Development of Naringin Extract from Pomelo Peel Using Natural Deep Eutectic Solvent System as Green Technology for Antidiabetic Purpose: Box-Behnken design approach

Development of Naringin Extract from Pomelo Peel Using Natural Deep Eutectic Solvent System as Green Technology for Antidiabetic Purpose: Box-Behnken design approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of Naringin Extract from Pomelo Peel Using Natural Deep Eutectic Solvent System as Green Technology for Antidiabetic Purpose: Box-Behnken design approach Thongtham Suksawat, Natnicha boonthaworn, Yanisa Junseedeechai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6892292/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Journal of Pharmaceutical Innovation → Version 1 posted You are reading this latest preprint version Abstract Diabetes remains a critical public health issue, intensifying the demand for safe, affordable, and eco-friendly therapeutic alternatives. Pomelo peel, rich in the flavonoid naringin, offers promising antidiabetic potential. This study introduces a novel green extraction approach using natural deep eutectic solvents (DES), offering a safer alternative to organic solvents. Among six Thai pomelo cultivars, Khao Nam Phueng exhibited the highest naringin content (4.28% w/w dry weight), significantly surpassing Khao Yai (2.74% w/w DW). Regional variation was also observed: peels from Nakhon Pathom yielded 4.01%, compared to only 1.62% from Pathum Thani. Seasonal decline was evident, with early-harvest fruit showing nearly double the naringin content of late-season samples. Initial screening identified choline chloride:citric acid (1:1) as the most effective DES, extracting 0.19%w/w DW naringin, followed by malic acid (0.18% w/w DW) and oxalic acid (0.13%w/w DW). Optimization using Box–Behnken design improved yield to 0.28% w/w. The extract displayed potent α-glucosidase inhibition (IC₅₀=9.99 µg/mL) and strong antioxidant activity (FRAP=192.3 FEAC/µg DW). Antiglycation activity was moderate (IC₅₀=104.75 µg/mL), lower than pure naringin (IC₅₀=36.77 µg/mL), likely due to solvent-matrix interactions. Predictive response surface models showed high accuracy, and the extract remained chemically stable over six months with only slight degradation (0.24–0.25% w/w). This work demonstrates a novel, scalable method to valorize agro-industrial pomelo peel waste into standardized, high-quality antidiabetic extracts using sustainable DES technology. These findings support broader applications of DES in green phytopharmaceutical development and highlight cultivar and harvest timing as key factors in maximizing bioactive compound recovery. Anti-α-glucosidase activity assay Antiglycation assay DES Naringin pomelo peel Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Diabetes is a rapidly growing global health concern, particularly in countries with diets rich in sugar and fat. While modern medications are effective in managing diabetes, their high cost and risk of severe side effects have led to the exploration of alternative treatments, including herbal products. Herbal remedies are generally more affordable and pose fewer risks than conventional medications, making them an attractive option for diabetes management [ 1 ]. However, challenges such as the high cost of purified herbal compounds and the lack of quality control in herbal extracts, particularly in standardizing active compound concentrations, limit their widespread adoption. Addressing these issues requires the development of cost-effective, high-yield extraction methods and robust quality control measures to ensure the safety and efficacy of herbal products for diabetes patients [ 2 ]. Pomelo ( Citrus grandis L.) peel, a traditional ingredient in Thai medicine, has shown promise as a natural remedy for diabetes. It has been used in formulations like "Eight Peel Compound" and "Yahom Thepachit" and contains bioactive compounds such as naringenin and naringin, which belong to the flavonoid group. These compounds exhibit antidiabetic properties, including lowering blood sugar levels, reducing insulin resistance, improving glucose tolerance, and inhibiting key enzymes like α-glucosidase and α-amylase [ 3 ]. However, the production of purified forms of these compounds is associated with high costs and often relies on organic solvents that pose environmental and health hazards. Thailand is a significant producer of pomelo, with widely cultivated varieties such as Khao Thong Dee, Khao Nam Pueng, and Tubtim Siam. Among these, Khao Thong Dee is the most prevalent, with major growing areas in provinces like Nakhon Pathom, Nakhon Si Thammarat, and Pathum Thani. Research indicates that the naringin content in pomelo peel decreases after harvest, highlighting the importance of timely and efficient extraction processes. Traditional extraction methods, such as liquid-liquid or solid-liquid extraction using solvents like ethanol and methanol, have been effective but are increasingly being replaced by modern, eco-friendly techniques [ 4 ]. Advances in extraction technologies, including ultrasonic-assisted extraction, supercritical fluid extraction, and deep eutectic solvents (DES), offer sustainable and efficient alternatives. These methods reduce solvent usage, enhance yield, and minimize environmental impact, aligning with green technology principles. For example, DES, composed of ionic liquids acting as hydrogen bond donors and acceptors, provide superior extraction efficiency and bioactivity enhancement compared to traditional solvents [ 5 ]. This study focuses on the development of a novel high-yield extraction methods for naringin from pomelo peel using green technologies and Box-Behnken design approach. The aim is to enhance the quality and value of herbal raw materials and extracts, paving the way for the production of standardized and eco-friendly herbal products for diabetes management. 2. Materials and methods 2.1. Chemicals Fresh Citrus grandis (pomelo) fruits were sourced from various regions across Thailand, as detailed in the Supplementary Material. The cultivars included Khao Hom (KH), Khao Nam Phueng (KN), Khao Pan (KP), Khao Tang Kwa (KT), Khao Thong Dee (KD), and Khao Yai (KY). The KN variety was additionally sourced from three distinct locations: Nakhon Pathom (NP), Nakhon Si Thammarat (NS), and Pathum Thani (PT). The naringin standard (purity > 98%) was purchased from Sigma Aldrich (Merck, Germany). All other reagents and chemicals used in this study were of analytical grade and were purchased from Tokyo Chemical Industry Co., Ltd. (TCI, Japan). 2.2. Plant material preparation Fruits of C. grandis (pomelo) were collected from local farms located in various regions across Thailand over three separate batches during May, June, and July 2024, as detailed in the Supplementary Material. Each cultivar was authenticated based on its geographical origin and morphological characteristics, following the criteria established in a previous study [ 4 ]. The fruits were thoroughly washed, and the pericarps were manually peeled and collected. The pericarps were then dried in a hot air oven at 50°C for 24 hours, ground using a mechanical grinder, and passed through a No. 45 mesh sieve. The resulting powder was stored in airtight, light-resistant containers until further analysis. For subsequent experiments, the pomelo variety with the highest naringin content; determined from the three collected batches; was selected. This variety was also sourced from different regions in Thailand, as documented in the Supplementary Material. 2.3. Pomelo peel extraction The pomelo peel powder from each source was extracted using 80% ethanol at a solvent to powder ratio of 20:1. The extraction was performed using the microwave-assisted technique with a microwave extractor (800 watts) until the temperature reached 72°C. This method was compared with the traditional maceration technique, where the samples were extracted with 80% ethanol over 7 days. The extracts were then analyzed for naringin content in each sample using high-performance liquid chromatography (HPLC). 