays an essential role in the induction of diseases [49].In the present study, twelve leaf extracts were obtained from diferent plants and evaluated for the total phenolic, favonoid, tannin content, antioxidant activity, antiarthritic, and lipoxygenase inhibition activity.
DPPH is a stable nitrogen-centered free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule.When DPPH radicals react with reducing agents, DPPH radicals receive an electron or hydrogen radical from an antioxidant, and corresponding hydrazine is formed.Te solution becomes purple to yellow color [50].Te IC 50 value of 42.48 μg/mL for the DPPH scavenging activity of ethanolic extract of C. arietinum by Parithy M et al. 2013 was compared with the present investigation for the same plant extract IC 50 value of 9.41 ± 0.07 μg/mL, which was found to be higher as compared with previous studies [51].Similarly, the extract of C. album by Debski B. et al., 2018 showed 65 ± 8% signifcant p < 0.05 inhibition, and the present study showed 6.13 ± 1.23% inhibition.Tis comparison resulted in signifcantly higher inhibitory activities of present plant extract [52].
At physiological pH, sodium nitroprusside spontaneously generates nitric oxide, which reacts with oxygen to form nitrite ions that may be measured using Griess' reagent.Nitrite ion production is decreased due to competition between nitric oxide scavengers and oxygen [53].Te previous activity by Ghosh et al. on the bulb extract of Dioscorea bulbifera showed nitric oxide scavenging activity of 49.85 ± 0.16% at the concentration of 1000 μg/mL and 9.72 ± 0.15% for the recent study on leaf extract of the same plant.Tus, it resulted in higher antioxidant activities of leaves than bulbs of Dioscorea bulbifera [54].Te nitric oxide scavenging activity was performed by Griess Illosvoy reaction, and values were represented as mean ± SEM.Te result by Alam et al. showed that the nitric oxide scavenging activity at the concentration of 2000 μg/mL of ethanolic extract of leaf of A. tricolor was 57.4 ± 2.11, and in the present study, the value of the extract of A. viridis at the concentration of 2 mg/mL is 9.51 ± 1.21.Tis verifes that the species viridis is better than tricolor [55].
Reactive oxygen species are molecules that generate free oxygen radicals, including hydroxyl and superoxide.Tese molecules are highly reactive and participate in mitochondrial dysfunctions to prevent the production of ATP and promote cellular disruption [56,57].Te hydrogen peroxide scavenging activity for the extract of C. album was done by Devi et al. and revealed that bay leaf extract showed concentration-dependent inhibition at 1000 µg/mL concentration of 72% and had the highest scavenging activity as compared with BHT.In the present study, the scavenging activity at the same concentration was most efective at 11.73 ± 1.58% but concentration-dependent as compared to the previous study [58].
Potassium ferricyanide (Fe 3+ ) combines with substances having reduced potential to generate potassium ferrocyanide (Fe 2+ ) at 700 nm.Te increased absorbance of the reaction mixture indicates increasing reducing power [59].Candel et al. evaluated the reducing ability of ethanolic extract of Anthocephalus cadamba and found a signifcant positive correlation between ferric reducing antioxidant power (FRAP) and total favonoids.It also concluded that extracts enriched with favonoids and phenolics act like ferric-reducing agents and protect from oxidative stress [60].For the FRAP activity of methanolic extract of seeds, fowers, stem, and leaves of Amaranthus caudatus, FRAP reagent was prepared by Karamac et al. and observed in the Amaranthus extracts (1000 µg/mL).It showed the highest absorbance in the following order: leaves ≥ fowers >> stem > seeds.Similarly, the present study of FRAP ethanolic extract of the leaf of A. viridis (11.61 ± 2.62 µg/mL) shows increasing activity with increasing absorbance, i.e., concentrationdependent [61].
Hydroxyl (OH • ) radical is a robust reactive oxygen species.Tis moiety directly interacts with polyunsaturated fatty acid found in the cell membrane, damaging all the essential components of the cells and destroying the biological system, which promotes diferent pathological conditions such as cancer [53].Te earlier study of Ghosh et al. on a bulb of D. bulbifera at 1000 μg/mL concentration showed the % inhibition of hydroxyl radical by 64.23 ± 1.25% in the ethanolic extract [62].But the present study on leaves of D. bulbifera revealed the percentage hydroxyl radical scavenging activity of 15.57 ± 0.91% at the same concentration, which was found to be better in the leaf than in the bulb extract.Te same scavenging activity was performed by thiobarbituric acid (TBA) method described by Sun et al. 2017.It was carried out in sweet potato leaves, which were extracted using ethanol.At 2000 μg/mL concentration, the % hydroxyl scavenging activity was 65.99 ± 2.07, expressed as mean ± standard deviation.In the present study, the leaf extract of I. batatas at the same concentration was 40.9 ± 2.92, which is comparatively higher compared to past studies [63].
Reactive oxygen species are formed from various processes, and the metal oxidation reaction is one of them.Te transition of metal ions ferrous (Fe 2+ ) possesses the generation of free radicals by gaining or losing the electron.Terefore, diferent plant extracts were used to determine the inhibition capacity of free radicals via metal chelating activity [64].Te ferrous ion chelating activity was carried out using ferrozine by Vu et al. on the methanolic corn extract of C. esculenta and was found to be 61.9%[65].Te present study of ethanolic leaf extract of the same plant is 14.61 ± 0.08%.Tis shows a higher analysis of leaves than the corn of C. esculenta.Te previous research carried out by Beevi et al. on the root of R. sativus using various solvents such as methanol, water, ethyl acetate, and hexane.It showed 32.15%, 28.54%, 21.98%, and 20.86% of chelating activity, respectively.Te present study on leaves of R. sativus using ethanolic extract showed 4.76 ± 0.68% inhibition at 1 mg/mL of concentration [66].
