The plant was collected from District Dera Ghazi Khan Punjab Pakistan (30° 2’ N and 70° 38’ E). The solvents used for the extraction were ethanol, ether, and chloroform, whereas sodium carbonate, aluminum chloride, folin reagent, and sodium nitrate were used for TPC and TFC (Handa, 2005). About 50 g of plant sample was weighed and grinded in pester mortar with a small amount of ethanol. The paste of plant sample was packed into Soxhlet apparatus for extraction. The greenish extract was the product of solvent extraction, which further proceeds for decolorization by different concentrations of activated charcoal. Steps involved in the extraction and analysis are presented in Figure 1 .

The TPC of the samples was estimated using the folin-Ciocalteu reagent method described by (Ozturk et al., 2022). Briefly, the total phenolic content of the plant extract was determined on the basis of a dilution method about to 0.2 N in the presence of folin-Ciocalteu reagent, and kept for 2 h in the culture tube. This material was further oxidized using sodium carbonate at approximately 75/g per liter. The absorbance of the sample was measured using a spectrophotometer at 750–760 nm using gallic acid as the standard reagent (Ozturk et al., 2022).

TFC contents were measured according to the method described by Sen et al (Sen et al., 2013). A volume of 250 ml plant extract added in culture tube containing distilled water. Then, 76 µL sodium nitrate was added to the culture tube. Further AlCl₃ was added in the culture tube for 6–7 minutes. After this, the plate was placed in the spectrophotometer and reading was recorded at 500 nm. Three readings were taken. Quercetin was used as a standard reagent in the calibration curve (Dewanto et al., 2002).

The extracts were subjected to UV-VIS scanning and diluted 30 times with the appropriate extraction solvent (ethanol or methanol) at room temperature before analysis. Absorbance spectra were acquired between 200 and 400 nm using a UV-VIS spectrophotometer.

The extracts of Pseudoconyza viscosa (Mill.) were analyzed using an HPLC system (YL 9100, Korea), which included an autosampler (YL 9150) with a 100 µL fixed loop and a YL 9120 UV-visible detector. Separation was carried out on an SGE Protecol PC18GP120 column (250 mm × 4.6 mm, 5 µm) at ambient temperature. The mobile phase consisted of methanol and water in a 70:30 (v/v) ratio, and the separations were conducted in iso-cratic mode with an elution flow rate of 1 mL/min. Standards were used to validate the peaks.

Molecular docking studies explained the behavior of ligands isolated from Pseudoconyza viscosa (Mill.) against functional residues involved in the antioxidant activity of the human peroxiredoxin 5 protein using a dataset of 11 phytochemicals ( Table 1 ). The structure of target protein was retrieved from the Protein Data Bank (PDB ID:1HD2) having high resolution 3.78 Å, ligand bound in the active site and no mutation as shown in Figure 2 . AutoDock Vina was used to perform molecular docking analysis. This standalone offline tool works in two modes: (i) MGL tools GUI based system, used for receptor (protein) and ligand Pseudoconyza viscosa (Mill.) compounds preparation. (ii) Command based mode used to execute the ligand -receptor docking (Gupta et al., 2018).

The results of the UV-VIS Analysis showed the presence of several aromatic compounds. The absorption of electromagnetic radiation at 200–400 nm indicates the presence of heteroatoms. Compounds containing σ bonds, π bonds, a lone pair of electrons, chromophores, and aromatic rings were identified in UV UV-visible spectra. The spectra of the resultant compounds are shown in Figure 2 .

The extract revealed the presence of various phenolic and flavonoid compounds including phenanthrenes (peak 1), dicaffeyl derivatives (peak 2), hydroxycinnamic acids (peaks 3-7), flavonoid glycosides (peaks 8-12), and dicaffeoylquinic acids (peaks 13-15). These findings are in consistent with earlier phytochemical research on other medicinal plants, which have reported it as a rich source of polyphenolic secondary metabolites (Lobo et al., 2010; Sen et al., 2013). Additionally, these compounds have demonstrated potent antioxidant and neuroprotective effects (Al-Khayri et al., 2022).

