Abstract Pilea microphylla (L.) Liebm. is a well-known medicinal plant belonging to the Urticaceae family. Despite its widespread use in traditional medicine, the chemical composition and biological activities of P. microphylla extracts collected in Vietnam have not been fully documented. The chemical components of the ethanolic extract obtained from P. microphylla whole plants are reported here for the first time using gas chromatography-mass spectrometry (GC-MS) analysis, revealing 62 constituents that account for 95.4% of this extract, with β-sitosterol (14.3%), cis-phytol (13.1%), tetrahydroedulan B (12.6%), hentriacontane (5.8%), and neophytadiene (5.6%) as dominant compounds. Additionally, approximately 45 metabolites have been reported from P. microphylla in the literature. The extract exhibited significant xanthine oxidase (XO) inhibition (IC₅₀ = 74.12 ± 1.58 μg/mL) and nitric oxide (NO) production inhibition (IC₅₀ = 61.29 ± 1.79 μg/mL). Molecular docking studies examined the binding interactions between 105 phytochemicals and the active sites of inducible nitric oxide synthase (iNOS) (PDB ID: 3E7G), interleukin (IL)-1β (PDB ID: 8C3U), and XO (PDB ID: 3NVY), with binding energies ranging from -5.4 to -11.3 kcal/mol, -5.0 to -8.8 kcal/mol, and -5.0 to -10.0 kcal/mol, respectively, indicating the anti-inflammatory and anti-XO potential of this species. These findings underscore the promising therapeutic applications of P. microphylla extracts for treating inflammatory and gout-related conditions.

Keywords: Anti-inflammation, GC-MS, Molecular docking, Pilea microphylla, Phytoconstituents

Funding: This research was funded by the University of Medicine and Pharmacy at Ho Chi Minh City under contract number 469/2025/HĐ-ĐHYD, dated 30/9/2025.

Citation:  Quynh, T.T.T., Tu, V.L., Duy, H.T., Huy, N.D., and Chen, T.V. 2027. Exploring the phytochemical composition and biological activities of Pilea microphylla (L.) Liebm. (Urticaceae): A combined experimental and in silico molecular docking investigation. Natural and Life Sciences Communications. 26(1): e2027006.

Graphical Abstract:

INTRODUCTION

Natural resources provide valuable plant-derived natural products or their derivatives that are highly sought after as alternatives to synthetic drugs for treating communicable and non-communicable diseases (Atanasov et al., 2015; Thomford et al., 2018). In the field of drug research and development, natural products derived from traditional medicinal plants continue to play a crucial role as multi-target agents for the treatment of metabolic disorders. In recent years, the use of traditional medicinal plants has increased significantly, and species from the Urticaceae family have been widely studied for their pharmacological effects based on their traditional uses. Among them, Pilea microphylla (L.) Liebm. is of particular interest due to its diverse phytochemical composition and potential biological activities.

Pilea microphylla (L.) Liebm. is a species of flowering plant in the Urticaceae family (Poudel et al., 2025). It is commonly known as “Cỏ Lăn tăn” (in Vietnamese), “Artillery plant”, “Pistol plant”, or “Gunpowder plant”. P. microphylla represents one of the approximately 614 species within the Pilea Lindl. genus. Macromorphologically, P. microphylla is recognized by its morphological characteristics, which include a glabrous succulent annual, monoecious with stems erect or ascending, 3-17 cm tall, and cystoliths dense; stipules persistent, triangular, membranous; inflorescences often androgynous, compactly cymose-capitate with male flowers pedicellate; perianth lobes ovate, subapically corniculate; achene ovoid, compressed, smooth (Poudel et al., 2025). Ethnopharmacological investigations conducted in various countries have revealed numerous traditional applications of P. microphylla. Traditionally, P. microphylla whole plant has been used to treat diabetes, diarrhea, asthma, ulcers, bruises, infertility, inflammation, and urinary problems, as well as for women in labor and to cleanse the uterus (Fathimathul Nisa and Aswathi, 2023). According to modern pharmacological evidence, P. microphylla is reported to possess significant biological properties, including antioxidant, radioprotective, anti-inflammatory, antidiabetic, antihyperlipidemic, antidepressant, and antimicrobial activities (Prabhakar et al., 2007; Modarresi Chahardehi et al., 2010; Bansal et al., 2012; Chahardehi et al., 2013; Singh et al., 2022). These biological activities may be attributed to their chemical constituents, particularly the abundance of secondary metabolites such as flavonoids (e.g., quercetin, quercetin-3-O-rutinoside, 3-O-caffeoylquinic acid, luteolin-7-O-glucoside, apigenin-7-O-rutinoside, apigenin-7-O-β-D-glucopyranoside, etc.) (Bansal et al., 2011, 2012) and essential oils (e.g., (E)-caryophyllene, (E)-nerolidol, α-pinene, methyl salicylate, α-humulene, etc.) (Singh et al., 2022).

