Green Extraction of Corn Silk Flavonoids Using NADES-UAE: Optimizing Conditions for Agricultural Waste Valorization

Carotenoids from marigold flowers (Tagetes erecta L.) are commonly extracted using conventional solvents like hexane and ethanol, which may pose environmental and health risks. Based on these considerations, this study aimed to evaluate the potential of Natural Deep Eutectic Solvents (NADES) as greener alternatives and to determine the optimal NADES composition and extraction conditions using Ultrasound-Assisted Extraction (UAE). The most effective NADES was selected based on total carotenoid content, statistically analyzed using one-way ANOVA. Optimization was carried out using Response Surface Methodology (RSM) with a Box–Behnken Design (BBD) to assess the effects of ultrasonic power, extraction time, and temperature. Total carotenoid content was determined by UV-Vis spectrophotometry. Among the tested solvents, the hydrophobic NADES composed of menthol:lactic acid (8:1) showed the highest efficiency (98.48 ± 0.20 mg β-carotene equivalent (BCE)/g dry sample (DS)), outperforming the hydrophilic NADES, hexane, and 80% ethanol. Optimization using Response Surface Methodology with a Box–Behnken Design identified optimal UAE conditions at 100% ultrasonic power, 40 minutes, and 60°C, yielding 304.01 ± 1.60 mg BCE/g DS. These results confirm the effectiveness of NADES as sustainable and efficient solvents for carotenoid extraction from marigold flowers.

Flavonoid compounds have attracted considerable interest due to their promising potential applications cosmetic products, particularly as skin-lightening agents, while efficient and sustainable extraction remains a major challenge. This study aimed to optimize the extraction of flavonoid compounds and their tyrosinase inhibitory activity from Rhodomyrtus tomentosa leaves using ultrasound-assisted extraction (UAE) using natural deep eutectic solvents (NADESs). Initially, several choline chloride-based NADESs were evaluated for their extraction efficiency to identify the most effective solvent, which was subsequently used in the next phase involving the optimization of extraction conditions. The extraction variables, including extraction time, water addition to the NADESs, and the solid-to-liquid ratio were optimized using Response Surface Methodology (RSM) with a Box-Behnken design. Both total flavonoid content and tyrosinase inhibitory activity were determined spectrophotometrically. Choline chloride-propylene glycol (1:1) was identified as the optimal NADES compared to choline chloride-based NADESs combined with various other hydrogen bond donors. The developed models showed high R² value (0.9944 for flavonoids; 0.9998 for tyrosinase inhibition) with non-significant lack-of-fit values, indicating the excellent predictive performance of the response model. The optimal extraction conditions were obtained at an extraction time of 60 min, 20% water addition, and a solid-to-liquid ratio of 0.05 g/mL, resulting in a total flavonoid content of 14.3729 mg QE/ g dried leaves and tyrosinase inhibition of 83.56%. These findings highlight the potential of NADES-UAE as an efficient and environmentally friendly platform for producing bioactive extracts of R. tomentosa suitable for skin-lightening applications.

This study establishes an efficient and eco-friendly ultrasound-assisted extraction (UAE) method for total flavonoids present in Cercis chinensis seeds using natural deep eutectic solvents (NADES). Among nine NADES formulations screened, choline chloride–levulinic acid (ChCl–Lev, 1:2) demonstrated optimal performance, yielding 112.1 mg/g total flavonoids. Through Response Surface Methodology (RSM), the ultrasound-assisted extraction (UAE) parameters were explored. Under the optimized conditions (water content of 30%, time of 28 min, temperature of 60 °C, and solvent-to-solid ratio of 1:25 g/mL), the total flavonoid yield reached 128.5 mg/g, representing a 195% improvement compared to conventional ethanol extraction. The recyclability of NADES was successfully achieved via AB-8 macroporous resin, retaining 80.89% efficiency after three cycles. Extraction kinetics, modeled using Fick’s second law, confirmed that the rate constant (k) increased with temperature, highlighting temperature-dependent diffusivity as a key driver of efficiency. The extracted flavonoids exhibited potent antioxidant activity, with IC50 values of 0.86 mg/mL (ABTS•+) and 0.69 mg/mL (PTIO•). This work presents a sustainable NADES-UAE platform for flavonoid recovery and offers comprehensive mechanistic and practical insights for green extraction of plant bioactives.