The identified flavonoid glycosides, such as quercetin, kaempferol, and their rutinoside derivatives, are well-known for their potent antioxidant, anti-inflammatory, and anti-cancer properties (Al-Khayri et al., 2022). Similarly, the dicaffeoylquinic acids detected (peaks 13, 15) demonstrate a broad spectrum of pharmacological activities, including anti-inflammatory, neuroprotective, and anti-diabetic effects (Boukhatem and Setzer, 2020; Marcos-Gómez et al., 2024). The presence of these derivatives in the P. Pseudoconyza viscosa (Mill.) extract suggests that they may contribute significantly to the observed biological activities of this medicinal plant. ( Table 1 ).

The HPLC analysis of the ethanolic extract of Pseudoconyza viscosa (Mill.) revealed the presence of several phenolic and flavonoid compounds ( Figure 3 ). The extraction of active components consists of different steps which can affect both the quality and quantity of the extracted components. The diversity of peaks suggests a complex chemical profile, which may explain the broad-spectrum antioxidants, antimicrobial, and enzyme inhibitory activities previously reported for Pseudoconyza viscosa (Mill.). Compounds eluting at earlier retention times are likely hydrophilic, such as phenolic acids, whereas those eluting later may be hydrophobic, including flavonoids or other secondary metabolites. In our analysis, Quercetin-O-dihexoside was the most abundant compound, with a concentration of 48.7 mg/L. It was closely followed by 5-Hydroxy-7,8,6’-trimethoxy-2’-hexoside(acetyl) flavone at 35.5 mg/L. The other compounds we measured included: Malonyl-1,4-O-dicaffeoylquinic acid (18.3 mg/L), Kaempferol-O-caffeoylhexoside (21.7 mg/L), and 5-Hydroxy-6,8-dimethoxy-7-hexoside flavone (23.5 mg/L) (Table 2).

Quercetin is well known for its antioxidant properties, primarily due to its ability to scavenge free radicals and mitigate oxidative stress (Qi et al., 2022; Deepika and Maurya, 2022). This observation is consistent with earlier studies suggesting that quercetin-rich compounds can offer various health benefits, including anti-inflammatory and anticancer effects (Shamsudin et al., 2022; Saeed et al., 2012).

Malonyl-1,4-O-dicaffeoylquinic acid and Kaempferol-O-caffeoylhexoside further enhance the antioxidant profile of Pseudoconyza viscosa (Mill.). Malonyl-1,4-O-dicaffeoylquinic acid, a derivative of chlorogenic acid, is linked to various pharmacological properties, including potent antioxidant activity and potential protective effects against neurodegenerative diseases (Szwajgier et al., 2017; Jang et al., 2022). Kaempferol and its derivatives have been found to have impact on inflammation and promoting cardiovascular health (Kamisah et al., 2023). 5-Hydroxyl-6,8-dimethoxy-7-hexoside flavone is not much studies however flavonoids with similar structures have been noted for their antimicrobial and antioxidant properties (Al-Khayri et al., 2022).

The ethanolic extract of Pseudoconyza viscosa (Mill.) demonstrates exceptional phytochemical richness, with quantitative analysis revealing remarkably high total phenolic content (TPC) of 311.74 mg GAE/g and total flavonoid content (TFC) of 208.2 mg QE/g ( Figure 4 ). These values represent a significant phytochemical profile, substantially exceeding previously reported values for related species such as Inula viscosa, which showed TPC of 236.78 ± 7.63 mg GAE/g and TFC of 94.36 ± 1.86 mg QE/g in methanolic extract. The considerable difference-approximately 32% higher phenolic content and 121% higher flavonoid content-positions P. viscosa as an exceptionally potent source of these bioactive compounds among medicinal plants, suggesting enhanced therapeutic potential.

The high total phenolic and flavonoid contents observed in the Pseudoconyza viscosa (Mill.) ( Figure 4 ).

Several studies have reported the presence of diverse phenolic and flavonoid compounds in Pseudo Conyza and related species. For example, a study on Pseudoconyza anastomosans, a closely related plant, found significant levels of phenolic acids, flavonoids, and other polyphenols (Karak, 2019; Mrid et al., 2022). Similarly, research on Conyza species, which are closely related to Pseudoconyza, has shown the abundance of phenolic and flavonoid constituents (Jung et al., 2009). The TPC and TFC values are notably higher compared to a previous study, which reported TPC at 236.78 ± 7.63 mg GAE/g and TFC at 94.36 ± 1.86 mg QE/g in the methanolic extract of Inula viscosa (Eruygur et al., 2024). The significant levels of phenolics and flavonoids in P. viscosa indicates its potential for various applications in the nutraceutical industry. Phenolic compounds are not only for its antioxidant properties but also for their antimicrobial and anti-inflammatory properties. Flavonoids also provide additional health benefits, such as enzyme inhibition and modulation of cell signaling pathways. These bioactive compounds in P. viscosa position it as a promising candidate for developing natural antioxidant supplements or therapeutic agents aimed at managing disorders related to oxidative stress.