Despite these promising properties, investigation into the potential role of P. microphylla in gout-related targets, especially xanthine oxidase (XO) inhibition and inflammation-related pathways, remains limited. Furthermore, it is yet unclear how the molecular mechanisms behind these biological activities relate to the phytochemical composition. Thus, integrating bioactive chemical compounds with biological and computational approaches may provide valuable insight into the therapeutic potential of this species.

To contribute to filling identified research gaps and leveraging modern methodological integration, this study was designed with the following specific goals: (i) to identify phytochemical compounds from P. microphylla; (ii) to evaluate in vitro xanthine oxidase (XO) inhibitory and anti-inflammatory activities of P. microphylla extract; and (iii) to provide molecular docking support for the potential interactions between the identified compounds and target proteins associated with gout and inflammatory (iNOS and IL-1β) responses. Based on this comprehensive approach, our work aims to establish a phytochemical profile and biological activities of P. microphylla, providing a scientific foundation for traditional applications of P. microphylla. These findings indicate that P. microphylla could be considered a potential candidate for further studies in the prevention and management of XO- and inflammation-related diseases.

MATERIALS AND METHODS

Plant materials

The whole plant of Pilea microphylla (L.) Liebm. (Urticaceae) was collected in September 2025 in Hiep Binh Ward, Ho Chi Minh City, Vietnam (altitude: 10 m, coordinates: 10°50'22"N, 106°42'53"E), and was identified by Dr. Tran Thi Thuy Quynh. A voucher specimen (No. TVC-LT06.25) has been deposited at the Department of Traditional Pharmacy, Faculty of Traditional Medicine, University of Medicine and Pharmacy at Ho Chi Minh City, Vietnam (Figure S1). The morphology of P. microphylla is shown in Figure 1.

Figure 1.  Morphology of Pilea microphylla (L.) Liebm. (A. Whole plants; B. Flowers and Fruits) (Photos by Tran Van Chen).

Preparation of plant extract

Fresh whole plants of P. microphylla (approximately 4.0 kg) were collected, cleaned, dried at temperatures below 45°C, and subsequently ground into powder to obtain a total of 300 g of dried plant material. The ethanolic extract of P. microphylla was obtained by soaking 300 g of roughly crushed whole plant in 96% ethanol (1.0 L × 3) for 4 hours (Bansal et al., 2011). The filtered solution was concentrated under reduced pressure at a temperature below 45°C using a rotary evaporator (DLAB Re 100-Pro, USA). The crude extract was characterized by a dark green color, a uniform consistency, and a mild aroma. The extraction yield (%) was calculated as the ratio of the mass of the dried extract to the initial mass of the material, whereas the moisture content (%) was determined using a moisture analyzer (OHAUS-MB23). The extraction yield and moisture content of the ethanolic extract of P. microphylla were 8.33% and 7.9 ± 0.4%, respectively. Finally, the crude extract was stored at 4°C for use in determining chemical composition and evaluating biological activity.

Chemical compositions

Phytochemical screening

A preliminary qualitative phytochemical screening was performed to identify phytochemical compounds (polyphenols, flavonoids, tannins, coumarins, triterpenes, steroids, saponins, cardiac glycosides, alkaloids, organic acids, essential oils, carbohydrates, and fatty acids) from the crude extract of P. microphylla whole plants according to the standard protocols (María et al., 2018; Mannoubi et al., 2025; Okeke et al., 2026).

Isolated phytochemical compounds retrieved from the literature

The relevant information on chemical profiling of this plant was gathered from various scientific databases, including Web of Science, PubMed, and Google Scholar. The keywords used included “Pilea microphylla” and “phytochemistry”, or “phytocompounds”, or “chemical compounds”.

Analysis of the chemical compositions by gas chromatography-mass spectrometry (GC-MS) 

The chemical constituents of P. microphylla extract were analyzed by GC-MS employing the Shimadzu GC-MS-QP2010 Plus system (Kyoto, Japan) with an Equity-5 capillary column (30 m length × 0.25 mm internal diameter × 0.25 μm film thickness) for the separation. P. microphylla extract (1.0 mg) was diluted with dichloromethane (ratio 1:100). The ion source, interface, and GC’s injector temperatures were set at 280°C. The GC oven temperature was held at 60°C for 2 min, then ramped to 240°C at a rate of 3°C/min (10 min hold) and subsequently increased to 280°C at 5°C/min (40 min hold). The electron ionization energy was 70 eV. The scan range of the MS was set at 40–500 m/z with a scan rate of 1 scan/s. Helium was used as a carrier gas at a constant flow of 1.5 mL/min. The injected volume was 1.0 μL with splitless injection mode. The retention indices (RIs) were calculated using the retention times of alkanes (C₈–C₃₈, Merck). The chemical compositions were tentatively identified by comparison with standard samples in the Wiley 7, NIST 11, Adams Book databases (Adams, 2007), and in the literature. The data were quantified using the relative peak area percentage of the total ion chromatogram (TIC) peak (Borah et al., 2019; Likittrakulwong et al., 2023). The formula used to calculate RI was:

RI= 100 × [n + (N-n) × [logRT(unknown) - logRT(n)]/[(logRT(N) - logRT(n)]]               (1)                      

Where, RI = retention index determined with reference to homologous series of n-alkanes (C₇–C₃₀); RT = retention time of the respective component; N = no. of carbon atoms in the larger alkane; n = no. of carbon atoms in the smaller alkane (Borah et al., 2019).

