Formulation and Characterization of Sonchus arvensis L. Nanosuspension for Enhanced Antioxidant and Lipid-Lowering Activities

Introduction

Hyperlipidemia is a medical disorder characterized by elevated lipid or fat levels in the bloodstream. These lipids, primarily cholesterol and triglycerides, play vital roles in maintaining numerous physiological functions. However, when present in excessive amounts, they can contribute to a heightened risk of cardiovascular conditions, including atherosclerosis, heart attacks, and stroke. Various medications have shown promise in lowering serum cholesterol. Ezetimibe combined with statins has yielded moderate benefits, whereas bempedoic acid has demonstrated a 15% LDL cholesterol reduction in trials. For patients with persistently high lipid levels despite statin therapy, FDA-approved PCSK9 inhibitors are now recommended. Newer options, such as inclisiran, an siRNA therapy administered biannually, show the potential to improve compliance, especially when used with pelacarsen, which targets lipoprotein (a). Additionally, Volanesorsen and its successor olezarsen target chylomicrons to reduce triglyceride levels, and ANGPTL3 inhibitors, such as evinacumab, have been approved for familial hypercholesterolemia, highlighting significant advances in lipid-lowering treatments.¹

Herbal remedies are viable alternatives to commercially available cholesterol-lowering medications, particularly when cardiovascular illnesses are the leading cause of mortality.^2,3 CVD risk stratification should be considered when selecting the appropriate medications. The risk is determined by age, severity, and presence of additional personal or familial CVD risk factors. Lifestyle changes are recommended, with a particular incidence of dietary treatment for 3–6 months before pharmacological therapy reassessment and decision-making.^4,5

In recent years, there has been a growing interest in the therapeutic potential of natural products and plant-based compounds.^6,7 Natural compounds derived from medicinal plants have demonstrated significant promise in treating various ailments due to their bioactive constituents, which offer a wide range of pharmacological activities with minimal side effects compared to synthetic drugs. These natural products are often rich in polyphenols, flavonoids, and alkaloids, which contribute to their therapeutic efficacy. Sonchus arvensis L., commonly known as field sow thistle, is a vascular plant belonging to the phylum Tracheophyta and the class Magnoliopsida (dicotyledons). It is systematically classified under the order Asterales, within the family Asteraceae (daisy family), and the genus Sonchus. It is a medicinal plant that has been used in traditional medicine because of its diverse pharmacological properties, including antioxidant, anti-inflammatory, and hypolipidemic effects.^8,9 The plant has been reported to exhibit additional pharmacological benefits, such as antimicrobial, hepatoprotective, nephroprotective, and antidiabetic properties. The presence of bioactive compounds, including flavonoids, sesquiterpene lactones, and phenolic acids, enhances its medicinal value, making it a potential candidate for novel drug formulations.

These characteristics make Sonchus arvensis L. a promising candidate for developing innovative drug delivery systems. Unlike many other herbal formulations, which suffer from issues such as poor solubility, rapid degradation, and inconsistent bioavailability, Sonchus arvensis L. has been recognized for its high stability and potent bioactivity, making it particularly well-suited for advanced pharmaceutical applications. Building on the therapeutic potential of Sonchus arvensis L.¹⁰

The development of poorly soluble drugs presents a major challenge in the pharmaceutical industry because of their limited solubility and bioavailability, which can significantly reduce their therapeutic efficacy.^11,12 One promising strategy for addressing these limitations is the formulation of nanosuspensions.¹³ Nanosuspensions are colloidal dispersions of submicron-sized drug particles stabilized by surfactants, which enhance the solubility, stability, and bioavailability of poorly soluble drugs.¹⁴ By reducing the particle size to the nanometer range (<100 nm),¹⁵ nanosuspensions increase the surface area available for dissolution, which improves both the rate and extent of drug absorption. Additionally, these formulations can be designed to achieve controlled and sustained drug release, thereby improving patient compliance and therapeutic outcomes.^16–19 Sonchus arvensis L.-based nanosuspensions offer several advantages over other herbal nanosuspensions. First, its strong antioxidant and anti-inflammatory properties provide synergistic therapeutic effects, enhancing the overall efficacy of the nanosuspension. Second, its diverse range of bioactive compounds allows for multifaceted therapeutic applications, targeting multiple pathways in disease treatment. Furthermore, the inherent stability and low toxicity profile of Sonchus arvensis L. make it a superior choice for nanosuspension formulations, ensuring better safety and efficacy for long-term use. These advantages underscore its potential as a groundbreaking component of modern herbal nanomedicine. This study aimed to formulate and evaluate a nanosuspension-based drug delivery system utilizing Sonchus arvensis L.