To improve the deep processing and utilization of wild blueberries, this study presents a green and highly efficient method for extracting flavonoids from blueberries. The approach combines natural deep eutectic solvents (NADESs) with ultrasound-assisted extraction. Among the 22 tested NADES, Betaine/urea (BU), was the most effective solvent for extracting flavonoids from blueberries. The extraction parameters of ultrasound-assisted betaine/urea (UABU) were optimized using a response surface methodology (RSM). This optimization procedure yielded the optimized conditions outlined below: a molar ratio of urea to betaine of 3.3:1, a water content of 60% (m/v), an ultrasonic power of 330 W, a solid-to-liquid ratio of 1:30, an extraction temperature of 50 °C, and an ultrasonic extraction duration of 30 min. Under these conditions, the total flavonoid content (TFC) extracted using UABU reached 6.06 ± 0.024 mg_RE/g_DW, a 1.44-fold increase compared to ultrasound-assisted 70% (v/v) ethanol (UAE). Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) nontargeted metabolomics analysis revealed that the flavonoids extracted by UABU had highly relative content (RC) of Oenin, 3'-methoxy-4',5,7-trihydroxyflavonol, Isorhamnetin-3-O-glucoside and Isoquercitrin. Significant disparities exist regarding the types and RC of flavonoids obtained via UAE. Results from in vitro antioxidant assays demonstrated that UABU has superior antioxidant activity relative to UAE. This study demonstrated the feasibility of using NADESs, specifically BU, as an efficient and eco-friendly extraction medium for flavonoids from wild blueberries. The yield of flavonoids was increased by this method, and bioactive compounds were also protected-findings that underscore the potential of green solvents for application in the food industry.

Ultrasonic-assisted extraction and biological activities of traditional Chinese/Mongolian medical herb Artemisia ordosica Krasch. using natural deep eutectic solvents

ABSTRACTS This study extracted flavonoids and phenolics from watermelon fruit by microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE) combined with a natural deep eutectic solvent (NADES). The effectiveness of NADES combined with UAE and MAE was evaluated for extracting these compounds from watermelon rinds. The study chose a 2:1 molar ratio of hydrogen bond donors (HBDs) to hydrogen bond acceptors (HBAs). Among the tested combinations, 1,2-propanediol-lactic acid showed the highest solubility for flavonoids and phenolics within the extraction system. Experiments with a single factor were used to examine the effects of different parameters on total phenolic content (TPC) and total flavonoid content (TFC). The Box-Behnken design (BBD) model optimized the NADES-based UAE and MAE processes based on TPC and TFC responses. The results of the experiment and the predicted values from the BBD agreed well. Optimal conditions for recovering phenolics and flavonoids from watermelon rinds with NADES-based UAE were determined as 1/60 g/ml for SLR, 300 W, 20% water content, and 15 min, while NADES-based MAE: 1/50 g/ml for SLR, 400 W, and 3 min. This study presents an efficient approach for extracting biologically active compounds from watermelon.

Eco-friendly extraction of six different rosemary (Rosmarinus officinalis L.) genotypes-using natural deep eutectic solvents: Optimization and modeling via response surface methodology (RSM)