XO inhibition assay

The XO inhibitory activity of P. microphylla ethanolic extract was evaluated spectrophotometrically under aerobic conditions, following reported guidance by Umamaheswari et al. (2007) and Chen et al. (2009). The reaction mixture consisted of 35 μL of 0.1 mM phosphate buffer (pH 7.5), 30 μL of XO solution (0.01 IU/mL), and 50 μL of sample solutions at different concentrations (12.5–200 μg/mL). This mixture was pre-incubated for 15 minutes at 25°C. Continuously, 60 μL of xanthine (150 mM) was added and incubated at 25°C for 30 minutes. Finally, the reaction was stopped by the addition of 25 μL of 1N hydrochloric acid. The absorbance (A) of the reaction mixture was measured at 290 nm using the ELISA Plate Reader (Biotek). Phosphate buffer, negative well (the reaction mixture without the test sample), and allopurinol were used as blank control (A_blank), negative control (A_negative control), and positive control, respectively. One unit of XO was defined as the amount of enzyme required to produce 1.0 mmol of uric acid/minute at 25°C. IC₅₀ values were calculated from the mean values of data from triplicate determinations by using Microsoft Excel 2023. The XO inhibitory activity is expressed by the following formula:

LPS-induced inflammation model of RAW264.7 cells

Measurement of nitric oxide (NO) production

The anti-inflammatory activity of the ethanolic extract of P. microphylla was evaluated by inhibiting nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages using previously described methods with slight modifications (Van Chen et al., 2025; Ayub et al., 2026). Murine macrophage RAW264.7 cells were obtained from the American Type Culture Collection (ATCC, TIB-71; RRID: CVCL_0493) and cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 1.0 mM sodium pyruvate, 2.0 mM L-glutamine, 10 mM HEPES, and 10% fetal bovine serum (Sigma, USA) at the Biotechnology Center of Ho Chi Minh City, Vietnam. Every three days, cells were cultured at 37°C with 5% CO₂ and re-cultured at a ratio of 1:3. For the test, RAW 264.7 cells were cultured in 96-well plates at a density of 2 × 10⁵ cells per well and incubated for 24 hours. Before treatment, the cells were pre-incubated for 2 hours. Subsequently, cells were treated with the extract (25.0–150 µg/mL) in LPS at a final concentration of 0.5 µg/mL, and further incubated for 24 hours at 37°C. Nitrite production was then measured using a nitric oxide detection kit (Griess Reagent System, Promega, WI, USA). The positive and negative controls were diclofenac sodium (Lek Pharmaceuticals D.D., Slovenia; concentration range of 1.563–25.0 μg/mL) and dimethyl sulfoxide (1% DMSO), respectively. Absorbance was measured at 540 nm using a microplate reader (VersaMax, Molecular Devices, USA). A sodium nitrite standard curve was used to determine the nitrite concentrations in the samples. The inhibitory activity was expressed as IC₅₀ values, which were calculated using Microsoft Excel 2023 software.

Cell viability assessment

The cytotoxicity of the extract toward RAW 264.7 cells was performed using the MTT assay as previously reported by Van Chen et al. (2025) and Ayub et al. (2026). In short, 10 µL of MTT solution (5 mg/mL) was added to each well to achieve a final concentration of 0.5 mg/mL. After 4 hours incubation, the culture medium was carefully removed, and formazan crystals were dissolved in 100 µL of DMSO. Absorbance (OD) was measured at a wavelength of 540 nm using an ELISA microplate reader (VersaMax, Molecular Devices, USA). Cell viability (%) was calculated using the following formula:

% Cell viability = (OD of treated sample / OD of untreated control) × 100 (%)                             (3)

Molecular docking

To gain molecular-level insights into how the phytochemicals from P. microphylla (identified through GC-MS and extensive literature review) interact with key pathological targets, we performed in silico docking simulations. This study focused on three primary enzymes central to gout (XO) and inflammatory (Inducible Nitric Oxide Synthase (iNOS) and Interleukin (IL)-1β) responses. The crystallographic coordinates for the biological targets were sourced from the RCSB Protein Data Bank (https://www.rcsb.org/). We selected the human iNOS oxygenase domain (PDB ID: 3E7G) (Garcin et al., 2008), IL-1β (PDB ID: 8C3U) (Hommel et al., 2023), and bovine xanthine oxidase (PDB ID: 3NVY) (Cao et al., 2014) as the docking models. To ensure the catalytic integrity of the active sites, the molybdenum molybdopterin (Mo-pt) cofactor was retained for XO, while the heme group and tetrahydrobiopterin (BH4) cofactor were preserved for the iNOS structure.