Materials and Methods

Materials

Sonchus arvensis L. folium were harvested by the farmer from Cikanyere village, Sukanyere subdistrict, Cianjur, Indonesia. A voucher specimen (Herbarium No: 642) was deposited at the Herbarium Jatinangoriense, Biosystematics and Molecular Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran. The materials used in this investigation were distilled water, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Lieberman-Burchard reagent, phosphate buffer saline (PBS), acetic anhydride, aluminum chloride (AlCl₃), anhydrous sodium sulfate, chloroform, 70% ethanol, ferric chloride (FeCl₃), gallic acid, hydrochloric acid (HCl), potassium dihydrogen phosphate (KH₂PO₄), potassium acetate (CH₃CO₂K), polysorbate 80 (Tween 80^®), quercetin, sodium carbonate (Na₂CO₃), sodium hydroxide (NaOH), Folin-Ciocalteu reagent, and Dragendorff’s reagent, all sourced from Sigma-Aldrich Co., St. Louis, MO, USA.

Methods

This study was conducted in four stages. Initially, an extract of Sonchus arvensis L. was obtained and subjected to phytochemical screening. This was followed by the formulation of a nanosuspension and its characterization, including particle size analysis (PSA), polydispersity index (PI), and zeta potential measurements. Short-term stability evaluation was conducted for 28 days, after which transmission electron microscopy (TEM) was used for further analyses. The study concluded with an in vitro release assessment and evaluation of the antioxidant and antihyperlipidemic properties of the prepared nanosuspensions.

Extraction and Phytochemical Screening of Sonchus arvensis L.

The extract of Sonchus arvensis L. was prepared using the maceration technique. Initially, 1.5 kg of sifted Sonchus arvensis L. powder was added. Leaves prepared using a no. 40 mesh sifter were accurately weighed. This powder was then extracted with 16 liters of 70% ethanol solvent over three days. The extraction process was organized into three separate maceration vessels with intermittent stirring to prevent saturation. The resulting filtrate was concentrated using a rotary evaporator at 60°C. Subsequently, the concentrated extract was evaporated in a water bath to obtain a viscous extract was achieved.²⁰

Phytochemical Screening of Sonchus arvensis L. Extract

In this study, phytochemical screening was performed to determine the presence of alkaloids, flavonoids, tannins, and saponins in the extracts. Each extract exhibited positive indicators, including sediment and foam formation and color changes.

Measurement of Total Flavonoid Content (TFC)

The Total Flavonoid Content (TFC) was determined following the guidelines outlined in the Indonesian Herbal Pharmacopeia 2^nd edition 2017. The procedure involved preparing both the sample extract and standard quercetin solution. For the sample preparation, approximately 0.2 g of the extract was accurately weighed and placed into an Erlenmeyer flask, followed by the addition of 25 mL of ethanol P. The mixture was stirred for 30 min using a magnetic stirrer to ensure thorough extraction. Subsequently, the solution was filtered into a 25-mL volumetric flask, and ethanol P was added to reach the final volume.

For the preparation of the standard solution, approximately 10 mg of quercetin was weighed and dissolved in ethanol P in a 25-mL volumetric flask. A series of dilutions were prepared to obtain standard solutions with concentrations of 100, 75, 50, and 25 mg/mL, which were used to generate the calibration curve.

In the test procedure, 0.5 mL of both the test and standard solutions were separately pipetted into vials. To each vial, 1.5 mL of ethanol P, 0.1 mL of 10% aluminum chloride P, 0.1 mL of 1 M sodium acetate, and 2.8 mL of water were added. The mixture was then shaken and allowed to stand for 30 min at room temperature to allow for color development. The absorbance of each solution was measured at the maximum absorption wavelength, and a blank solution prepared without the addition of aluminum chloride was used as a reference. Finally, a calibration curve was constructed using standard quercetin solutions, and the flavonoid concentration in the test sample was determined based on this curve.