Te basic principle to assess the total antioxidant capacity through phosphomolybdenum assay includes the reduction of Mo (VI) to Mo (V) by the plant extract A monomeric protein called lipoxygenase (LOX) is produced when hydroperoxidase is produced by oxidizing polyunsaturated fatty acids such as linoleic, linolenic, and arachidonic acid.LOX is widely distributed in many animals, plants, fungi, and cyanobacteria species.Te 5-lipoxygenase is derived from the 5-carbon of arachidonic acid and is commonly found in mammalians.Te LOX is expressed in many immune, epithelial, and tumor cells.It is crucial for several physiological conditions, including stroke, neurological illness, skin disorders, tumorigenesis, and cardiovascular complications.Most signifcantly, it is a precursor to infammation [37,38].Sendangratri et al. carried out lipoxygenase inhibition activity of tuber extract of sweet potatoes of diferent varieties.Te tubers were macerated with 70% ethanol for the activity, and their ability to inhibit soybean lipoxygenase was assessed.Also, the result showed that the IC 50 values of purple, orange, and white sweet potato tuber extracts were 46.09, 52.12, and 63.69 μg/ mL, respectively.However, in recent study, the IC 50 value of ethanolic extract of leaves of I. batatas is 62.94 ± 3.53 μg/ mL at a higher extract concentration, i.e., 2000 μg/mL [70].Also, the in vitro anti-infammatory activity, i.e., lipoxygenase inhibition of the same species, was observed and found to be showing the IC 50 value in the given order spinosus > tricolor > dubis > viridis, 58.17 ± 3.49, 66.20 ± 2.67, 72.49 ± 3.44, and 73.93 ± 3.38 μg/mL, respectively.Te IC 50 value of A. viridis in the present study is 58.99 ± 3.84 μg/mL, which is comparatively higher than the past studies for the same species [71].
Rheumatoid arthritis is a chronic immunological infammatory condition.It initially afects the small joints of the hand and legs, which commonly start from the fgure joints.Te continuous development of this condition damages the joint cartilage, tendon, and ligaments, resulting in intense pain.Diferent antiarthritic drugs are available, but those drugs only showed symptomatic relief, which is unsuitable for the patient [72].Te primary goal was to look at alternative compounds for managing and preventing the onset of arthritis.Previously, Win KC et al. 2018 performed the protein denaturation and bovine serum albumin procedure to investigate the ethanolic extract of A. cadamba for its in vitro antiarthritic capabilities.Te percentage of arthritis protection was found to be 45.58% at a concentration of 500 µg/mL.In contrast, the present study shows that the rate of arthritis protection is 60.41 ± 3.88%; it concluded that the Anthocephalus cadamba has better antiarthritis activity than the Neolamarckia cadamba, and various species showed varying activities.
Arthritic responses have been linked to proteinases.Many serine proteinases are found in the lysosomal granules of neutrophils.Leukocyte proteinases signifcantly infuence the development of tissue damage during infammatory processes.Recent research has demonstrated that numerous favonoids contributed substantially to several plants' antioxidant and anti-infammatory properties.Tus, present research clarifes that bioactivity (favonoids and many more) may infuence anti-infammatory properties [73].In previous studies, Brassica nigra had the proteinase inhibitory capacity.It showed that the increase in the dose (µg/mL) increased the percentage of proteinase inhibition.At 50, 100, 150, 200, and 250 µg/mL, concentration expressed 15. 26, 22.14, 25.36, 32.89, and 42.57percentage inhibition, respectively.B. nigra had the favonoid content responsible for inhibiting trypsin (regulatory of infammation) enzyme and thus acted as an anti-infammatory agent [39].Prior research by Mahajan et al. on methanolic extract of D. bulbifera tubers demonstrated a signifcant % proteinase inhibition of 13.61% at 0.2 mg/mL concentrations.In contrast, the current study shows an inhibition of 35.9% at the same concentration, which is lesser to the previous study [40].
Anti-infammatory agents mainly inhibit the cyclooxygenase enzyme responsible for synthesizing infammatory mediators by converting arachidonic acid to prostaglandins.Prostaglandin G2 (PGG2) is converted into PGH2 together by the enzyme peroxidase, which creates long membrane channels.Due to the release of chemical mediators, membrane channel opening caused the release of arachidonic acid from the membrane and converted to prostaglandin.Tese enzymes' extracellular activity is considered to be related to both acute and chronic infammation.Sen et al. conducted in vitro anti-infammatory research in A. caudatus L by observing the inhibition of protein denaturation and HRBC membrane stabilizing assay.At a 500 μg/mL concentration, the methanolic extract of leaves from A. caudatus L impacted membrane stability and protein denaturation.But at an 800 μg/mL concentration, the ethanolic extract of A. viridis exhibits the same activity of membrane stabilization and protein denaturation.Hence, the % inhibition in the present study is comparatively higher than in the past studies [74].