The iNOS structure represents a catalytic pocket defined by residues such as Glu377, Tyr347, Gln263, Arg266, and Arg388. The IL-1β model features an allosteric site where the binding environment is stabilized by hydrogen bonds with Lys93 and Met95, alongside extensive hydrophobic contacts with Val3, Val47, Pro57, and Val100. Preparation of the protein receptors involved the systematic removal of non-essential water molecules and co-crystallized ligands, followed by the addition of polar hydrogens to reflect proper protonation states.

The chemical library, comprising compounds from both experimental GC-MS data and screened literature, was sourced from PubChem. These structures were converted into the .pdbqt format, with rotatable bonds assigned to allow for ligand flexibility during the simulation. Molecular docking were conducted using AutoDock Vina 1.2.3 (Trott and Olson, 2010; Eberhardt et al., 2021), the grid box for each simulation was carefully calibrated to encompass the established active or allosteric sites. Prior to the main screening, the docking protocol was calibrated by re-docking the native co-crystallized ligands into their respective active sites (PDB IDs: 3E7G, 8C3U, and 3NVY). The protocol's accuracy was verified by calculating the Root Mean Square Deviation (RMSD) between the re-docked and original crystal poses. The calculated RMSD values between the re-docked and native poses are 0.4623 Å (3E7G), 1.1191 Å (8C3U), and 1.0159 Å (3NVY). Since all values are well below the 2.0 Å threshold, the protocol’s reliability is confirmed. After removing duplicate entries, the final library of 105 compounds was used for docking studies.

ADME and drug-likeness prediction

The absorption, distribution, metabolism, excretion (ADME), and toxicity profiles and drug-likeness predictions for the 105 phytocompounds were evaluated using the ADMETlab 2.0 platform (https://admetmesh.scbdd.com). Canonical SMILES of the constituents served as inputs. Drug-likeness was assessed using Lipinski’s Rule of Five and the pan-assay interference compounds (PAINS) filter. Key pharmacokinetic and toxicity parameters were predicted.

Statistical analysis

All experiments were performed in triplicate with three technical replicates (n=3) per sample, and the results are presented as the mean ± standard deviation (SD). IC₅₀ values were determined by Microsoft Excel 2021. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test in Minitab 20 software, with statistical significance set at P ≤ 0.05.

RESULTS

Chemical profiling

P. microphylla crude extract was phytochemically evaluated through simple qualitative screening assays. As a result, this extract revealed the presence of secondary metabolites, including polyphenols, flavonoids, coumarins, tannins, triterpenes, steroids, alkaloids, organic acids, essential oils, carbohydrates, and fatty acids; however, it also revealed the absence of cardiac glycosides and saponins.

Literature screening reveals that the isolated phytochemical compounds of P. microphylla comprise a series of flavonoid derivatives, other phenols, steroids, triterpenoid, and essential oils (Bansal et al., 2011; Isaza Martínez et al., 2016; Singh et al., 2022; Linh et al., 2024). P. microphylla is a rich source of natural products with diverse chemical compositions. To date, a total of approximately 45 metabolites have been separated and identified from P. microphylla (Table 1). Therefore, the multiple pharmacological effects of P. microphylla may be attributed to its bioactive compounds.

Table 1. Chemical compositions of P. microphylla screened from literature.

GC-MS analysis

GC-MS analysis of P. microphylla extract reveals diverse chemical components (Figure 2, Table 2), with 62 identified compounds representing 95.4% of the total extract. The major chemical constituents identified were β-sitosterol (14.3%), cis-phytol (13.1%), tetrahydroedulan B (12.6%), hentriacontane (5.8%), and neophytadiene (5.6%).

Figure 2. Gas chromatogram of the ethanolic extract of P. microphylla whole plants and chemical structures of the major compounds.

Table 2. Chemical compositions of the ethanolic extract from P. microphylla whole plants.

Note: RT (min): Retention time in minutes; RI (E.): Experimental retention index; RI (L.): Corresponding retention index from the literature; MS: Mass spectrometry; SI: Similarity index.

XO inhibitory activity

In this study, the ethanolic extract of P. microphylla demonstrated moderate XO inhibitory activity with IC₅₀ values of 74.12 ± 1.58 μg/mL (y = 22.067ln(x) – 45.014; R² = 0.9886), indicating its potential as a natural uric acid reducing agent. This was significantly different compared to the allopurinol (control sample) with IC₅₀ = 1.11 ± 0.02 μg/mL (P < 0.05). The results are shown in Table 3.

Table 3. XO inhibitory activity of the ethanolic extract from P. microphylla whole plants.