Measurement of Total Phenolic Content (TPC)

The Total Phenolic Content (TPC) was determined using a standardized procedure to ensure accuracy and reproducibility. The process began with the preparation of the sample extract, where approximately 0.2 g of the extract was accurately weighed and placed in an Erlenmeyer flask. To facilitate extraction, 25 mL of methanol P was added, and the mixture was stirred for 30 min using a magnetic stirrer. Following extraction, the solution was filtered into a 25-mL volumetric flask, and methanol P was added to obtain the final volume.

For the preparation of the standard solution, approximately 10 mg of gallic acid was weighed and dissolved in methanol P in a 25-mL volumetric flask. A series of dilutions were prepared to obtain standard solutions with concentrations of 100, 80, 40, 20, and 10 µg/mL, which were subsequently used to generate the calibration curve.

In the test procedure, 1 mL of both the test and standard solutions were separately pipetted into vials. To each vial, 5.0 mL of diluted Folin-Ciocalteu reagent (7.5% in water) was added, and the mixture was allowed to stand for 8 min to ensure complete reaction. Subsequently, 4.0 mL of 1% NaOH was added, and the solutions were incubated for 1 h to allow color development. The absorbance of each solution was measured at the maximum absorption wavelength of approximately 730 nm. A blank solution prepared using the same procedure but without adding the test sample was used as a reference. Finally, a calibration curve was constructed using standard gallic acid solutions, and the phenolic content in the test sample was determined based on this curve.²¹

Formulation of Nanosuspension

The nanosuspension was prepared using the nanoprecipitation method, as shown in Figure 1, by combining the organic and aqueous phases, as presented in Table 1. Sonchus arvensis L. extract (0.25 g) was dissolved in 10 mL of ethanol (organic phase), and 0.25, 0.5, or 1 g of the stabilizer (Tween 80) was dissolved in 100 mL of distilled water (aqueous phase). The resulting organic layer was gradually (1mL/min) poured into the aqueous phase using a syringe with constant stirring at 1000 rpm for 6 h. at room temperature.^22–24

Table 1 Formulation of Nanosuspension Tween 80 to Extract Ratios

Figure 1 Nanoprecipitation method for Sonchus arvensis nanosuspension preparation.

Characterization of Utilized Nanosuspension

Particle size analysis (PSA) of the Sonchus arvensis L. formulations was conducted by assessing the z-average diameter and measuring the polydispersity index (PI) using photon correlation spectroscopy (PCS) with a Zetasizer (NanoSizer 3000, Malvern Instruments, Malvern, UK) at a scattering angle of 90°.²⁵ The measurements were performed in a cell with a width of 1 cm at a controlled temperature of 25.0 ± 0.1 °C. Additionally, the zeta potential (ZP) of the nanosuspension was measured in a capillary cell using the same instrument, which employed the Helmholtz–Smoluchowski equation to convert the observed particle electrophoretic mobility into zeta potential values. The zeta potential reflects the surface charge of the nanoparticles and serves as an indicator of their physical stability.²⁶

Transmission Electron Microscopy (TEM) Analysis

The surface morphologies of the prepared nanosuspensions and extracts were examined using transmission electron microscopy (TEM).²⁷ A small quantity of nanosuspensions and extracts was carefully deposited dropwise onto a specially designed copper grid for TEM analysis. Subsequently, the copper grid was placed in an oven at 45 °C to ensure complete drying of the sample. The dried samples were analyzed using an HT7700 TEM (Hitachi, Tokyo, Japan). Imaging was conducted at a voltage of 100 kV with a magnification of ×100,000, and video recordings were captured for 0.1 s. The resulting images were processed using ImageJ 2.0 software.^28,29

In vitro Release Studies of Sonchus arvensis L. Extract and Formulated Nanosuspension

An in vitro release study of the extracts and three optimized nanosuspensions was conducted using the dialysis bag technique (Figure 2).³⁰ The release experiments were performed at an intestinal pH of 7.4, utilizing phosphate-buffered saline (PBS) at the same pH level. In this procedure, pre-weighed samples of the extract and nanosuspensions were placed into pre-soaked dialysis bags with a molecular weight cut-off (MWCO) of 12–14 kDa (Carolina Dialysis Tubing, Burlington, NC). The release process was carried out in 100 mL of PBS at pH 7.4, maintained at a temperature of 37 ± 0.5°C with continuous stirring at 100 rpm using a magnetic stirrer. Samples were collected at predetermined time points (0, 1, 2, 3, 4, 5, 6, 8, and 24 h). Following each sampling, an equal volume of fresh medium was added to maintain sink conditions. The quercetin content in the collected samples was analyzed using a UV-Vis spectrophotometer at a wavelength of 345 nm.^24,30

Figure 2 The in vitro release study using the dialysis bag technique.