2.4. Analysis of naringin content in pomelo peel extracts using HPLC The naringin content in the pomelo peel extracts was analyzed using a validated HPLC method, following the protocol described in a previous study [ 6 ]. Naringin standard solutions were prepared at concentrations ranging from 3.125 to 100 µg/mL and filtered through a syringe filter. For sample preparation, 20 µL of each extract was dissolved in 1 mL of methanol for every 20 mg of sample, using 70% methanol as the solvent. The resulting solution was filtered through a syringe filter before analysis. The HPLC system was operated with a detection wavelength of 280 nm, a column temperature of 25°C, and a flow rate of 1 mL/min. The mobile phase consisted of 0.3% acetic acid in water (solvent A) and acetonitrile (solvent B) with the following gradient program: 0 min, 5% solvent B; 5 min, 25% solvent B; 10 min, 45% solvent B; and 35 min, 50% solvent B. The results were compared with a standard curve prepared using naringin standard; Y = 0.0852X + 2.183, r² = 0.9999 (Fig. 1 ). 2.5. DES extraction of pomelo peel 2.5.1. Preliminary of DES extraction from selected source Three different DES were prepared for pomelo peel extraction, each consisting of a 1:1 molar ratio of choline chloride combined with citric acid, oxalic acid, or malic acid. The hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) components were mixed with water and gently stirred on a hot plate at temperatures not exceeding 60°C until a homogeneous, transparent solution was obtained. The prepared DES mixtures were stored in sealed, light-protected containers at 4°C until use. For extraction, 1 g of dried pomelo peel powder was combined with 20 mL of each DES (solvent to powder ratio 20:1) and subjected to ultrasonic-assisted extraction at 60°C for 60 minutes. The resulting extracts were filtered through cotton using a syringe and stored at 4°C in light-protected containers for subsequent phytochemical analysis by HPLC. 2.5.2. Optimization of DES extraction 2.5.2.1. Experimental design A Box-Behnken central experimental design was applied to optimize the DES extraction of naringin from pomelo peel. This design utilized three independent variables, each tested at three levels (-1, 0, + 1): the molar ratios of malic acid (X1), the molar ratios of citric acid (X2) from prior studies, and the powder-to-solvent ratio (X3). The response variables measured were naringin content (Y1) and anti-α-glucosidase activity (Y2), antioxidant (FRAP) activity (Y3), and polarity (Y4) of the DES extract. The experimental setup (Supplement Material), varied the DES molar ratio (1:1, 1:2, and 1:3) and solvent to powder ratio (10:1, 15:1, and 20:1). The experimental data were analyzed using response surface methodology to fit a second-order polynomial model, as described in Eq. 1: Y n = β 0 + β 1 X 1 + β 2 X 2 + β 3 X 3 + β 12 X 1 X 2 + β 13 X 1 X 3 + β 23 X 2 X 3 + β 11 X 1 2 + β 22 X 2 2 + β 33 X 3 2 (1) Where Y represents the response variables, β 0 is the constant coefficient, β 1 , β 2 , and β 3 are the linear coefficients, β 12 , β 13 , and β 23 are the interaction coefficients, and β 11 , β 22 , and β 33 are the quadratic coefficients. The independent variables X 1 , X 2 , and X 3 represent the molar ratios of the DES components and the solvent to powder ratio. A regression model was constructed to describe the relationships between the input variables (DES component ratio and solvent to powder ratio) and the responses. Statistical analysis was performed to identify significant variables for optimizing the extraction conditions, with the goal of selecting extracts with the highest naringin content, anti-α-glucosidase activity, antioxidant (FRAP) activity, and polarity for antiglycation determination. 2.6. Bioactivity test 2.6.1. Anti-α-glucosidase activity assay The α-glucosidase enzyme inhibition activity was tested using 0.1 M sodium phosphate buffer (pH 6.9) as the primary solution. A stock solution of the enzyme was prepared at a concentration of 2 U/mL in the buffer and stored at -20°C. A working solution with a concentration of 0.1 U/mL was prepared from the stock solution. p-nitrophenyl α-D-glucopyranoside (pNPG) at 2 mM in buffer was used as the substrate, with acarbose as the positive control and the buffer as the negative control. All DES extracts were tested as sample groups, and placebo DES (DES without pomelo extract) was included to confirm the absence of inherent enzyme inhibition. The reaction was incubated and stopped by adding Na₂CO₃. Absorbance was measured at 405 nm, and the percentage inhibition and IC₅₀ values were calculated using the equation: \(\:\text{P}\text{e}\text{r}\text{c}\text{e}\text{n}\text{t}\text{a}\text{g}\text{e}\:\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}=[1-\frac{\left({OD}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\text{}-{OD}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{b}\text{l}\text{a}\text{n}\text{k}}\text{}\right)}{\left({OD}_{\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}\text{}-{OD}_{\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{b}\text{l}\text{a}\text{n}\text{k}}\right)\text{}\text{}}]\times\:100\) 2.6.2 Antiglycation assay The antiglycation assay was performed in a 96-well plate. Each well contained bovine serum albumin (BSA), glucose anhydrous, magnesium oxide, and DES extract. The glycated control contained BSA, glucose, and sodium phosphate buffer with NaN₃, while the blank control consisted of BSA and sodium phosphate buffer. Samples were incubated at 37°C for 14 days. Trichloroacetic acid (TCA, 100%) was added, and the mixture was centrifuged. The pellet was washed with 5% TCA, dissolved in phosphate-buffered saline (PBS), and analyzed for advanced glycation end-products (AGEs) using a spectrofluorometer with excitation at 370 nm and emission at 440 nm. Rutin served as the positive control. All DES extracts were tested as sample groups, and placebo DES (DES without pomelo extract) was included to confirm the absence of inherent enzyme inhibition. The percentage inhibition was calculated using the formula: \(\:\text{P}\text{e}\text{r}\text{c}\text{e}\text{n}\text{t}\text{a}\text{g}\text{e}\:\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}=[1-(\frac{\text{F}\text{l}\text{u}\text{o}\text{r}\text{e}\text{s}\text{c}\text{e}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}{\text{F}\text{l}\text{u}\text{o}\text{r}\text{e}\text{s}\text{c}\text{e}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{e}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}}\left)\text{}\right]\times\:100\) . 2.6.3. Ferric reducing antioxidant power (FRAP) assay The antioxidant capacity of the test samples was assessed using the FRAP assay, with Trolox serving as the reference antioxidant standard. A Trolox stock solution (1 mg/mL in methanol) was serially diluted to obtain standard concentrations ranging from 0.47 to 30 µg/mL. The FRAP reagent was freshly prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl, and 20 mM ferric chloride (FeCl₃) in a 10:1:1 (v/v/v) ratio. For each assay, 20 µL of the sample or standard was added to 180 µL of FRAP reagent in a 96-well microplate and incubated at room temperature for 30 minutes. Absorbance was measured at 593 nm using a microplate reader. Appropriate blanks and negative controls were included in each run. All DES extracts were tested as sample groups, and placebo DES (DES without pomelo extract) was included to confirm the absence of inherent enzyme inhibition. Antioxidant activity was calculated based on the Trolox calibration curve and expressed as Trolox equivalent antioxidant capacity (TEAC) per µg of dry sample weight. 