Note: IC₅₀: Half-maximal inhibitory concentration; Mean ± SD, n=3. Different superscript letters (^a and ^b) within the same row indicate statistically significant differences at P < 0.05.

NO production inhibitory activity

P. microphylla ethanolic extract was analyzed for its anti-inflammatory activity using an LPS-induced inflammation model in RAW264.7 cells. This result is presented in Table 4. A strong association between concentration and the percentage of inhibition of NO production was observed. As a result, the activity of P. microphylla ethanolic extract (IC₅₀ = 61.29 ± 1.79 µg/mL; y = 40.698ln(x) – 117.5; R² = 0.9855) against the inhibition of NO production was found to be lower than that of diclofenac sodium (IC₅₀ = 2.43 ± 0.46 µg/mL). Besides, a statistically significant difference was observed between these samples (P < 0.05).

Table 4. NO production inhibitory activity of the ethanolic extract from P. microphylla whole plants.

Note: IC₅₀: Half-maximal inhibitory concentration; Con.: Concentration; Different superscript letters (a and b) within the same row indicate statistically significant differences at P < 0.05.

Molecular docking study

In this study, in silico approaches were applied to two inflammation-related targets (iNOS and IL-1β) to identify potential compounds and generate hypotheses regarding their molecular mechanisms based on molecular docking. The molecular docking simulations revealed that compounds from P. microphylla exhibit diverse binding affinities and high selectivity toward specific biological targets. The binding affinities of identified compounds against iNOS, IL-1β, and XO are summarized in Table 5 and Table S1.     

Table 5. Binding affinity (kcal/mol) of the major compounds from P. microphylla against iNOS (3E7G), IL-1β (8C3U), and xanthine oxidase (3NVY) targets.

ADME profiling and drug-likeness of phytocompounds

The ADME analysis provided strong theoretical justification for the pharmacological potential of the 105 screened phytocompounds (Supplementary Table S1). The evaluation revealed an exceptional drug-likeness profile, with 101 out of 105 compounds (96.2%) successfully complying with Lipinski’s Rule of Five (allowing for a maximum of one violation). Only four bulky flavonoid glycosides (Luteolin-7-O-glucoside, Isorhoifolin, Rutin, and Diosmetin-7-O-rutinoside) exhibited two or three violations, primarily due to their high molecular weights and numerous hydrogen bond donors/acceptors; nevertheless, these compounds demonstrated the most potent docking affinities. Furthermore, a substantial proportion of the smaller constituents, particularly monoterpenes, sesquiterpenes, and simple aglycone flavonoids (e.g., Quercetin and Kaempferol), displayed high predicted gastrointestinal (GI) absorption. These robust pharmacokinetic profiles reinforce the validity of the selected hits and justify the large-scale computational screening approach.

DISCUSSION

Chemical profiling

Compared with the previous report, our Vietnamese sample contained a markedly lower amount of (E)-caryophyllene (0.2%) than that reported for the Uttarakhand population (48.1%) (Singh et al., 2022). Additionally, the chemical components identified in the Vietnamese P. microphylla ethanolic extract differed in constituents from those reported for the P. microphylla essential oil (Singh et al., 2022). These findings indicate significant chemical variability, which may be influenced by geographical origin and extraction method (ethanolic extract vs. essential oil). In other words, the content and chemical composition of identified constituents from this species can vary considerably depending on various environmental factors, including the plant part used, developmental stage, maturity, harvest time, and genetic heritage. In particular, β-sitosterol, a bioactive phytosterol, was identified in the P. microphylla extract, with a notable abundance (14.3%). This compound has also been previously reported in P. microphylla whole plant (Linh et al., 2024). However, other major constituents, including cis-phytol, tetrahydroedulan B, hentriacontane, neophytadiene, and cholestanol, are reported herein for the first time from P. microphylla. Moreover, P. microphylla ethanolic extract revealed several different long-chain aliphatic hydrocarbons and fatty acid derivatives, such as nonacosane, hentriacontane, squalene, isoamyl dodecanoate, neophytadiene, methyl linoleate, methyl linolenate, ethyl palmitate, methyl palmitate, etc. It is noteworthy that these constituents play significant roles in various biological activities and have been reported for their diverse potential pharmacological benefits, particularly anti-inflammatory and antimicrobial activities (Yore et al., 2014; Nyemb et al., 2018). The presence of triterpenoid (lupeol) and steroid (β-sitosterol) in the ethanol extract further underscores its significance in phytochemical studies. These phytocompounds were renowned for their antioxidant, anti-XO, antidiabetic, anticancer, anti-inflammatory, antibacterial, acetylcholinesterase inhibitory activities (Siddique and Saleem, 2011; Munvera et al., 2021; Khan et al., 2022; Nyemb et al., 2022; Adhimoolam et al., 2024; Bindebe et al., 2025; Diguir et al., 2025), highlighting the species’ potential for medicinal use (Wilairatana and Thiruvengadam, 2022).