In vitro Antioxidant Activity Assay of Formulated Nanosuspensions and Crude Extract

The antioxidant properties of the nanosuspension formulations were evaluated using the DPPH scavenging assay, which measures the ability of antioxidant compounds to donate hydrogen atoms to neutralize the DPPH radicals. For the assay, various concentrations (50, 100, 150, 200, and 250 ppm) of each sample, including the Sonchus arvensis L. extract and its formulated nanosuspension, were prepared by dilution in 96% ethanol. A 1 mL aliquot of 0.2 mm DPPH solution was then added to 1 mL of each sample, followed by thorough mixing. After a 30 min incubation period in the dark, the absorbance was recorded at 517 nm using a UV-Vis spectrophotometer.³¹ All experiments were performed in triplicates. Serial dilutions of ascorbic acid were used as positive controls. The percentage of DPPH radical-scavenging activity was calculated using the following equation:

In vitro Anti-Hyperlipidemic Studies of Sonchus arvensis L. Extract and Prepared Nanosuspensions

The anti-cholesterol activity of the extract and prepared nanosuspension was evaluated using a photometric method, wherein cholesterol was allowed to react with the Liebermann-Burchard reagent. The crude extract and nanosuspension formulations were tested across a concentration range of (15.625, 31.25, 62.5, 125, and 250 mg/L). A volume of 4 mL was collected and subsequently transferred to a capped test tube, to which 1 mL of a 1000 ppm cholesterol stock solution was added, shaken for 2 min, and 2 mL of the Liebermann-Burchard reagent was then added. The mixture was incubated for 15 min in the dark, the experiment was conducted in triplicate, and the colorimetric results were quantified using a UV-Vis spectrophotometer at a peak wavelength of 620 nm. A blank control was established using a solution of 5 mL chloroform combined with 2 mL of Liebermann-Burchard reagent. The negative control consisted of 1 mL of 1000 ppm cholesterol solution in 5 mL chloroform supplemented with 2 mL Liebermann–Burchard reagent. A positive control was prepared using simvastatin at concentrations of (15.625, 31.25, 62.5, 125, and 250 mg/L). The residual cholesterol concentration was determined from the absorbance data measured at 620 nm.^32,33 The percentage inhibition was calculated using the following formula:

Results

Sonchus arvensis L. Extraction

The yields obtained from the maceration method for the extraction of Sonchus arvensis L. are presented in Table 2. The resulting yield was 12.38% (w/w), meeting the specified standard of the Indonesian herbal pharmacopeia for Sonchus arvensis L. extract, which requires a minimum yield of 7.5%.²¹

Table 2 Sonchus arvensis Standardization Results

Phytochemical Screening of Sonchus arvensis L. Extract

The phytochemical composition of the Sonchus arvensis L. extract was assessed using different reagents to identify the presence of various secondary metabolites (Table 3). The observed visual changes in the extract following each treatment are shown in Figure 3.²¹

Table 3 Phytochemical Screening of Sonchus arvensis Extract Results

Figure 3 Phytochemical screening results. (A and B) Alkaloids forming white precipitate using Mayer reagent, (C) Dragendorff reagent forming an Orange-brown precipitate, (D) using FeCl3 1% forming a blue-black color for polyphenols, (E) Mg powder + 5 mL HCl 2N forming an Orange/red color for flavonoids; (F) Amyl alcohol reagent forming a yellow-red color for flavonoids (G) Gelatin 1% for tannins forming white precipitate or turbidity, (H) saponins after shaking yield persistent foam.

Measurement of Total Flavonoid Levels of Sonchus arvensis L. Extract

The total flavonoid content of the Sonchus arvensis L. extract is presented in Table 1. The extract was found to have a flavonoid concentration equivalent to 33.9 ± 1.5 mg of quercetin equivalents (QE) per gram of extract.²¹

Measurement of Total Phenol Levels of Sonchus arvensis L. Extract

The phenolic content of the Sonchus arvensis L. extract, as presented in Table 2, was determined to be 30.3 ± 0.06 mg GAE/g gallic acid.²¹

Characterization of Prepared Nanosuspensions

The prepared nanosuspensions were characterized in terms of particle size distribution, polydispersity index, zeta potential, and short-term stability (28 days).