2.7 Stability study of DES extracts The stability of the extracts was evaluated under different conditions: 30°C/60% RH and 40°C/75% RH for 1, 2, 3, and 6 months. After each period, the extracts were analyzed for naringin content using HPLC. 2.8 Statistical analysis All experimental data were analyzed using SPSS version 28.0 (IBM Corp., USA) and Design-Expert version 13.0 (Stat-Ease Inc., USA). Analysis of variance (ANOVA) was employed to determine the significance of the model and the individual factors. The coefficient of determination (R²) and the adjusted R² (Adj-R²) were used to assess the model's goodness-of-fit and predictive capability. Differences between means were considered statistically significant at p < 0.05. All experiments were performed in triplicate, and the results are presented as mean ± standard deviation (SD). 3. Results and discussion 3.1 Effect of different sources and variety of pomelo on naringin content Highly significant differences in peel naringin content (%w/w dry weight, DW) were observed among pomelo varieties and locations, along with a significant variety-by-month interaction (Figure 2A). Interestingly, certain varieties consistently accumulated higher levels of naringin across all locations, and fruits harvested earlier in the season exhibited greater naringin content. Specifically, the KN variety showed the highest mean naringin content (4.28%w/w DW), significantly exceeding that of KY (2.74%w/w DW). Overall, the mean naringin content across varieties followed the order: KN > KP ≈ KT > KD ≈ KY > KH. Location also had a modest but statistically significant effect: peels from NP (4.01%w/w DW) contained approximately 2.5 times more naringin than those from PT (1.62%w/w DW), with NS (2.33%w/w DW) yielding intermediate values. NP was the further analysis for location and harvesting period influnce. Notably, NP varitey, a clear decreasing trend in naringin content was observed across the three sampling months (from early to late harvest), with content at the first sampling approximately twice that of the final one. However, this seasonal trend was not evident in the other two locations (Figure 2). These results indicate that naringin accumulation in pomelo peel is influenced both by genetic (varietal) factors and by environmental or phenological factors. The higher naringin content in KN suggests intrinsic genetic differences in flavanone biosynthesis among varieties. This pattern is consistent with other citrus studies showing large cultivar-to-cultivar variation in flavonoid profiles. For instance previous study reported that Thai pomelo cultivars exhibited naringin levels in the pulp ranging from 25.20 to 39.42 mg per 100 g fresh weight, implying corresponding peel levels in the range of 0.8–3.5%w/w DW when extrapolated [7]. Our observed peel concentrations (approximately 3.28–4.28%w/w DW) fall within the same order of magnitude as those prior reports. Moreover, the finding that naringin is concentrated in the peel aligns with established citrus chemistry: one study reported that pummelo peel contained up to 3910 μg/mL of naringin, compared to only 220 μg/mL in the juice, reflecting the compound’s accumulation in rind tissues [8]. The observed seasonal variation in naringin content is also well-documented in the literature. Immature fruit tends to accumulate higher flavonoid levels, which decline as the fruit matures. Similar seasonal dynamics suggested that naringin content in immature honey pomelo peel decreased from 3.45%w/w DW in April to 1.77%w/w DW in May. Such dramatic reductions are typically due to developmental regulation of flavonoid biosynthesis, wherein juvenile peel tissues accumulate more naringin (imparting bitterness), which is subsequently diluted or metabolized during fruit ripening. This aligns with broader reviews noting that "immature fruit has a higher concentration of naringin" and emphasizing the importance of maturity stage in determining citrus fruit bitterness [9]. Location-based differences in naringin content may reflect microclimatic and edaphic influences, possibly due to cooler night temperatures, different soil properties, or differences in orchard management. Environmental factors such as light intensity, temperature, and water availability are known to influence phenolic and flavonoid metabolism in citrus. Plants exposed to higher UV light often upregulate antioxidant flavonoids like naringin. Conversely, lower sunlight exposure or nutrient-deficient soils can reduce such biosynthetic activity [10]. Although this study did not directly assess climatic variables, the consistent trends across sampling months suggest that environmental and agronomic factors, alongside genetics, influence naringin variation. In summary, naringin content in pomelo peel varies significantly by variety, location, and harvest time, supporting earlier findings in citrus research. Selecting high-naringin cultivars like Khao Nam Phueng and optimizing harvest timing can help maximize yields. Future work should focus on identifying the molecular and environmental mechanisms regulating naringin biosynthesis. 3.2 Preliminary of DES extraction from selected source Preliminary screening of three choline chloride-based DES revealed notable differences in their efficiency to extract naringin from the selected pomelo peel source (Table 1). Among the solvents tested, the combination of choline chloride and citric acid yielded the highest naringin content at 0.191%w/w dry weight (DW), followed by choline chloride:malic acid (0.183%w/w DW) and choline chloride:oxalic acid (0.126%w/w DW). These results suggest that the choice of HBD in the DES formulation significantly affects the solubilization and extraction efficiency of flavonoids such as naringin. Citric acid, being a tricarboxylic acid with a relatively higher molecular weight and multiple carboxyl and hydroxyl groups, may form more stable hydrogen bonding networks with choline chloride, enhancing its solvating power toward glycosylated flavonoids [11]. Malic acid, while also effective, exhibited greater variability in extraction efficiency, possibly due to its lower acidity and smaller molecular structure. In contrast, the oxalic acid-based DES demonstrated the lowest extraction capacity. Oxalic acid’s strong acidity and smaller molecular size may lead to a more compact solvent network with reduced solubilization capacity for large polar molecules like naringin. The relatively higher extraction efficiency of ChCl:citric acid is consistent with previous studies reporting its favorable performance in extracting flavonoids and polyphenols from plant materials. Furthermore, its biocompatibility and low toxicity make it a promising candidate for food-grade or nutraceutical applications [12]. These findings indicate that ChCl:citric acid is a suitable DES system for further optimization in the green extraction of naringin, potentially offering an eco-friendly alternative to conventional organic solvents such as methanol or ethanol. Table 1. Naringin content extracted from pomelo peel using different DES components DES Naringin content (%w/w DW) ChCl: citric acid 0.191 ± 0.002 a ChCl: oxalic acid 0.126 ± 0.001 b ChCl: malic acid 0.183 ± 0.05 a Note. ChCl = Choline chloride; HBD = hydrogen bond donor; DW = dry weight. Different superscript letters within the same column indicate statistically significant differences (p<0.05). 