The pharmacophore model provides a valuable framework for investigating the structure-activity relationships and potential mechanisms of action of the phytochemicals found in the Pseudoconyza viscosa (Mill.) extract. This exploration may aid in developing therapeutic applications for this medicinal plant (Muhammed and Akı-Yalcın, 2021; Gao et al., 2010). Eleven compounds from Pseudoconyza viscosa (Mill.) were docked with human peroxiredoxin 5 protein and results of the top-scored 10 compounds were shown with their binding energies ranging from -7.8 to -5.7 kcal/mol and binding interaction within the range of 4.0 Å. Human peroxiredoxin 5 (PRDX5) is known to have various pathological functions associated with oxidative stress, including neurodegenerative diseases, cancer, and inflammatory disorders. The inhibitors of Human peroxiredoxin 5 were analyzed using LigPlot to determine the amino acids residues involved in protein-ligand interactions. Dicaffeoylquinic acid IV shows least binding affinity and maximum binding interactions as shown in Figures 5a, b . The pharmacophore model provides a valuable foundation for further optimization and structural modifications of the bioactive compounds isolated from Pseudoconyza viscosa (Mill.). The insights gained can guide the design of novel derivatives with improved pharmacological properties, such as enhanced potency, selectivity, and bioavailability (Muhammed and Akı-Yalcın, 2021; Kumar and Bora, 2014). The structural features of 3,4-dicaffeoylquinic acid that enable its strong binding affinity include its dual caffeoyl substituents ester-linked at positions 3 and 4 of the quinic acid backbone, which introduce polarity and aromatic π-systems for hydrogen bonding and π-π stacking interactions. The catechol motifs in the caffeoyl groups enhance redox activity, stabilizing interactions in oxidative environments, while the quinic acid ring’s hydroxyl groups facilitate extensive hydrogen bonding with biological targets like enzymes or plasma proteins. The ester linkages contribute to structural rigidity and planarity, optimizing spatial alignment for interactions, and the molecule’s conformational flexibility allows steric complementarity with binding sites. Compared to other isomers (e.g., 1,4- or 4,5-DCQA), the 3,4 configuration balances steric strain and hydrophilicity, maximizing interaction diversity through both hydrophobic (aromatic) and hydrophilic (hydroxyl, ester) regions. This combination of functional groups and spatial arrangement enables broad interaction capacity, making dicaffeoylquinic acid IV highly effective in forming strong binding interactions in biological matrices. The number of hydrogen bond interactions ranged from 0 to 7, with compound Dicaffeoylquinic acid having the most at 7 interactions. The distances of these hydrogen bonds vary from 2.70 Å to 3.33 Å. Key interacting residues include Val70, Gly82, Gly92, Val94, Ala90, Glu91, Leu96, Arg86, Gly17, Arg95, and Lys22 ( Figure 5 ). Previous studies have also demonstrated the potent antioxidant, anti-inflammatory, antimicrobial, and neuroprotective activities of Conyza and Pseudo Conyza extracts, which have been attributed to their rich phytochemical composition (Veber et al., 2002; Jung et al., 2009; Wong-Paz et al., 2015; Ouamnina et al., 2024). Furthermore, plant-derived metabolites like dicaffeoylquinic acid and flavonoids, widely targeted metabolite modificomics technologies have emerged as powerful tools for enhancing the analysis of complex biological samples. Technologies like targeted liquid chromatography-mass spectrometry (LC-MS) and stable isotope labeling empower researchers to concentrate on specific metabolite classes while expanding detection capabilities. This allows for the simultaneous identification and quantification of hundreds of compounds in a single assay. For example, modifications such as derivatization or enzymatic treatments enhance the ionization efficiency and stability of metabolites, facilitating their detection in diverse matrices like plant extracts. In these extracts, flavonoids, secondary metabolites recognized for their antioxidant and health-promoting properties are often found in low concentrations (Yang et al., 2024).