Particle Size Analysis

Initially, the particle size decreased with increasing Tween concentration, ranging from 13.133 nm for NS-Sa-1:1 to 11.533 nm for NS-Sa-4:1 (Table 4). This reduction reflects surfactant-induced stabilization, as higher Tween concentrations reduced the particle size. However, during the stability study, a slight increase in particle size was observed across all formulations. Specifically, after storage, the particle size of the NS-Sa-1:1 ratio increased to 14.23 nm, the NS-Sa-2:1 ratio increased to 13.13 nm, and the NS-Sa-4:1 ratio increased slightly to 12.9 nm. NS-Sa-4:1 exhibited the most stable particle size over time (Figure 4), indicating that a higher concentration of Tween could effectively limit particle growth.³⁴

Table 4 Characterization of Prepared Nanosuspension (PSA, PI, Zeta Potential, n=3)

Figure 4 Stability studies of three formulations of nanosuspension (A) Particle size, (B) Polydispersity Index (PI), (C) zeta potential.

Polydispersity Index

In terms of PI, all formulations initially exhibited low values (0.145–0.161), suggesting a narrow and favorable particle size distribution. However, after 28 days of storage, the PI values increased across all formulations, likely reflecting a broadening of the size distribution due to particle aggregation. Specifically, the PI for NS-Sa-1:1 increased from 0.161 to 0.324 for NS-Sa-2:1, from 0.145 to 0.292 for NS-Sa-4:1, and from 0.146 to 0.317. Although NS-Sa-4:1 showed slightly improved stability, all ratios showed an increase in polydispersity, which may indicate minor instability during prolonged storage.

Zeta Potential

Zeta potential measurements initially varied with Tween concentration, with values decreasing from −6.26 mV at the NS-Sa-1:1 ratio to −15.033 mV at the NS-Sa-2:1 ratio, before rising slightly to −12.5 mV at the NS-Sa-4:1 ratio. More negative zeta potential values suggest greater electrostatic stability, and these values changed accordingly over the storage period. For NS-Sa-1:1, the zeta potential increased from −6.26 mV to −13.1 mV, implying improved stability. However, NS-Sa-2:1 decreased from −15.033 mV to −9 mV, indicating diminished electrostatic repulsion and a greater risk of aggregation (Figure 4). The NS-Sa-4:1 zeta potential remained stable at approximately −12.5 mV,³⁵ underscoring the effectiveness of higher Tween concentrations in maintaining electrostatic stability over time.

In the characterization and stability study (28 days), NS-Sa-4:1 demonstrated the most consistent stability across particle size, PI, and zeta potential. Overall, the particle sizes across all formulations remained within the nanoscale range (1–100 nm),³⁶ reaffirming their suitability for nanosuspension applications in drug delivery systems.

Transmission Electron Microscopy (TEM) Analysis

TEM analysis revealed that the raw Sonchus arvensis L. extract (Figure 5A) displayed an irregular and variable morphology, which is common in unprocessed plant extracts because of their natural diversity in particle size and shape. In contrast, (Figure 5B–D), representing the three different formulations, showed consistent spherical morphology, indicating that each formulation successfully produced a nanosuspension. This spherical shape suggests that the extract components were effectively transformed into nanoparticles, likely enhancing the stability and homogeneity of the formulations compared to the original extract.

Figure 5 TEM analysis of Sonchus extract and three NS formulations shows the morphology of spherical shape of NSs and Sonchus extract (A) Sa-Extract, (B) NS-Sa-1:1, (C) NS-Sa-2:1, (D) NS-Sa-4:1.

Each formulation was prepared using varying concentrations of Tween 80, a surfactant that plays a crucial role in stabilizing nanoparticles by preventing particle aggregation. The consistent spherical morphology across all three formulations, regardless of the Tween 80 ratio, indicated that each formulation achieved a stable nanosuspension. This nanosuspension formation is promising for improving the solubility, stability, and pharmaceutical availability of the extract, which are key factors in applications that require controlled delivery and enhanced therapeutic efficacy of the extract.

