Metabolic syndrome is a cluster of disorders that increases the risk of cardiovascular disease and type 2 diabetes and it represents a growing public health concern. These conditions include obesity, insulin resistance, hypertension and dyslipidemia. Prevention strategies primarily focus on lifestyle modifications, including dietary changes and increased physical activity. Calorie-restricted diets that emphasize unsaturated fats, whole grains, fruits and vegetables (Castro-Barquero et al., 2020) can effectively prevent and treat metabolic syndrome. Regular physical activity, especially aerobic exercise, helps manage conditions associated with metabolic syndrome, such as obesity and hypertension and helps preserve lean body mass during weight loss (Myers et al., 2019). However, outcomes may vary due to individual responses and the need for personalized treatment strategies (Castro-Barquero et al., 2020; Myers et al., 2019).
       
Functional foods are increasingly used as dietary interventions for hyperlipidemia and hyperglycemia, as they regulate lipid and glucose metabolism through the inhibition of key enzymes, including pancreatic lipase, α-amylase and α-glucosidase and by modulating protein expression related to metabolic pathways. Recent studies have identified various natural and synthetic compounds with significant inhibitory effects on these enzymes. Functional foods contain high levels of bioactive components, such as fibers, phytosterols and antioxidants, which reduce oxidative stress and the risk of diabetes and metabolic syndrome-associated cardiovascular diseases (Permatasari et al., 2024; Bintanah et al., 2023; Permatasari et al., 2023; Alkhatib et al., 2021). Nevertheless, further research is required to understand the long-term effects and underlying mechanisms of individual functional foods.
       
Wolffia globosa, commonly known as duckweed, offers several health benefits due to its rich nutritional composition and bioactive compounds. This plant is a valuable source of protein, essential amino acids, dietary fiber and antioxidants. In addition, it is one of the few plant-based sources of vitamin B12, which can improve serum B12 levels, especially in vegetarian and vegan diets (On-Nom et al., 2023; Sela et al., 2020; Monthakantirat et al., 2022). The protein extract from W. globosa has exhibited antimicrobial and other health-promoting properties (Duangjarus et al., 2022; Tipnee et al., 2017). Furthermore, the rapid growth and high adaptability of W. globosa make it well suited for cultivation in aquaculture systems (Said et al., 2022). Its protein content can reach up to 45.54% of dry weight, with consistently high yields under aquaculture conditions. These characteristics make W. globosa a promising raw material for nutraceutical and functional food development (Desfita et al., 2025; Hu et al., 2024; Bong-Hyun et al., 2024).
       
Recently, nutritious snacks have been developed as functional food supplements, owing to their cost-effectiveness and consumer acceptability (Sharun et al., 2024; Min et al., 2024). Thus, W. globosa has potential as a functional food for the management of metabolic syndrome, particularly in controlling glycemic status through dietary intervention. In a randomized controlled trial, a shake containing W. globosa resulted in significantly lower postprandial glucose peaks and a faster return to baseline levels compared with a traditional yogurt shake, indicating improved glycemic responses in abdominally obese individuals (Zelicha et al., 2019). The high protein and fiber content of W. globosa is associated with reduced fat absorption and improved satiety, which may contribute to weight management in metabolic conditions (On-Nom et al., 2023; Zelicha et al., 2019).
       
Despite these promising findings, the long-term effects and molecular mechanisms of action of W. globosa remain insufficiently explored. In particular, reports on its role in regulating lipid and glucose metabolism through the inhibition of key digestive enzymes are limited. Therefore, the present study aimed to evaluate the antioxidant properties of W. globosa using various antioxidant assays and to investigate its inhibitory effects on key metabolic enzymes involved in lipid and glucose metabolism, including pancreatic lipase, α-amylase and α-glucosidase.