Although diisooctyl phthalate and other phthalate derivatives are components of plasticizers, they have also been reported in some plant extracts, such as Allium fistulosum L., Brassica oleracea L., Campanula colorata Wall., Cirsium japonicum DC., Dalbergia odorifera T.C. Chen, Lilium brownii Lehm, Myrica rubra (Lour.) Siebold & Zucc., Eichhornia crassipes (Mart.) Solms, Solanum lycopersicum L., Nephelium lappaceum L. (Huang et al., 2021), Kaempferia champasakensis Picheans. & Koonterm (Hieu et al., 2023), and Curcuma sahuynhensis Škorničk. & N.S. Lý (Tran et al., 2024), etc. Therefore, these components were also detected in the whole plant of P. microphylla. However, further confirmatory analyses are necessary to verify their origin in future studies.

XO inhibitory activity

XO is a crucial enzyme responsible for uric acid production in the liver, and inhibition of this enzyme represents an established therapeutic approach for the treatment of gout and related disorders (Kim et al., 2017). Chemical profiling showed that β-sitosterol, cis-phytol, tetrahydroedulan B, neophytadiene, and hentriacontane are the main constituents. These compounds may notably contribute to the diverse bioactivity of the extract, as they together accounted for more than half of the identified components. Indeed, β-sitosterol exhibited greater binding interactions than the reference XO inhibitor febuxostat by interacting with key amino acid residues within the XO structure, according to the docking analysis (Pothipongsa et al., 2024). In addition, XO is considered a major source of reactive oxygen species (ROS) produced during the synthesis of uric acid, thereby contributing to cellular damage caused by oxidative stress (Jomova et al., 2023). Thus, compounds with both XO inhibitory and antioxidant properties may be considered prospective treatments for treating hyperuricemia and its related diseases (Dhammaraj et al., 2024). In this case, phytol has been proved to exhibit significant antioxidant activity via hydroxyl radical scavenging, which may assist in reducing oxidative DNA damage (Islam et al., 2018). Besides, studies on XO inhibition in P. microphylla and Urticaceae species remain limited; however, they are known to contain a variety of phenolic compounds, particularly flavonoids such as quercetin, kaempferol, rutin, luteolin, and apigenin, which have been reported to inhibit XO activity (Fathimathul Nisa and Aswathi, 2023; Xue et al., 2023).

In a screening study of 288 extracts from 96 traditional medicinal plants used in Vietnam for the treatment of gout and related symptoms, 188 extracts exhibited XO inhibitory activity at 100 μg/mL, of which 46 showed more than 50% inhibition. At 50 μg/mL, 168 extracts showed inhibitory activity, of which 21 exhibited more than 50% inhibition (Nguyen et al., 2004). In comparison, the IC₅₀ value obtained for P. microphylla (IC₅₀ = 74.12 ± 1.58 μg/mL) indicates an effective inhibitory ability at lower concentrations than those of the other plants. Overall, these findings highlight P. microphylla as a promising natural source of XO inhibitors and provide a foundation for future research to identify active compounds and clarify their anti-hyperuricemic properties.

NO production inhibitory activity

Previous studies have shown that the majority of naturally occurring compounds with anti-inflammatory properties belong to flavonoids, phenolics (Serafini et al., 2010; Zhang et al., 2011), steroids (Bakrim et al., 2022), etc. From the literature and GC-MS results, β-sitosterol appeared as the major bioactive constituent in plant samples from our study and a previous study by Linh et al. (2024). Earlier studies have reported significant anti-inflammatory effects of β-sitosterol in experimental models. In J774A1 macrophages induced by LPS, β-sitosterol increased the activity of anti-inflammatory factor IL-10, decreased the activities of chemokines and pro-inflammatory factors, enhanced the activity of tyrosine phosphatase, and inhibited the migration of NF-κB (Valerio and Awad, 2011). In a study by Liao et al. (2018), β-sitosterol exhibited significant anti-inflammatory activity by inhibiting the secretion of pro-inflammatory mediators in keratinocytes and macrophages stimulated by PGN, TNF-α, or LPS, including TNF-α, IL-1β, IL-6, IL-8, and ROS. In addition, it reduced the expression of NLRP3, a key component of the inflammasome complex, suppressed caspase-1 activation, and partially inhibited NF-κB signaling in macrophages (Liao et al., 2018). Zhang et al. (2023) demonstrated that β-sitosterol reduced ROS production in a CuSO₄-induced inflammation model in zebrafish and increased the expression of sod and gpx4b to inhibit oxidative stress. It inhibited neutrophil migration to the lateral line and decreased the expression of inflammatory genes (IL-8 and MyD88) to suppress inflammation (Zhang et al., 2023). These findings suggest that β-sitosterol may contribute to the anti-inflammatory activity of P. microphylla ethanolic extract.