3.3 Effect of independent variables on response variables This study employed response surface methodology (RSM) based on a Box–Behnken design to optimize the extraction conditions of naringin from pomelo peel using DES (Figure 3). The independent variables included the molar ratio of malic acid (X₁), citric acid (X₂), and the solvent to powder ratio (X₃), while the responses were naringin content (Y₁), anti-α-glucosidase activity (Y₂), antioxidant activity by FRAP assay (Y₃), and polarity (Y₄). The relationship between each response and the three independent variables was described using a second-order polynomial regression model. The predicted and experimental values were closely matched, confirming model accuracy. Naringin content (Y 1 ) The quadratic regression model for naringin content (Y₁) was found to be highly significant, exhibiting an excellent coefficient of determination (R²) and adjusted R², which together confirm the strong predictive performance of the second-order model. Moreover, the lack-of-fit test was not significant, indicating that the model adequately fits the experimental data. While the linear terms for malic acid ratio (X₁) and citric acid ratio (X₂) did not significantly influence naringin content, the solvent-to-powder ratio (X₃) contributed positively to the model. These findings align with previous DES optimization studies, where the solvent-to-powder ratio has been shown to exert both strong linear and nonlinear effects [13]. As illustrated in Figure 3, the 3D response surface plot demonstrated that naringin content increased with decreasing solvent-to-powder ratios. This trend reflects the combined interaction and quadratic effects, with a slight decline in yield observed at excessively high solvent-to-powder ratios. The quadratic model in actual factor terms was Y₁=0.99−0.07X 3 +0.002X 3 2 Anti-α-glucosidase activity (Y₂) The linear model for anti-α-glucosidase activity (Y₂) was also statistically significant, with a high R², adjusted R², and non-significant lack-of-fit. Significant linear terms, particularly solvent to powder ratio revealed diminishing returns at high activity against α-glucosidase activity. Since α-glucosidase inhibition is directly related to naringin concentration, the trends in Y₂ closely mirrored Y₁. The 3D response surface showed that maximum inhibition occurred in the high X₁ and X₂ region, with a broad plateau. Unlike Y₁, inhibition approached 100% without strong decline, though curvature remained evident. X₃ had a moderate positive effect. These results agree with previous reports highlighting naringin as a major contributor to bioactivity [14]. The linear model in actual factor terms was Y₂=31.02−1.16X 3 Antioxidant activity (FRAP)(Y₃) The antioxidant activity, measured using the FRAP assay (Y₃), was effectively modeled using a statistically significant second-order polynomial equation. The model demonstrated strong explanatory power and passed the lack-of-fit test, confirming its adequacy. The quadratic terms for malic acid ratio (X₁) and citric acid ratio (X₂) were significant contributors, with both the interaction term (X₁X₂) and the quadratic terms (X₁² and X₂²) showing significant influence. These findings suggest the existence of an optimal concentration range for both acids. While increasing acid content initially enhanced antioxidant activity, excessive levels led to a plateau or slight decline, possibly due to the degradation of acid-sensitive antioxidant compounds. In particular, the negative coefficient of X₂² indicates that very high citric acid concentrations may be detrimental to antioxidant efficacy. The 3D response surface plot for Y₃ displayed a characteristic hill-shape, with peak antioxidant activity occurring at moderate-to-high values of X₁ and X₂. Increasing the solvent to powder ratio (X₃) also improved antioxidant activity, likely by enhancing mass transfer; however, the benefit diminished at higher levels. Notably, the highest FRAP values did not coincide with maximum acid concentrations, but rather overlapped with the optimal regions for naringin content (Y₁) and extraction yield (Y₂), indicating a balance between these variables. Interestingly, these results diverge from those observed for α-glucosidase inhibitory activity, suggesting that the antioxidant effects may be attributable to other bioactive flavonoids beyond naringin, such as hesperidin, narirutin, and eriocitrin [15]. The quadratic model in actual factor terms was Y 3 =456.46−180.00X 1 −58.97X 2 −9.52X 3 +1.25X 1 X 3 −2.04X 2 X 3 +39.43X 1 2 +20.64X 2 2 +0.32X 3 2 Polarity (Y₄) The quadratic regression model developed to predict solvent polarity based on malic acid, citric acid, and solvent-to-powder ratio was found to be statistically non-significant , indicating that the model lacks predictive strength for this particular response. Additionally, none of the linear, interaction, or quadratic terms reached statistical significance, although the quadratic term for citric acid approached borderline significance, suggesting a potential nonlinear relationship worth further exploration. While the model's predictive utility is limited, the equation offers insight into the general directionality of the effects. For example, the positive coefficients for the linear terms of malic acid, citric acid, and solvent-to-powder ratio suggest that increases in these factors may correspond to slight increases in polarity. However, the negative quadratic term for citric acid (–0.42) implies that extremely high concentrations may reduce solvent polarity, which is chemically plausible due to changes in hydrogen bonding and ionic strength at elevated acid levels. These results suggest that solvent polarity, as measured or defined in this context, is not strongly influenced by the selected formulation variables within the tested range. It is possible that the measurement method for polarity was either too insensitive or not directly affected by these components. Alternatively, the design space may have been too narrow to observe meaningful variation in this response. Overall, the response surface models developed in this study effectively captured the relationships between extraction variables and key response outcomes. The solvent to powder ratio consistently emerged as a critical factor across bioactivity responses, while citric and malic acid ratios exhibited more complex, nonlinear effects. In contrast, the model for polarity was not statistically significant, suggesting that polarity, as measured here, may not be strongly influenced by the studied formulation parameters or that its measurement method lacked sensitivity. These findings underscore the potential of DES optimization for enhancing functional compound extraction, while also highlighting the need for further investigation into physicochemical descriptors like polarity. Table 2. The ANOVA results of response surface model for naringin content, anti-a-glucosidase activity, antioxidant, and Polarity of pomelo peel DES extract Source Df naringin content (%w/w DW) anti-a-glucosidase activity (IC 50 , mg/mL) antioxidant (FRAP) (FEAC/mg DW) Polarity Model Quadratic Linear Quadratic Linear F-value p -value F-value p -value F-value p -value F-value p -value Model 9 49.3 <0.05 * 26.67 < 0.05 * 393.99 <0.05 * 1.13 0.4708 X 1 -Malic acid 1 1.86 0.2312 0.1346 0.7206 72.14 <0.05 * 0.5009 0.5107 X 2 -Citric acid 1 2.67 0.1630 0.5729 0.4650 217.86 < 0.05 0.0500 0.8319 X 3 - C-Solvent to powder ratio 1 395.72 <0.05 * 79.29 <0.05 * 151.17 <0.05 * 0.3920 0.5587 X 1 X 2 0.5941 0.4757 0.5098 0.5072 0.4465 0.5336 X 1 X 3 1.34 0.2999 64.35 <0.05 * 0.5476 0.4926 X 2 X 3 0.5941 0.4757 171.71 <0.05 * 1.39 0.2915 X 1 2 0.0952 0.7701 2370.23 <0.05 * 0.0553 0.8234 X 2 2 0.0038 0.9532 649.35 <0.05 * 6.15 0.0559 X 3 2 40.40 <0.05 * 100.55 <0.05 * 0.7761 0.4187 Lack of fit 9 7.75 0.1164 3.58 0.2373 1.07 0.5155 0.1279 0.9354 R 2 Adj R 2 0.9688 0.8461 0.9961 0.7602 *Significant at p<0.05, Df = degree of freedom. 