In vitro Drug Release Study

Owing to the presence of numerous bioactive phytoconstituents in plants, it is challenging to precisely quantify all of these components. Therefore, in the present study, a single key bioactive compound was selected as a reference for the analysis of quercetin, which was identified as the primary constituent in the Sonchus arvensis L. extract and was employed as the standard compound for evaluating the dissolution study. The concentrations of the active constituents were determined using a quercetin calibration curve. The dissolution profiles of both the crude plant extract and nanosuspensions were analyzed in a phosphate buffer solution (pH 7.4) as the dissolving medium. The results indicated that the release of both the plant extract and nanosuspensions increased over time, with a significant difference in the release rates between the two. According to theoretical principles, the in vitro release rate of a drug can be enhanced by decreasing its particle size, thereby increasing its surface area.

The slower release of the crude extract can be attributed to its larger particle size, lack of stabilization, and reduced solubility, which hinder its dissolution.

The enhanced release rate of NS-Sa-4:1 demonstrated the advantages of nanosuspension technology, such as improved particle size reduction, stability, and solubility, achieved in the presence of a high concentration of Tween 80.

The in vitro release study clearly showed that NS-Sa-4:1 outperformed the crude extract in terms of release rate and overall drug release. The superior performance of NS-Sa-4:1 can be attributed to its smaller and more stable particle size, uniform morphology, as observed in the TEM analysis, and better long-term stability. The higher concentration of Tween 80 effectively enhanced drug dissolution and release, making it the best candidate for improving therapeutic efficacy.

The crude extract exhibited a slower and less regulated release profile than the nanosuspension formulations, achieving a cumulative release of approximately 28.9% over 24 h. In contrast, NS-Sa-4:1 displayed a markedly faster and more sustained release profile, reaching a cumulative release of approximately 92.51% within the same period (Figure 6).

Figure 6 In vitro release of crude extract and nanosuspension over 24 hrs.

Antioxidant Profile Based on Percentage of Inhibition and IC₅₀ Values

The analysis of IC₅₀ values, derived from the inhibition percentages presented in Figure 7, revealed that NS-Sa-4:1 demonstrated the highest antioxidant activity among the tested formulations, exhibiting efficacy similar to that of ascorbic acid, a standard antioxidant. The superior antioxidant capacity of this formulation is attributed to its reduced particle size, enhanced stability, and uniform particle distribution, which differentiates it from both the raw extract and other nanosuspension formulations. These results highlight the potential of nanosuspension technology for improving the antioxidant properties of bioactive compounds.

Figure 7 Percentage DPPH inhibition of various formulations of Sonchus arvensis L. extract, including the raw extract (Sa-Extract), nanosuspensions prepared with different polymer ratios (NS-Sa-1:1, NS-Sa-2:1, NS-Sa-4:1), and ascorbic acid as a control, at concentrations of 50, 100, 150, 200, and 250 ppm. Data are presented as mean ± standard deviation (SD). ** indicates a significant difference compared to all other groups at P < 0.01; *indicates P < 0.05; and #denotes non-significant differences (P > 0.05).

Figure 7 illustrates the percentage inhibition of various formulations of Sonchus arvensis L. extract (Sa-Extract, NS-Sa-1:1, NS-Sa-2:1, and NS-Sa-4:1) at different concentrations (50, 100, 150, 200, and 250 ppm) compared to ascorbic acid as the control. This comparison underscores the differences in antioxidant efficacy between the raw extract, nanosuspension formulations, and standard antioxidant.

At all concentrations, ascorbic acid consistently demonstrated the highest percentage of inhibition, with statistical significance (P < 0.01, denoted by **), reaffirming its superior antioxidant activity. Among the tested formulations, the nanosuspension NS-Sa-4:1 generally exhibited the highest percentage of inhibition, although it remained significantly lower than ascorbic acid at all concentrations (P < 0.01). These findings suggest that while nanosuspension technology improves antioxidant activity, it does not reach the efficacy level of the standard.

The raw extract (Sa-Extract) exhibited the lowest percentage of inhibition across all concentrations, emphasizing the limitations of the unprocessed form. Comparisons among the nanosuspension formulations (NS-Sa-1:1, NS-Sa-2:1, and NS-Sa-4:1) revealed no significant differences in their inhibition percentages (P > 0.05, denoted by #), indicating comparable antioxidant efficacies among these formulations. This similarity suggests that while nanosuspension technology enhances activity over the raw extract, the polymer ratio within the tested range has a limited impact on antioxidant potential.