Limitation of the Study.
In the present study, we only carried out the in-vitro study of selected plant extracts.Due to limited resources, we were unable to do in-vivo assay, molecular pathway analysis, and quantitative structure-activity relationship (QSAR) modeling of identifed compounds through mass spectroscopy.In future, computation and molecular analysis should be carried out for validation of our study.

−
indicates absence of phytoconstituents, + indicates presence of phytoconstituents, and * indicates no determination of phytoconstituents.
Te experimental study was conducted in the Department of Pharmacology, Universal College of Medical Science (UCMS), Bhairahawa, Rupandehi, from December 2021 to May 2022.Before starting this study, the project registration was completed by the Institutional Review Committee of Universal College of Medical Sciences, Bhairahawa, Rupandehi, Nepal Registration Number: UCMS/IRC/032/22.2.1.Plant Material Collection and Verifcation.In the present study, most of medicinal plants used were seasonal; therefore, the fresh leaves of all those twelve medicinal plants, namely, Cicer arietinum, Murraya koenigii, Colocasia esculenta, Amorphophallus paeoniifolius, Ipomoea batatas, Amaranthus viridis, Raphanus sativus, Neolamarckia cadamba, Dioscorea bulbifera, Chenopodium album, Cinnamomum tamala, and Brassica nigra, were collected from Rupandehi district in December 2021.Te plants were verifed and certifed by Mr. Subodh Khanal, Assistant Professor and Course In-charge of Medicinal and Aromatic Plants, Department of Soil and Environmental Science, Institute of Agriculture and Animal Science (IAAS), Paklihawa, Rupandehi, Nepal.Te specimen number of individual plants is listed in Table

Table 1 :
List of plant names, specimen numbers, percentage yield, moisture, and chlorophyll content of selected plants.
showed the presence of a high number of bioactive metabolites, and the remaining other plants leaves extract also showed good but not similar bioactive secondary metabolites profle.Te absence and presence of diferent secondary metabolites were identifed by performing different tests and reported the presence (+) and absence (− ) in the form of symbols (Table2).
1. From the above table, Neolamarckia cadamba has the highest yield percentage, and Colocasia esculenta has the lowest yield percentage, which was found to be 17.62% and 7%, respectively.3.2.Preliminary Phytochemical Screening.Te preliminary phytochemical screening of twelve plant leaf extracts showed that C. arietinum, M. koenigii, A. viridis, A. paeoniifolius, and N. cadamba 3.3.Moisture and Chlorophyll Content.Table

Table 2 :
Preliminary phytochemical screening of twelve sessional medicinal plants.

Table 3 :
Bioactive compounds present in diferent leaves plant extracts detected through GC-MS analysis.

Table 5 :
Anti-infammatory and antiarthritic activities of twelve sessional medicinal plants.Khandayataray  etal.and Rajeshwari et al. revealed diferent activities of leaf extract of C. album and B. nigra at diferent concentrations of 100 and 200 µg/m [68, 69].Te C. album represented that ethyl acetate, methanol, and aqueous extract had the highest antioxidant capacity of 0.812 ± 0.06, 0.891 ± 0.05, and 0.816 ± 0.11, respectively, compared to the present study at the same concentration of 16.3 ± 0.16 while the B. nigra leaves extract from acetone showed the least activity of 95.26 ± 07.85 compared with the present (15.79 ± 0.21) study at the same concentration.
C. arietinum expressed highest moisture content, and A. viridis had highest chlorophyll content.All plant extracts showed good phenolic, favonoid, and tannin content.While N. cadamba and D. bulbifera have highest total phenolic 12 Journal of Tropical Medicine content, I. batatas and C. album have favonoid highest value.Similarly, R. sativus and N. cadamba reported highest total tannin content.Also, diferent phytoconstituents were identifed from several plant extracts through gas chromatography and mass spectroscopy (GC-MS) methods.Te present study used twelve leaves extracts for the antioxidant and anti-infammatory activities.Tis study concluded that all the selected medicinal plants contain a good amount of phytoconstituents and show signifcant antioxidant and anti-infammatory activities.