3.4. Verification of predictive model To validate the reliability and predictive accuracy of the developed quadratic models, an experimental formulation was prepared using the optimized conditions: Malic acid molar ratio at 1, citric acid molar ratio at 1, and solvent to powder ratio at 12.24. The experimental values obtained for all three response variables; naringin content, anti-α-glucosidase activity, and antioxidant activity (FRAP); were compared against the model-predicted values and their 95% prediction intervals (Table 3). The observed naringin content (0.28% w/w DW) was closely aligned with the predicted value (0.30% w/w DW) and fell within the prediction interval of 0.26–0.34, indicating good model accuracy. Similarly, the observed anti-α-glucosidase activity (IC₅₀= 9.99 µg/mL) showed acceptable agreement with the predicted value (16.10 µg/mL), although it was near the upper bound of the prediction interval (12.74–19.46 µg/mL). This slight deviation could be attributed to biological variability or matrix effects during formulation. In contrast, the antioxidant activity (FRAP assay) showed excellent correlation between predicted (199.50 FEAC/µg DW) and observed (192.31 FEAC/µg DW) values, though the observed mean slightly undershot the lower bound of the prediction interval, possibly due to degradation of labile antioxidants during extraction. Overall, the results confirm the robustness and validity of the optimization models in predicting key bioactive properties under practical formulation conditions. Minor discrepancies highlight the complexity of physicochemical interactions in DES but remain within acceptable experimental margins. The present model reliably predicts extraction outcomes for naringin and related activities from pomelo peel. Its adequacy is supported by both the internal validation and consistency with literature reports of RSM-optimized DES extractions [16]. These findings reinforce the value of combining DES and RSM for green extraction: not only are DES themselves sustainable solvents, but the optimized process yields high concentrations of target compounds while minimizing trial-and-error. Future work could further leverage this model to explore other DES formulations or scale-up extraction, confident in the predictive power demonstrated here. In conclusion, the Box–Behnken RSM model proved to be a robust tool for maximizing naringin recovery from pomelo peel, aligning with contemporary green extraction strategies and prior successful validations. Table 3. Confirmation of model predictions at malic acid molar ratio=1, citric acid molar ratio=1, solvent to powder ratio=12.24 (n=3) Response Predicted mean Observed mean 95% prediction interval naringin content (%w/w DW) 0.30 0.28 0.26-0.34 anti-a-glucosidase activity (IC 50 , mg/mL) 16.10 19.99 12.74-19.46 antioxidant (FRAP) (FEAC/mg DW) 199.50 192.31 195.08-203.92 Note: All observed values represent the average of three replicates. 3.5. FT-IR spectrum analysis of DES extract The FTIR spectra presented in Figure 4 compare (A) naringin extracted with DES, (B) the DES solvent alone (choline chloride, malic acid, and citric acid), (C) pure naringin, and (D–F) individual components of the DES: choline chloride, malic acid, and citric acid, respectively. The spectrum of pure naringin (Figure 4C) exhibits characteristic peaks, including a broad O–H stretching vibration around 3416 cm⁻¹, attributed to phenolic and hydroxyl groups, and C–H stretching bands in the region of 2957–2859 cm⁻¹. A distinct carbonyl stretching band appears near 1630 cm⁻¹, corresponding to the flavanone structure of naringin, while the peaks at approximately 1470 cm⁻¹ and 1361 cm⁻¹ are related to aromatic C=C stretching and phenolic ring vibrations, respectively. The region between 1240–1139 cm⁻¹ displays C–O stretching, including contributions from ether and glycosidic linkages in the naringin molecule [17]. Importantly, the spectrum of the naringin extract in DES (Figure 4A) retains all major characteristic peaks of pure naringin with no observable shifts in wavenumber, indicating that the molecular structure of naringin remains intact after extraction. The O–H stretch, C=O vibration, and aromatic peaks are all clearly visible and match those of the reference compound (Figure 4C), confirming that the DES system effectively extracted naringin without causing structural modification. The lack of any new peaks or significant shifts further supports the absence of chemical interaction between naringin and DES components at the molecular level. In contrast, the FTIR spectrum of the DES mixture without naringin (Figure 4B) shows a broad O–H stretching band around 3360 cm⁻¹, attributed to strong hydrogen bonding among citric acid, malic acid, and choline chloride. Carbonyl stretching bands from the carboxylic acids appear around 1743 and 1691 cm⁻¹, and C–O stretching bands are observed in the 1240–1139 cm⁻¹ range [18]. These peaks are distinct from those of naringin and do not interfere with the identification of naringin in the extract. This comparison confirms that the functional groups of naringin in the extract (Figure 4A) originate from the flavonoid itself and not from the DES matrix. The individual spectra of choline chloride (D), malic acid (E), and citric acid (F) also exhibit their respective characteristic peaks, such as broad O–H stretching, C–H bending, and C=O stretching, yet none show the distinct aromatic or flavanone-related bands present in pure naringin [18]. Thus, the FTIR results clearly demonstrate the successful extraction of naringin by the DES system, with the compound retaining its structural integrity. These findings align with previous studies where unchanged spectral positions indicated the absence of chemical transformation post-extraction. 3.6. Antiglycation activity The BSA–glucose assay revealed that pure naringin exhibited potent antiglycation activity with an IC₅₀ value of 36.77 µg/mL. In contrast, the naringin formulated in DES showed significantly reduced activity, with an IC₅₀ of 104.75 µg/mL. The DES placebo, which contained no naringin, exhibited negligible antiglycation activity (IC₅₀>1000 µg/mL). The differences between pure naringin and its DES formulation were statistically significant and both were significantly different from the placebo group. The positive control, aminoguanidine, effectively inhibited protein glycation at low concentrations, consistent with its established use as a standard glycation inhibitor. Flavonoids, including naringin, are known to inhibit the formation of advanced glycation end-products (AGEs) primarily through their antioxidant and free-radical scavenging activities. Certain flavonoids, such as luteolin, quercetin, and rutin, can inhibit AGE formation more effectively than aminoguanidine at 10 mM concentration, suggesting that natural polyphenols can be superior antiglycation agents [19]. The IC₅₀ value obtained for pure naringin in the current study compares favorably with previous findings, highlighting its strong potential as an inhibitor of protein glycation. However, the reduced antiglycation activity observed in the naringin–DES formulation may be attributed to physicochemical interactions within the solvent matrix. DES are widely recognized as green solvents capable of enhancing the solubility, stability, and even bioavailability of poorly water-soluble compounds. Despite these benefits, in the context of the in vitro BSA–glucose assay, the DES matrix may have limited naringin’s accessibility or reactivity. This could occur through molecular sequestration via hydrogen bonding, or by modifying key microenvironmental factors such as pH or viscosity. Another possible explanation is that the DES formulation diluted the effective concentration of naringin available to exhibit antiglycation activity [20]. Notably, the placebo DES (without naringin) showed no antiglycation effect, confirming that the solvent components themselves were biologically inert in this context. While DES can enhance solubility and protect bioactive compounds from degradation, the current findings highlight a potential drawback: they may also interfere with the interaction of active compounds and biological targets in vitro . Therefore, the selection of an appropriate solvent system is critical when formulating plant-derived compounds for biological assays or therapeutic use. Future research should focus on evaluating the pharmacokinetics and in vivo bioavailability of DES-formulated flavonoids to better understand their true therapeutic potential. 