In addition to steroids, the anti-inflammatory activity of P. microphylla extract may also be attributed to phytocompounds, such as flavonoids (e.g., chlorogenic acid, rutin, kaempferol, quercetin, etc.) and other constituents. Several in vitro and in vivo studies have reported that chlorogenic acid, rutin, quercetin, and kaempferol have significant anti-inflammatory activities. Hwang et al. (2014) demonstrated that chlorogenic acid exhibited significant anti-inflammatory activity by inhibiting nitric oxide (NO) production and suppressing COX-2 and iNOS expression. It also reduced pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) and inhibited the nuclear translocation of NF-κB (Hwang et al., 2014). In 2023, Huang et al. reported that chlorogenic acid executed anti-inflammatory functions by regulating the synthesis and secretion of inflammatory mediators (TNF-α, IL-1β, IL-6, IL-8, NO, and PGE2), as well as modulating key signaling pathways and related factors (NF-κB, MAPK, and Nrf2), protecting cells and tissues against inflammation-related diseases, such as cardiovascular disease, cerebrovascular disease, and diabetes mellitus (Huang et al., 2023). In a report by García-Mediavilla et al. (2007), quercetin and kaempferol significantly reduced the protein and mRNA levels of iNOS, COX-2, and CRP. They also inhibited NF-κB activation and decreased the protein levels of phosphorylated IκBα and IKKα. These findings suggest that the anti-inflammatory activities of quercetin and kaempferol in Chang Liver cells may be related to the suppression of the NF-κB signaling pathway (García-Mediavilla et al., 2007). In RAW 264.7 cells, quercetin and rutin significantly reduced NO levels and the expression of COX-2 and TNF-α, suggesting their potential anti-inflammatory mechanisms (Lee et al., 2024). Moreover, the anti-inflammatory properties of rutin and quercetin were previously evaluated in mice using both acute and chronic inflammation models. The results showed that intraperitoneal administration of rutin and quercetin at daily doses of 80 mg/kg inhibited both phases of inflammation, with rutin exhibiting stronger activity in the chronic phase (Guardia et al., 2001).

The P. microphylla extract showed moderate inhibition of NO production, suggesting its anti-inflammatory potential in the LPS-induced inflammation model in RAW264.7 cells; therefore, further research is needed to explore its mechanism of action and isolate its active ingredients.

Molecular docking study

During the inflammatory response, several key indicators can represent different levels of inflammation. Numerous studies suggest that certain pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-2, IL-6, and IL-8, are involved in pathological inflammation (Shaikh, 2011; Kany et al., 2019; Al-Qahtani et al., 2024). Immunostimulatory cytokines or bacterial pathogens activate iNOS through the activation of inducible nuclear factors, including NFkB signaling, resulting in the production of high amounts of nitric oxide (NO)–a key marker and mediator of inflammatory responses (Aktan, 2004).

For the two protein targets associated with inflammatory responses, compounds belonging to the flavonoid glycoside group demonstrated absolute dominance in binding energy. Isorhoifolin and Diosmetin-7-O-rutinoside emerged as the most promising candidates, with docking scores on iNOS of -11.3 and -10.7 kcal/mol, respectively. In the iNOS binding site (PDB ID: 3E7G), both established a dense network of hydrogen bonds with key residues, including Gln263, Tyr347 (with an exceptionally short distance of 1.57 Å), Arg381, and Arg388. Notably, they formed strong interactions (Hydrogen bonding, Pi-Sigma, and Pi-Pi T-shaped) with the Heme group (HEM901), effectively blocking the enzyme's catalytic center Figure 3. Furthermore, at the IL-1β target (PDB ID: 8C3U), Diosmetin-7-O-rutinoside formed a Pi-Cation interaction with Lys94 and hydrogen bonds with Lys97, Asn102, Ser45, while Isorhoifolin was stabilized by hydrogen bonds with Asn102, Ser114 and hydrophobic interactions with Pro57 and Val100.

Notably, Steroids such as Cholest-4-ene-3,6-dione displayed multi-target inhibitory potential, achieving impressive scores on both iNOS (-9.2 kcal/mol) and IL-1β (-7.7 kcal/mol). At the iNOS site, Cholest-4-ene-3,6-dione formed a hydrogen bond with Gln387 and established a system of Alkyl and Pi-Alkyl interactions with Pro350, Tyr347, and Tyr373, along with a Pi-Sigma interaction with HEM901. At the IL-1β site, it bound via a hydrogen bond with Lys55 and hydrophobic interactions with Lys94 and Val100 (Figure 4).

Figure 3. Binding modes and 2D interaction diagrams of flavonoid glycosides with iNOS and IL-1β.

Figure 4. Binding modes and 2D interaction diagrams of cholest-4-ene-3,6-dione with iNOS and IL-1β.