Plant collection and extraction
 
Five hundred grams of fresh duckweed (Wolffia globosa) were obtained from the Golden Banana Community Enterprise, Sang Khom District, Udon Thani Province, Thailand. Plant identification was performed based on morphological characteristics and known geographical distribution. The whole plants were shade-dried at 50°C and pulverized into a fine powder. Ten grams of the dried powder were macerated in 1 L of 95% (v/v) ethanol (RCI Labscan, Thailand) with sonication at room temperature for 72 h. The extraction was performed in triplicate (n = 3). The pooled W. globosa extract (WE) was concentrated to constant weight using a rotary vacuum evaporator, followed by a water bath and stored at 4°C until further analysis.
 
Total phenolic content
 
Total phenolic content (TPC) of WE was determined using the Folin-Ciocalteu (FC) method and results were expressed as gallic acid equivalents (GAE). Briefly, 20 µL of WE (5 mg/mL, dissolved in dimethyl sulfoxide, DMSO; RCI Labscan, Thailand) was mixed sequentially with 100 µL of diluted FC reagent (1:10, v/v) and 80 µL of 7.5% (w/v) Na₂CO₃ (RCI Labscan, Thailand) in distilled water. The mixture was incubated in the dark at room temperature for 30 min and absorbance was measured at 765 nm using a microplate reader. TPC was calculated from a gallic acid standard curve (y = 77.442x + 0.0031, R² = 0.9998) and expressed as mg GAE/g WE. All measurements were conducted in triplicate (n = 3) (Hussein et al., 2010).
 
Total flavonoid content
 
Total flavonoid content (TFC) was determined using the aluminum chloride (AlCl₃) colorimetric method, with quercetin as the reference standard. Fifty microliters of WE (2.0 mg/mL in ethanol) or standard solution was added to a 96-well microplate, followed by AlCl₃ (Loba Chemie, India), ethanol and sodium acetate solution in a ratio of 10:96:10 (v/v/v). The mixture was incubated in the dark at room temperature for 40 min and absorbance was measured at 415 nm. A quercetin standard curve (y = 3.5717x + 0.0644, R² = 0.9955) was used for quantification. Results were expressed as mg quercetin equivalent (QE)/g WE, based on triplicate measurements (n = 3) (Biju et al., 2014).
 
Antioxidant assays
 
WE was dissolved in absolute ethanol to obtain concentrations of 0.001, 0.01, 0.1, 1.0 and 10 mg/mL. Antioxidant activity was evaluated using four established assays: DPPH radical scavenging, nitric oxide (NO) radical scavenging, ferrous ion-ferrozine metal chelation and ferric thiocyanate lipid peroxidation inhibition assays. These assays measured DPPH radical reduction, NO radical reduction using Griess reagent (Sigma-Aldrich, USA), metal ion chelation capacity and inhibition of lipid peroxidation, respectively. Absorbance was recorded at assay-specific maximum wavelengths (lmax) using a microtiter plate reader (BIO-RAD, USA). All assays were performed in triplicate (n = 3). Antioxidant activity was expressed as IC50  values (mg/mL), calculated from dose-response curves. Vitamin C (ascorbic acid; Sigma-Aldrich, USA), vitamin E (α-tocopherol; Sigma-Aldrich, Germany) and EDTA (Sigma-Aldrich, USA) were used as positive controls, as appropriate (Yen and Duh, 1994; Manosroi et al., 2012; Aktas et al., 2013).
 
Assays of metabolic enzymatic inhibition
 
Inhibition of pancreatic lipase
 
WE was dissolved in 10% (v/v) DMSO to obtain concentrations ranging from 0.001 to 10 mg/mL. Orlistat (0.0005-5 mg/mL in 10% DMSO) served as the positive control. Lipase inhibitory activity was determined by measuring the release of p-nitrophenol at 415 nm, following enzymatic hydrolysis of p-nitrophenyl butyrate (Sigma-Aldrich, USA) by pancreatic lipase (Sigma, USA). All measurements were performed in triplicate (n = 3) (Bustanji et al., 2010).
 
Inhibition of α-glucosidase
 
α-Glucosidase inhibitory activity was assessed by monitoring the release of p-nitrophenol from p-nitrophenyl-α-D-glucopyranose (Sigma, Switzerland). WE (0.001-10 mg/mL in 10% DMSO) was tested, while acarbose (0.0005-5 mg/mL in 30 mM phosphate buffer, pH 6.5) was used as the positive control. Absorbance was measured at 415 nm and assays were conducted in triplicate (n = 3) (Alam et al., 2017).
 