Lime leaves (Citrus aurantifolia) have health benefits as anti-inflammatory, appetite stimulant, antidiarrheal, and weight loss. This activity cannot be separated from the secondary metabolite compounds contained in lime leaves, one of which is flavonoids. Flavonoid compounds from lime leaves can be maximized using appropriate extraction methods. This study aims to determine the effect of extraction methods on total flavonoid content in lime leaves. Lime leaves were extracted with 96% ethanol (1:10) using the maceration and Ultrasound Assisted Extraction (UAE) method. Qualitative tests were carried out using phytochemical screening tests and quantitative tests by measuring the total flavonoid content of macerated and UAE lime leaf extract using UV-Vis spectrophotometry. The quantitative data obtained was then analyzed statistically using the SPSS Independent Samples T-Test with a confidence level of 95%. The yield value of the maceration method was 14.43% and the UAE method was 8.8%. The total flavonoid content produced by the UAE method was 38.626 ± 0.321 mg QE/g extract, and the maceration method was 36.876 ± 0.368 mg QE/g. Based on the statistical analysis of the Independent Samples T-Test, it was found that there was a significant difference between the total flavonoid levels of lime leaf extract from maceration and UAE (p<0.05). The UAE extraction method produces higher flavonoid content than the maceration method. These results indicate that the extraction method influences the total flavonoid content of lime leaves.

Natural Deep Eutectic Solvents (NADES) have emerged as promising, environmentally friendly alternatives to conventional organic solvents for the extraction of bioactive compounds. However, their application to Erythrina crista-galli, a species known for its rich flavonoid content and therapeutic potential, has not yet been systematically investigated. This study addresses this research gap by developing an ultrasound-assisted extraction (UAE) process using NADES, statistically optimized through Central Composite Design (CCD). Among thirteen NADES formulations tested, the NADES11 formulation, composed of lactic acid, citric acid, and glycerol in a molar ratio of 4.5:4.5:1, demonstrated the highest total flavonoid content (TFC) and was selected for further optimization using Response Surface Methodology (RSM). The optimal extraction conditions were established at 97 min, 64 °C, and a solvent-to-material ratio of 1:25. Under these optimized parameters, the antioxidant activities of NADES11 and ethanol extracts were evaluated using DPPH, ABTS, and FRAP assays. The NADES11 extract exhibited significantly higher antioxidant activities, with values of 0.3690 ± 0.0039, 0.2965 ± 0.0118 mmol Trolox/g, and 0.6095 ± 0.0059 mmol Fe²⁺/g, respectively, compared to the ethanol extract, which yielded 0.0532 ± 0.0022, 0.0439 ± 0.0009 mmol Trolox/g, and 0.1043 ± 0.0018 mmol Fe²⁺/g. These results confirm the superior capability of NADES11 not only in efficiently extracting flavonoid compounds but also in preserving their antioxidant properties. HIGHLIGHTS This study explores natural deep eutectic solvents (NADES) as eco-friendly alternatives to conventional solvents for extracting flavonoids from crista-galli twigs. Thirteen NADES formulations were tested, with NADES-11 comprising od lactic acid, citric acid, and glycerol with a ratio of 4.5:4.5:1 showing the highest flavonoid yield. NADES-based extraction was combined with Ultrasound-Assisted Extraction (UAE) to improve efficiency. Extraction parameters were optimized using Response Surface Methodology (RSM) and Central Composite Design (CCD), yielding optimal conditions at 97 min, 64 °C, and a 1:25 solvent-to-material ratio. NADES-11 extract exhibited significantly higher antioxidant activity (DPPH, ABTS, and FRAP assays) compared to the ethanol extract. This work represents the first systematic and statistically optimized NADES-UAE protocol for flavonoid extraction from crista-galli, highlighting its potential as a sustainable green extraction strategy. GRAPHICAL ABSTRACT