In contrast to the trends observed with inflammatory cytokines, the XO target (PDB ID: 3NVY) favored compounds with less bulky structures and higher hydrophobicity. Squalene exhibited the strongest affinity (-10.0 kcal/mol). Interaction analysis showed that Squalene fills the hydrophobic pocket of XO through an extensive network of Alkyl and Pi-Alkyl interactions with Leu648, Phe649, Leu873, His875, Phe914, Phe1009, Val1011, Leu1014, and Ala1079. Specifically, the Pi-Sigma interaction with Phe914 plays a crucial role in anchoring the long hydrocarbon chain of Squalene within the catalytic center. Vitamin E derivative group showed similar interaction patterns. DL-α-Tocopherol (-9.6 kcal/mol) was stabilized by a Pi-Pi T-shaped interaction with Phe649, while γ-Tocopherol (-9.3 kcal/mol) established additional critical hydrogen bonds with Ser876 and Glu802, combined with Pi-Pi Stacked/T-shaped interactions with Phe914 and Phe1009 (Figure 5).

Figure 5. Visualization of squalene and tocopherols within the XO catalytic site.

An interesting finding was that flavonoid glycosides (such as Diosmetin-7-O-rutinoside), despite being potent anti-inflammatories, showed poor results on XO (scores > -5 kcal/mol) due to steric hindrance in the deep pocket. Conversely, flavonoid aglycones such as Quercetin (-8.4 kcal/mol) and Kaempferol (-8.3 kcal/mol) maintained good inhibitory activity on this target.

By integrating traditional knowledge, phytochemical evidence, experimental studies, and computational analyses, this work provides scientific evidence for the XO-inhibitory and anti-inflammatory effects of P. microphylla.

While this molecular docking study provides valuable structural insights, it relies on static protein conformations. Due to the large-scale nature of the initial screening, molecular dynamics (MD) simulations and binding free-energy calculations were not performed to assess the dynamic stability of the complexes. Therefore, the docking results reported herein should be considered strictly predictive and exploratory. Future studies incorporating MD simulations and targeted experimental validations are required to conclusively establish the underlying mechanism of action.

CONCLUSION

This study presents a comprehensive phytochemical profile of P. microphylla extracts, targeting both volatile and non-volatile secondary metabolites based on GC–MS analysis and previously reported literature, respectively. A total of 62 chemical components and 45 secondary metabolites were identified, revealing a diverse range of bioactive compositions in the extract. The XO inhibitory and NO production inhibition assays showed that the ethanolic extract exhibited notable anti-XO (IC₅₀ = 74.12 ± 1.58 μg/mL) and anti-inflammatory activities (IC₅₀ = 61.29 ± 1.79 μg/mL). These bioactivities are attributed to the richness of secondary metabolite compounds detected in this species. Molecular docking studies examined the binding interactions between phytochemical compounds and the active sites of iNOS (PDB ID: 3E7G), IL-1β (PDB ID: 8C3U), and XO (PDB ID: 3NVY), with binding energies ranging from -5.4 to -11.3 kcal/mol, -5.0 to -8.8 kcal/mol, and -5.0 to -10.0 kcal/mol, respectively, suggesting the anti-XO and anti-inflammatory potential of this species. These findings provide experimental support for the traditional use of P. microphylla and contribute to a better understanding of its application in folk medicine for inflammatory and gout-related diseases.

ACKNOWLEDGEMENTS

This research was funded by the University of Medicine and Pharmacy at Ho Chi Minh City under contract number 469/2025/HĐ-ĐHYD, dated 30/9/2025.

AUTHOR CONTRIBUTIONS

Tran Thi Thuy Quynh: Conceptualization (Lead), Data Curation (Lead), Formal Analysis (Equal), Investigation (Equal), Project Administration (Lead), Funding Acquisition (Lead), Writing – Original Draft (Lead); Vo Linh Tu: Data Curation (Equal), Software (Lead), Validation (Equal), Investigation (Equal); Huynh Thanh Duy: Data Curation (Equal), Formal Analysis (Equal), Supervision (Equal), Writing – Review & Editing (Equal); Nguyen Duc Huy: Data Curation (Supporting), Formal Analysis (Supporting), Writing – Original Draft (Supporting); Tran Van Chen: Conceptualization (Equal), Formal Analysis (Lead), Project Administration (Equal), Funding Acquisition (Equal), Investigation (Lead), Supervision (Lead), Validation (Equal), Visualization (Equal), Writing – Review & Editing (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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Supplementary

Exploring the phytochemical composition and biological activities of Pilea microphylla (L.) Liebm. (Urticaceae): A combined experimental and in silico molecular docking investigation.

Figure S1. A voucher specimen (No. TVC-LT06.25) of Pilea microphylla (L.) Liebm. (Urticaceae) collected in Vietnam.

Table S1. Binding affinities (kcal/mol) of P. microphylla phytocompounds against iNOS (3E7G), IL-1β (8C3U), and xanthine oxidase (3NVY) targets, and their corresponding ADME profiles.