3.7. Stability study of DES containing naringin The average naringin content in DES extract stored at 30°C/75%RH and 40°C/75%RH over a 6-month period was presented in Figure 5. At time 0, the naringin content was 0.28%w/w DW. A progressive decline in naringin content was observed under both storage conditions. After 6 months, the content had decreased to 0.25%w/w DW at 30°C/75%RH and 0.24%w/w DW at 40°C/75%RH. Each storage condition indicated a significant effect of time on naringin content. At 6-month, naringin content values were significantly lower than the baseline and 1-month values under both storage conditions. This trend demonstrates that both temperature and duration significantly influenced the chemical stability of the DES extract. The bar graph (Figure 4) further illustrates this trend, showing a steeper decline under the 40°C/75%RH condition. The pattern of naringin degradation appeared consistent with first-order kinetics, as supported by a linear fit of ln (concentration) versus time (data not shown). This is in agreement with prior studies indicating that flavonoid degradation, including that of naringin and related glycosides, follows first-order kinetics under accelerated stability conditions [21]. The estimated half-lives under 30°C and 40°C conditions were approximately 8–10 months and 3–4 months, respectively, based on extrapolation. While DES are known to enhance the stability of phenolic compounds due to strong hydrogen bonding and reduced water activity, the observed degradation over 6 months was still substantial, particularly under accelerated conditions. These findings align with previous study which reported degradation of phenolic compounds in DES-based orange peel extracts over time, at a slower rate than in conventional solvents [22]. It is important to note that under the ICH Q1A(R2) guideline, a decrease greater than 5% in the assay value is considered a significant change during stability testing [23]. In this study, the extract lost more than 5% of its original naringin content by 3 months at 40°C and by 6 months at 30°C, indicating potential challenges in long-term storage stability. Therefore, product formulations using this DES extract would require stability-enhancing strategies, such as inclusion of antioxidants, encapsulation, or packaging under inert conditions to prolong shelf life. The practical implications are critical, particularly for botanical ingredients intended for pharmaceutical, nutraceutical, or cosmeceutical applications, which must comply with regulatory standards for shelf-life and active compound stability. Further kinetic modeling using Arrhenius equations and real-time stability testing would be valuable in determining accurate expiration dates. 4. Conclusions This study introduces a novel green extraction method using DES to recover naringin from pomelo peel, offering an environmentally safe alternative to conventional solvents. The approach efficiently converts pomelo peel; an agro-industrial byproduct; into a stable, multifunctional extract suitable for nutraceutical applications. Significant variation in naringin content was observed across different pomelo cultivars, growing regions, and harvest periods. The Khao Nam Phueng variety consistently exhibited the highest levels, while location and early-season harvesting were also critical factors influencing yield. These findings highlight the importance of cultivar selection and harvest timing in maximizing bioactive compound recovery. The optimized DES extract demonstrated strong in vitro bioactivities, including enzyme inhibition, antioxidant capacity, and antiglycation potential, while maintaining compound integrity and extract stability. The use of green solvents enables direct application of the extract in functional health products without the need for solvent removal. This work not only establishes a sustainable, scalable extraction method but also underscores the value of integrating agricultural biodiversity and harvest strategy into raw material standardization. Future research should focus on in vivo efficacy, formulation optimization, and regulatory assessment, as well as extending this green extraction platform to other phytochemical-rich plant residues. Declarations Funding sources This research project was also supported in part by the Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. Ethical statement This study does not contain any human or animal study. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. References M.M. Rahman, M.R. Islam, S. Shohag, et al., The multifunctional role of herbal products in the management of diabetes and obesity: a comprehensive review, Molecules 27 (2022) 1713 https://doi.org/https://doi.org/10.3390/molecules27051713. H. Wang, Y. Chen, L. Wang, et al., Advancing herbal medicine: enhancing product quality and safety through robust quality control practices, Frontiers in pharmacology 14 (2023) 1265178 https://doi.org/https://doi.org/10.3389/fphar.2023.1265178. L.-Y. Lin, C.-Y. Huang, K.-C. Chen, R.Y. Peng, Pomelo fruit wastes are potentially valuable antioxidants, anti-inflammatories, antihypertensives, and antihyperglycemics, Horticulture, Environment, and Biotechnology 62 (2021) 377-395 https://doi.org/https://doi.org/10.1007/s13580-020-00325-8. W. Makkumrai, Y. Huang, Q. Xu, Comparison of pomelo ( Citrus maxima ) grown in China and Thailand, Frontiers of Agricultural Science and Engineering 8 (2021) 335-352 https://doi.org/https://doi.org/10.15302/J-FASE-2021391. R.F. González-Laredo, V.I. Sayago-Monreal, M.R. 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Shin, A Comprehensive review of naringenin, a promising phytochemical with therapeutic potential, Journal of Microbiology and Biotechnology 34 (2024) 2425 https://doi.org/https://doi.org/10.4014/jmb.2410.10006. N.A. Estrada-Sierra, G. Rincon-Enriquez, J.E. Urías-Silvas, et al., Impact of ripening, harvest season, and the nature of solvents on antioxidant capacity, flavonoid, and p-synephrine concentrations in Citrus aurantium extracts from residue, Future Foods 6 (2022) 100153 https://doi.org/https://doi.org/10.1016/j.fufo.2022.100153. Y. Deng, S. Lu, Biosynthesis and regulation of phenylpropanoids in plants, Critical reviews in plant sciences 36 (2017) 257-290 https://doi.org/https://doi.org/10.1080/07352689.2017.1402852. O.S. Hammond, D.T. Bowron, A.J. Jackson, et al., Resilience of malic acid natural deep eutectic solvent nanostructure to solidification and hydration, The Journal of Physical Chemistry B 121 (2017) 7473-7483 https://doi.org/https://doi.org/10.1021/acs.jpcb.7b05454. I. Ahmad, S.O.P. Shakti, W.C. Prabowo, et al., Citric acid-glycerol-based NADES for microwave-assisted extraction enhances the polyphenols level of Eleutherine bulbosa mill. Urb. Bulbs, J. Appl. Pharm. Sci. 14 (2024) 189-197 https://doi.org/https://doi.org/10.7324/JAPS.2024.180291. N.C. Predescu, C. Papuc, V. Nicorescu, et al., The influence of solid-to-solvent ratio and extraction method on total phenolic content, flavonoid content and antioxidant properties of some ethanolic plant extracts, Revista de Chimie 67 (2016) 1922-1927. K. Zhang, Z. Ding, W. Duan, et al., Optimized preparation process for naringenin and evaluation of its antioxidant and α‐glucosidase inhibitory activities, Journal of Food Processing and Preservation 44 (2020) e14931 https://doi.org/https://doi.org/10.1111/jfpp.14931. R. Tocmo, J. Pena‐Fronteras, K.F. Calumba, et al., Valorization of pomelo ( Citrus grandis Osbeck) peel: A review of current utilization, phytochemistry, bioactivities, and mechanisms of action, Compr. Rev. Food Sci. Food Saf 19 (2020) 1969-2012 https://doi.org/ https://doi.org/10.1111/1541-4337.12561. I. Gupta, S.N. Adin, M. Aqil, M. Mujeeb, Qbd based extraction of naringin from Citrus sinensis L. Peel and its antioxidant activity, Pharmacognosy research 15 (2023) https://doi.org/ https://doi.org/10.5530/097484900241. A. Semalty, M. Semalty, D. Singh, M. Rawat, Preparation and characterization of phospholipid complexes of naringenin for effective drug delivery, Journal of inclusion phenomena and macrocyclic chemistry 67 (2010) 253-260 https://doi.org/https://doi.org/10.1007/s10847-009-9705-8. M.A. Ali, M.A. Kaium, S.N. Uddin, et al., Elucidating the structure, dynamics, and interaction of a choline chloride and citric acid based eutectic system by spectroscopic and molecular modeling investigations, ACS omega 8 (2023) 38243-38251 https://doi.org/https://doi.org/10.1021/acsomega.3c04570. Y. Xiao, J. Shen, J. Zhan, et al., Antiglycating effects of citrus flavonoids and associated mechanisms, Food Science and Human Wellness 13 (2024) 2363-2372 https://doi.org/https://doi.org/10.26599/FSHW.2022.9250247. G. Zengin, M.d.l.L. Cádiz-Gurrea, Á. Fernández-Ochoa, et al., Selectivity tuning by natural deep eutectic solvents (NADESs) for extraction of bioactive compounds from Cytinus hypocistis —studies of antioxidative, enzyme-inhibitive properties and LC-MS profiles, Molecules 27 (2022) 5788 https://doi.org/https://doi.org/10.3390/molecules27185788. L.M. Cordenonsi, N.G. Bromberger, R.P. Raffin, E.E. Scherman, Simultaneous separation and sensitive detection of naringin and naringenin in nanoparticles by chromatographic method indicating stability and photodegradation kinetics, Biomedical chromatography 30 (2016) 155-162 https://doi.org/https://doi.org/10.1002/bmc.3531. M. Panić, M. Andlar, M. Tišma, et al., Natural deep eutectic solvent as a unique solvent for valorisation of orange peel waste by the integrated biorefinery approach, Waste management 120 (2021) 340-350 https://doi.org/https://doi.org/10.1016/j.wasman.2020.11.052. D.J. Mazzo, The ICH stability guideline. International Stability Testing, CRC Press, 2020, pp. 1-13. Additional Declarations No competing interests reported. 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The geographic sources of pomelo are abbreviated as follows: NP=Nakhon Pathom, NS=Nakhon Si Thammarat, and PT=Pathum Thani.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6892292/v1/03d1c508e70d8a3f8c0b7ad7.png"},{"id":85205760,"identity":"20b7ae81-9a58-49a5-93c5-67009e847187","added_by":"auto","created_at":"2025-06-23 11:27:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":340529,"visible":true,"origin":"","legend":"\u003cp\u003e3D surface plot of (A-C) naringin content (%w/w DW); (D-F) anti-a-glucosidase activity (IC\u003csub\u003e50\u003c/sub\u003e, mg/mL); (G-I) antioxidant (FRAP) (FEAC/mg DW); and (J-L) Polarity\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6892292/v1/cdbcf75362ea85fc7b01fb71.png"},{"id":85205997,"identity":"4be73f81-dce2-44f2-b9ad-9d903cdb122a","added_by":"auto","created_at":"2025-06-23 11:35:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":85853,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectrum analysis of A: naringin DES extract, B: DES of choline chloride, malic and citric acid, C: pure naringin, D: choline chloride, E: malic acid, and F: citric acid\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6892292/v1/bf15a2f2e40849c4ab104de5.png"},{"id":85204476,"identity":"c4e0e458-5911-4c38-99ea-0943155ac396","added_by":"auto","created_at":"2025-06-23 11:11:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":46367,"visible":true,"origin":"","legend":"\u003cp\u003eStability of naringin content in DES extract stored at 30°C/75%RH and 40°C/75%RH Over a 6-month period\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e*\u003c/strong\u003eIndicates statistical differences (p\u0026lt;0.05) among time points within each condition, as denoted by different letters above the bars.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6892292/v1/7a4b0d2bf5d7428b02c29747.png"},{"id":92884656,"identity":"d55be01f-a5c8-42c8-93ef-bdf3088fc29e","added_by":"auto","created_at":"2025-10-06 16:13:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1356287,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6892292/v1/69dc9c72-1981-4e42-9b0e-eeac25a05402.pdf"},{"id":85204640,"identity":"8f805ea4-02b8-47be-991f-236cb44971ec","added_by":"auto","created_at":"2025-06-23 11:19:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19446,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6892292/v1/6034a3c574b76cae3ac1b423.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of Naringin Extract from Pomelo Peel Using Natural Deep Eutectic Solvent System as Green Technology for Antidiabetic Purpose: Box-Behnken design approach","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDiabetes is a rapidly growing global health concern, particularly in countries with diets rich in sugar and fat. While modern medications are effective in managing diabetes, their high cost and risk of severe side effects have led to the exploration of alternative treatments, including herbal products. Herbal remedies are generally more affordable and pose fewer risks than conventional medications, making them an attractive option for diabetes management [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, challenges such as the high cost of purified herbal compounds and the lack of quality control in herbal extracts, particularly in standardizing active compound concentrations, limit their widespread adoption. Addressing these issues requires the development of cost-effective, high-yield extraction methods and robust quality control measures to ensure the safety and efficacy of herbal products for diabetes patients [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePomelo (\u003cem\u003eCitrus grandis\u003c/em\u003e L.) peel, a traditional ingredient in Thai medicine, has shown promise as a natural remedy for diabetes. It has been used in formulations like \"Eight Peel Compound\" and \"Yahom Thepachit\" and contains bioactive compounds such as naringenin and naringin, which belong to the flavonoid group. These compounds exhibit antidiabetic properties, including lowering blood sugar levels, reducing insulin resistance, improving glucose tolerance, and inhibiting key enzymes like α-glucosidase and α-amylase [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the production of purified forms of these compounds is associated with high costs and often relies on organic solvents that pose environmental and health hazards. Thailand is a significant producer of pomelo, with widely cultivated varieties such as Khao Thong Dee, Khao Nam Pueng, and Tubtim Siam. Among these, Khao Thong Dee is the most prevalent, with major growing areas in provinces like Nakhon Pathom, Nakhon Si Thammarat, and Pathum Thani. Research indicates that the naringin content in pomelo peel decreases after harvest, highlighting the importance of timely and efficient extraction processes. Traditional extraction methods, such as liquid-liquid or solid-liquid extraction using solvents like ethanol and methanol, have been effective but are increasingly being replaced by modern, eco-friendly techniques [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdvances in extraction technologies, including ultrasonic-assisted extraction, supercritical fluid extraction, and deep eutectic solvents (DES), offer sustainable and efficient alternatives. These methods reduce solvent usage, enhance yield, and minimize environmental impact, aligning with green technology principles. For example, DES, composed of ionic liquids acting as hydrogen bond donors and acceptors, provide superior extraction efficiency and bioactivity enhancement compared to traditional solvents [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This study focuses on the development of a novel high-yield extraction methods for naringin from pomelo peel using green technologies and Box-Behnken design approach. The aim is to enhance the quality and value of herbal raw materials and extracts, paving the way for the production of standardized and eco-friendly herbal products for diabetes management.