At higher concentrations (200 and 250 ppm), all nanosuspension formulations showed increased percentage inhibition, with NS-Sa-4:1 exhibiting a slight superiority. However, the differences among the nanosuspension formulations were not statistically significant (P > 0.05). Despite these improvements, the percentage inhibition for all formulations consistently remained significantly lower than that of ascorbic acid (P < 0.05 or P < 0.01), reinforcing the unmatched potency of the standard.

Further analysis of IC₅₀ values, derived from the inhibition percentages presented in Figure 8, reveals that NS-Sa-4:1 demonstrates the highest antioxidant activity among the tested formulations. However, its activity was significantly lower than that of ascorbic acid, which was the positive control used in this experiment. The enhanced antioxidant capacity of this formulation can be attributed to its reduced particle size, improved stability, and uniform particle distribution. These properties likely differentiate it from both the raw extract and other nanosuspension formulations. Overall, the findings underscore the potential of nanosuspension technology to significantly improve the antioxidant properties of bioactive compounds, making it a promising strategy for enhancing the efficacy of natural extracts.

Figure 8 IC50 Values of DPPH antioxidant assay.

Antihyperlipidemic Based on Percentage of Inhibition and IC₅₀ Values

Figure 9 illustrates the percentage of cholesterol inhibition of various formulations, including Sa-Extract (raw extract of Sonchus arvensis L.), nanosuspensions (NS-Sa-1:1, NS-Sa-2:1, and NS-Sa-4:1), and simvastatin as a reference standard, across concentrations ranging from 15.625 to 250 ppm. The results highlighted a consistent pattern of performance among the formulations, with simvastatin demonstrating superior inhibitory activity at all concentrations (P < 0.01, denoted by **).

Figure 9 Percentage cholesterol inhibition of Sa-Extract (Sonchus arvensis L. raw extract), nanosuspensions (NS-Sa-1:1, NS-Sa-2:1, and NS-Sa-4:1), and Simvastatin as a standard at various concentrations (15.625, 31.5, 62.5, 125, and 250 ppm). Simvastatin consistently exhibited the highest percentage inhibition at all concentrations, with statistically significant differences (P < 0.01, denoted by **). Among the nanosuspensions, NS-Sa-4:1 showed the highest inhibition percentage, particularly at higher concentrations, while differences among nanosuspensions were not statistically significant at most concentrations (P > 0.05, denoted by #). At 250 ppm, NS-Sa-4:1 demonstrated a slight but significant improvement over NS-Sa-2:1 (P < 0.05, denoted by *). Error bars represent standard deviation (SD).

Among the nanosuspension formulations, NS-Sa-4:1 consistently exhibited the highest percentage of inhibition, followed by NS-Sa-2:1 and NS-Sa-1:1. At lower concentrations (15.625 and 31.5 ppm), the nanosuspensions displayed significantly higher inhibition percentages than the Sa-Extract (P < 0.01), yet all remained significantly less effective than simvastatin. At 62.5 ppm, NS-Sa-4:1 showed a slightly higher inhibition percentage than NS-Sa-2:1 and NS-Sa-1:1; however, these differences were not statistically significant (P > 0.05, denoted by #).

As the concentration increased (125 and 250 ppm), the percentage inhibition of all nanosuspension formulations improved markedly. NS-Sa-4:1 showed significantly higher inhibition than the Sa extract at both concentrations (P < 0.01). However, at 125 ppm, no significant differences were observed among the nanosuspensions (P > 0.05). At 250 ppm, NS-Sa-4:1 demonstrated a slight but statistically significant improvement over NS-Sa-2:1 (P < 0.05, denoted by *). Despite these improvements, all nanosuspension formulations remained significantly less effective than simvastatin (P < 0.01).

The analysis of IC₅₀ values (Figure 10), derived from the percentage inhibition data, further illustrates the relative performance of the formulations. IC₅₀ values represent the concentration required to achieve 50% inhibition, with lower values indicating greater efficacy. Among the nanosuspensions, NS-Sa-4:1 demonstrated the most pronounced antihyperlipidemic effect, approaching the efficacy of simvastatin. This enhanced performance is attributed to the smaller particle size, improved stability, and uniform particle distribution, which collectively enhance pharmaceutical availability and lipid interaction.