Inhibition of α-amylase
 
This assay was determined the reducing sugar, the product cleaved from starch, catalyzed by α-amylase present in WE or control. Each concentration of WE dissolved in 10%DMSO (0.001 to 10.0 mg/ml) and starch in phosphate buffer (pH 6.9) was mixed. The reaction was started with pancreatic α-amylase (Fluka, Germany) and incubated at 37°C. The reaction mixture was prepared with dinitrosalicylic acid (DNSA) solution (1% w/v DNSA, 30% w/v sodium potassium tartarate and 20%v/v 2N NaOH in distilled water) and kept in boiling water for stopping reaction. The mixture was diluted with distilled water and absorbance was measured at 540 nm by spectrophotometer (T80, Oasis Scientific, USA). Positive control was acarbose (Sigma-Aldrich, Germany) dissolved in 30 mM phosphate buffer, pH 6.5 (0.00025, 0.0025, 0.0025, 0.025, 0.25, 2.5 mg/ml) (Keerthana et al., 2013). The calibration curve between the inhibition of enzyme and WE concentration was compared with calibration curve between the inhibition of enzyme and positive control. The results arerepresented as IC₅₀.
 
Data analysis
 
Dose-response curves were constructed for WE and positive controls and IC₅₀ values were calculated by linear regression analysis. Results are presented as mean ±standard deviation (SD). Comparisons were based on calibration curves generated from enzyme inhibition percentages versus sample concentration.

The W. globose extract (WE) was semi-solid and brownish colored and the yield of extraction was about 5-10%. The WE contained only phenolic compounds (TPC = 28.19±0.79 mg GAE/g), whereas no detectable flavonoids were observed. DPPH and NO radical scavenging activity was observed and lipid peroxidation was inhibited (IC₅₀= 0.34±0.05, 0.23±0.06 and 80.51±16.02 mg/ml, respectively).
       
Therefore, this extract was unable to chelate metal ions (Table 1). WE strongly inhibited pancreatic lipase (IC₅₀ = 1.55±0.20 mg/ml) and α-amylase (IC₅₀= 0.02±0.01 mg/ml), while it poorly inhibited α-glucosidase (IC₅₀>1,000 mg/ml) (Table 2). The weak α-glucosidase inhibition may be due to the selective enzyme specificity and low levels of flavonoids, which are typically effective α-glucosidase inhibitors.




       
The TPC and TFC were calculated and applied from standard curves of gallic acid and quercetin, respectively. The relationship between antioxidant activities and sample concentration is represented as graphs with linear formulae and coefficients of determination, R² (Fig 1). The relationship of inhibition of key metabolic enzymes, including lipase, α-glucosidase and α-amylase, and sample concentration was also represented as graphs with linear formulae and coefficients of determination, R² (Fig 2).




       
The nutritional composition of W. globosa contains approximately 45.54% protein in dry weight, which is higher than many traditional foods, along with high dietary fiber content and essential amino acids. This duckweed species is a valuable food source with potential applications in human nutrition and aquaculture. It has been successfully integrated into snack products, enhancing their nutritional value and sensory appeal (Appenroth et al., 2018; Said et al., 2022; On-Nom et al., 2023). High-protein diets have been found to improve glucose tolerance and lipid profiles in mouse models by reduction of fatty acid synthesis and inflammatory markers in the liver and colon. In addition, high protein and dietary fiber diet significantly improved glycemic control and insulin resistance in diabetic mice that demonstrated protective effect against diabetes-related metabolic syndromes (Ni et al., 2022; Xu et al., 2024). High-fiber diets have been linked to improved insulin sensitivity and satiety, which is beneficial for the management of glucolipid metabolic disorders. The metabolites from high fiber diets can also alleviate neurodegenerative symptoms in obese individuals with diabetes (Dahiya et al., 2017; Luo et al., 2023). Moreover, high-protein and fiber-based supplements have led to weight loss and improved metabolic markers in clinical trial (Glynn et al., 2022).
       