The green extraction of bioactive compounds from date seeds was investigated using seven natural deep eutectic solvents (NADES) coupled with ultrasound-assisted extraction (UAE). The seven NADESs mainly consisted of choline chloride as hydrogen bond acceptors (HBA) and four sugars, two organic acids, and one polyalcohol as hydrogen bond donors (HBD) were utilized in this study. When the extraction efficiency of the NADESs was compared to that of the conventional solvents, all the NADESs showed superior bioactive compounds recovery efficacy. The lactic acid-based NADES had the highest extraction efficiency and was further optimized using the response surface method and Box-Behnken design. A four-factors including extraction time (10, 20, and 30 min), ultrasound amplitude (70, 80, and 90 %), % NADES content (30 %, 50 %, and 70 %) and solid-to-solvent ratio (1:30, 1.5:30, and 2:30 g/ml) each at three levels (-1, 0 and 1) using Box-Behnken design was applied. The % NADES content and the solid-to-solvent ratio were the major factors influencing the extraction efficiency of the total phenolic content (TPC) and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. The optimum extraction conditions included an extraction time of 15 min, ultrasound amplitude of 90 %, % NADES content of 70 % and solid-to-liquid ratio of 1:30 g/ml. The experimental values for TPC and DPPH at optimum extraction conditions were 145.54 ± 1.54 (mg GAE/g powder) and 719.19 ± 2.09 (mmol TE/g powder), respectively. The major phenolic compounds observed in the date seeds extracted using ChCl-LA were 3,4-dihydroxybenzoic acid, catechin and caffeic acid. This study reveals that the extraction of date seeds with NADES in combination with UAE technique was able to recover significantly higher amounts of phenolic compounds which could find useful applications in the food, pharmaceutical, and cosmetics industries.

In this study, an environmentally friendly and efficient method based on natural deep eutectic solvent (NADES) in combination with ultrasound-assisted extraction was used for the recovery of phenolic compounds from apple pomace. Acidic, alkaline, sugar-based and polyol-based NADESs were examined for extraction of total phenolic content from apple pomace under different conditions.NADES type, biomass percentage, extraction temperature, extraction time and water content were the testing parameters. NADES type and water content of the solvent were the most important parameters that affected the extraction yield. The most effective NADESs were sugar-based NADESs, which were followed by acidic NADESs. Up to 9.47 mgGAE/gapple pomacewas recovered from apple pomace with glucose:sucrose:water. The highest total flavonoid content was obtained with choline chloride:urea as 17.30 mgEPE/gapple pomace. On the other hand, NADES composed of choline chloride – lactic acid was found to exhibit a significant antioxidant activity. The novelty of this article is the investigation of acidic, alkaline, sugar-based and polyol-based NADESs in the recovery of phenolic compounds from apple pomace using ultrasound-assisted extraction, for the first time in the literature. The antioxidant activity of extracts were also explained in combination with that of NADESs.

RSM-based optimization of microwave- and ultrasound-assisted green extraction of iron-chelating and antioxidant natural products from Azadirachta indica leaves

Mangosteen (Garcinia mangostana L.) peel is a potential source of phenolic compounds with beneficial properties. Natural deep eutectic solvents (NaDES) have been considered an environmentally friendly and cheap alternative to conventional organic solvents. In this work, a green extraction methodology was developed using ultrasound-assisted extraction (UAE) and NaDES for the extraction of antioxidant non-extractable polyphenols (NEPs) from mangosteen peel. To select the best NaDES to extract NEPs from mangosteen peel, seven NaDES were studied. Antioxidant capacity and total phenolic and proanthocyanidin contents were determined for the extracts. The molecular weights for the NEPs present in those extracts were evaluated by size exclusion chromatography. Experimental results showed that choline chloride–lactic acid (1:2) was the NaDES allowing the highest antioxidant proanthocyanidin content in the extracts. A Box–Behnken experimental design was employed to optimize the main parameters in UAE with NaDES: water percentage, ultrasound amplitude, and extraction time. The optimal extraction conditions were 18.8% (v/v) water, 60% ultrasound amplitude, and 15 min as the extraction time. In addition, the cytotoxicity of the NEP extracts obtained under optimal extraction conditions was evaluated. Results indicated for the first time that the use of NaDES in combination with UAE could be a sustainable alternative for the extraction of antioxidant NEPs from mangosteen peel for important applications in the food, pharmaceutical, agrochemical, and cosmetic fields, as the extracts presented low cytotoxicity.

Green extraction of bioactive compounds from apple pomace by ultrasound assisted natural deep eutectic solvent extraction: Optimisation, comparison and bioactivity

Comparison, optimization and antioxidant activity of ultrasound-assisted natural deep eutectic solvents extraction and traditional method: A greener route for extraction of flavonoid from Moringa oleifera Lam. leaves