Figure 10 IC50 Values of antihyperlipidemic activity.

Overall, these findings underscore the therapeutic potential of nanosuspension technology in enhancing the efficacy of lipid-lowering agents, particularly when applied to bioactive compounds such as Sonchus arvensis L. The superior performance of NS-Sa-4:1 highlights the importance of optimizing the formulation to improve the clinical potential of natural extracts.

Discussion

This study highlights the use of a green synthetic approach for Sonchus arvensis L., marking a substantial step in sustainable nanotechnology. By eliminating the need for hazardous chemicals, this method aligns with global environmental initiatives and emphasizes the importance of eco-friendly practices in drug delivery systems.³⁷ Notably, this approach leverages the inherent bioactivity of Sonchus arvensis L., effectively bridging the gap between traditional medicinal approaches and advanced nanotechnological innovations.³⁸ In contrast to conventional methods, which often rely on toxic reagents, this strategy offers an environmentally safe alternative without compromising therapeutic efficacy.

This study breaks new ground by employing a Sonchus arvensis L. nanosuspension for antihyperlipidemic activity, offering an innovative, natural, and sustainable solution for managing lipid levels. The nanoprecipitation technique used in this study ensured uniform particle distribution, reduced synthesis time, and enhanced nanosuspension stability. These results were corroborated by Transmission Electron Microscopy (TEM) and zeta potential analysis, which confirmed the consistent morphology and stability of the formulation.³⁹

The results of this study showed that the Sonchus arvensis L. nanosuspension (NS-Sa-4:1) demonstrated remarkable antihyperlipidemic activity, with therapeutic effects comparable to those of simvastatin (p < 0.05). This efficacy can be attributed to its improved pharmaceutical availability and a controlled drug release profile. Furthermore, the nanosuspension exhibited strong antioxidant properties, with an IC₅₀ value indicating robust free radical scavenging activity.⁴⁰ These findings support the growing evidence of the advantages of nanosuspension formulations in enhancing the solubility and therapeutic performance of bioactive compounds.⁴¹

The observed improvements in antioxidant and lipid-lowering activities are directly linked to the physicochemical properties of the nanosuspension. The enhanced pharmaceutical availability, controlled drug release profile, and uniform particle distribution achieved through nanoprecipitation contribute to superior therapeutic efficacy. The smaller particle size of nanosuspensions increases the surface area, facilitating better solubility and cellular uptake, which in turn enhances their pharmacological effects. Additionally, the stable zeta potential observed in this study ensured the stability of the formulation, preventing aggregation and preserving its bioactivity. These physicochemical attributes collectively reinforce the potential of nanosuspensions as advanced and sustainable drug delivery systems.

Compared with previous studies, this study highlights the unique benefits of the nanosuspension formulation of Sonchus arvensis L. While earlier research predominantly focused on crude plant extracts, encapsulating Sonchus arvensis L. phytoconstituents within nanoparticles has demonstrated significantly improved stability and therapeutic potential. Supporting evidence from studies on gemfibrozil and curcumin nanosuspensions further validates the efficacy of nanoprecipitation in enhancing drug performance.^42,43 This approach not only amplifies the lipid-lowering and antioxidant potential of Sonchus arvensis L. but also redefines its application in the treatment of hyperlipidemia.

Conclusion

The nanosuspension formulation of Sonchus arvensis L. folium has been demonstrated to significantly enhanced its antihyperlipidemic and antioxidant effects compared to the crude extract. The optimized NS-Sa-4:1 formulation exhibited desirable physicochemical properties, including reduced particle size, improved stability, and enhanced solubility, which contributed to its superior in vitro release and bioactivity. These enhancements resulted in antihyperlipidemic and antioxidant effects comparable to those of conventional treatments, such as simvastatin and ascorbic acid, suggesting that nanosuspension technology effectively improves the pharmacokinetic and pharmacodynamic profiles of S. arvensis L. The observed improvements in lipid regulation and oxidative stress reduction highlight the potential of this formulation as a viable natural alternative for managing hyperlipidemia and oxidative stress-related disorders. These findings reinforce the therapeutic relevance of nanosuspension-based drug delivery for optimizing the efficacy of herbal bioactives, paving the way for further in vivo studies and potential clinical applications of this approach.