W. globose exhibits significant antioxidant activity due to the richness of bioactive compounds. Previous studies have indicated that various extraction methods, such as boiling, freeze-thawing and mechanical crushing, yield varying antioxidant activities. The boiling method had resulted in the highest total phenolic content, which may be a candidate for nutraceutical applications (Yadav et al., 2024). Therefore, the ethanolic extract has shown significant DPPH inhibition (75.77%) and contained the notable levels of carotenoids, flavonoids and phenolic compounds (Monthakantirat et al., 2022). In our study, we were extracted W. globose by the simple macerated with ethanol as WE. Compared with the previous study, the extract had a lower content of phenolic compounds and lacked flavonoids. The finding indicated the importance of the extraction methods and conditions on the yield of bioactive compounds. However, this extract exhibited preferrable antioxidant activities, including DPPH and NO radical scavenging activity and the inhibition of lipid peroxidation. It was implied that the antioxidant activities of WE were independent of levels of bioactive compounds. In addition, these antioxidant activities may relate to other bioactive compounds such as, phytosterols rather than phenolics and flavonoids in some antioxidant assays.
       
The W. globose plants contain high levels of antioxidants, such as phenolic compounds, which may support metabolic health by modulating key metabolic enzymes. The extracts of W. globosa have demonstrated significant radical scavenging activity, which has indicated the potential of protective effects against oxidative stress and the association of glucose and lipid metabolism (Tipnee et al., 2017; Monthakantirat et al., 2022). As our results, WE strongly inhibited pancreatic lipase and α-amylase, while there was poorly inhibited α-glucosidase. The finding corresponded to the previous study, which had reported that W. globosa Mankai had significantly reduced postprandial glucose peaks compared to traditional dairy shakes (Zelicha et al., 2019). In addition, W. globosa extract has demonstrated significant radical scavenging activity, indicating potential protective effects against oxidative stress related to lipid metabolism. There were contained phytosterols, such as β-sitosterol and stigmasterol, which have shown anti-inflammatory effects that may indirectly influence lipase activity and improve insulin sensitivity (Tipnee et al., 2017; Monthakantirat et al., 2022). This study supported that W. globosa extract can become the main ingredient of functional food, had potential for the management of metabolic conditions, such as anti-diabetic and anti-lipemic properties. Further study is necessary to conduct clinical trial of W. globosa snack products within overweight or obese people who have metabolic conditions.

Duckweed (Wolffia globosa) has emerged as a promising functional food ingredient for the management of metabolic disorders. Its value is attributed to its dense nutritional composition, characterized by high protein and dietary fiber levels, along with a diverse array of bioactive compounds, including phenolics, flavonoids and phytosterol. Our study supported that W. globose ethanolic extract exhibited DPPH and NO radical scavenging activity, inhibition of lipid peroxidation and inhibition of key metabolic enzymes, including pancreatic lipase and α-amylase.

We express our gratitude for the financial and technical assistance provided by Suan Sunandha Rajabhat University situated in Bangkok, Thailand. We are extending our appreciation to the Samut Songkhram Campus of Suan Sunandha Rajabhat University, Thailand, for their valuable support in plant identification and local collaborative efforts. Special Thanks to the Golden Banana Community Enterprise, Sang Khom District, Udon Thani Province, Thailand, for W. globosa sample providing and planting information.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Funding details
 
This research project has received funding support from the National Science, Research and Innovation Fund (NSRF), Thailand, under Grant No. 13681/2025, which is monitored by Suan Sunandha Rajabhat University, Bangkok, Thailand.
 
Authors’ contributions
 
All authors contributed toward data analysis, drafting and revising the paper and agreed to be responsible for all the aspects of this work.
 
Use of artificial intelligence
 
Not applicable.
 
Declarations
 
Authors declare that all works are original and this manuscript has not been published in any other journal.

Authors declare that they have no conflict of interest.