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eFresh \u003cem\u003eCitrus grandis\u003c/em\u003e (pomelo) fruits were sourced from various regions across Thailand, as detailed in the Supplementary Material. The cultivars included Khao Hom (KH), Khao Nam Phueng (KN), Khao Pan (KP), Khao Tang Kwa (KT), Khao Thong Dee (KD), and Khao Yai (KY). The KN variety was additionally sourced from three distinct locations: Nakhon Pathom (NP), Nakhon Si Thammarat (NS), and Pathum Thani (PT). The naringin standard (purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) was purchased from Sigma Aldrich (Merck, Germany). All other reagents and chemicals used in this study were of analytical grade and were purchased from Tokyo Chemical Industry Co., Ltd. (TCI, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Plant material preparation\u003c/h2\u003e \u003cp\u003eFruits of \u003cem\u003eC. grandis\u003c/em\u003e (pomelo) were collected from local farms located in various regions across Thailand over three separate batches during May, June, and July 2024, as detailed in the Supplementary Material. Each cultivar was authenticated based on its geographical origin and morphological characteristics, following the criteria established in a previous study [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The fruits were thoroughly washed, and the pericarps were manually peeled and collected. The pericarps were then dried in a hot air oven at 50\u0026deg;C for 24 hours, ground using a mechanical grinder, and passed through a No. 45 mesh sieve. The resulting powder was stored in airtight, light-resistant containers until further analysis. For subsequent experiments, the pomelo variety with the highest naringin content; determined from the three collected batches; was selected. This variety was also sourced from different regions in Thailand, as documented in the Supplementary Material.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Pomelo peel extraction\u003c/h2\u003e \u003cp\u003eThe pomelo peel powder from each source was extracted using 80% ethanol at a solvent to powder ratio of 20:1. The extraction was performed using the microwave-assisted technique with a microwave extractor (800 watts) until the temperature reached 72\u0026deg;C. This method was compared with the traditional maceration technique, where the samples were extracted with 80% ethanol over 7 days. The extracts were then analyzed for naringin content in each sample using high-performance liquid chromatography (HPLC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Analysis of naringin content in pomelo peel extracts using HPLC\u003c/h2\u003e \u003cp\u003eThe naringin content in the pomelo peel extracts was analyzed using a validated HPLC method, following the protocol described in a previous study [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Naringin standard solutions were prepared at concentrations ranging from 3.125 to 100 \u0026micro;g/mL and filtered through a syringe filter. For sample preparation, 20 \u0026micro;L of each extract was dissolved in 1 mL of methanol for every 20 mg of sample, using 70% methanol as the solvent. The resulting solution was filtered through a syringe filter before analysis. The HPLC system was operated with a detection wavelength of 280 nm, a column temperature of 25\u0026deg;C, and a flow rate of 1 mL/min. The mobile phase consisted of 0.3% acetic acid in water (solvent A) and acetonitrile (solvent B) with the following gradient program: 0 min, 5% solvent B; 5 min, 25% solvent B; 10 min, 45% solvent B; and 35 min, 50% solvent B. The results were compared with a standard curve prepared using naringin standard; Y\u0026thinsp;=\u0026thinsp;0.0852X\u0026thinsp;+\u0026thinsp;2.183, r\u0026sup2; = 0.9999 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. DES extraction of pomelo peel\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Preliminary of DES extraction from selected source\u003c/h2\u003e \u003cp\u003eThree different DES were prepared for pomelo peel extraction, each consisting of a 1:1 molar ratio of choline chloride combined with citric acid, oxalic acid, or malic acid. The hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) components were mixed with water and gently stirred on a hot plate at temperatures not exceeding 60\u0026deg;C until a homogeneous, transparent solution was obtained. The prepared DES mixtures were stored in sealed, light-protected containers at 4\u0026deg;C until use. For extraction, 1 g of dried pomelo peel powder was combined with 20 mL of each DES (solvent to powder ratio 20:1) and subjected to ultrasonic-assisted extraction at 60\u0026deg;C for 60 minutes. The resulting extracts were filtered through cotton using a syringe and stored at 4\u0026deg;C in light-protected containers for subsequent phytochemical analysis by HPLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Optimization of DES extraction\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003e2.5.2.1. Experimental design\u003c/h2\u003e \u003cp\u003eA Box-Behnken central experimental design was applied to optimize the DES extraction of naringin from pomelo peel. This design utilized three independent variables, each tested at three levels (-1, 0, +\u0026thinsp;1): the molar ratios of malic acid (X1), the molar ratios of citric acid (X2) from prior studies, and the powder-to-solvent ratio (X3). The response variables measured were naringin content (Y1) and anti-α-glucosidase activity (Y2), antioxidant (FRAP) activity (Y3), and polarity (Y4) of the DES extract. The experimental setup (Supplement Material), varied the DES molar ratio (1:1, 1:2, and 1:3) and solvent to powder ratio (10:1, 15:1, and 20:1). The experimental data were analyzed using response surface methodology to fit a second-order polynomial model, as described in Eq.\u0026nbsp;1:\u003c/p\u003e \u003cp\u003eY\u003csub\u003en\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e12\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e13\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e23\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e11\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e22\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e33\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e (1)\u003c/p\u003e \u003cp\u003eWhere Y represents the response variables, β\u003csub\u003e0\u003c/sub\u003e is the constant coefficient, β\u003csub\u003e1\u003c/sub\u003e, β\u003csub\u003e2\u003c/sub\u003e, and β\u003csub\u003e3\u003c/sub\u003e are the linear coefficients, β\u003csub\u003e12\u003c/sub\u003e, β\u003csub\u003e13\u003c/sub\u003e, and β\u003csub\u003e23\u003c/sub\u003e are the interaction coefficients, and β\u003csub\u003e11\u003c/sub\u003e, β\u003csub\u003e22\u003c/sub\u003e, and β\u003csub\u003e33\u003c/sub\u003e are the quadratic coefficients. The independent variables X\u003csub\u003e1\u003c/sub\u003e, X\u003csub\u003e2\u003c/sub\u003e, and X\u003csub\u003e3\u003c/sub\u003e represent the molar ratios of the DES components and the solvent to powder ratio. A regression model was constructed to describe the relationships between the input variables (DES component ratio and solvent to powder ratio) and the responses. Statistical analysis was performed to identify significant variables for optimizing the extraction conditions, with the goal of selecting extracts with the highest naringin content, anti-α-glucosidase activity, antioxidant (FRAP) activity, and polarity for antiglycation determination.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Bioactivity test\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Anti-α-glucosidase activity assay\u003c/h2\u003e \u003cp\u003eThe α-glucosidase enzyme inhibition activity was tested using 0.1 M sodium phosphate buffer (pH 6.9) as the primary solution. A stock solution of the enzyme was prepared at a concentration of 2 U/mL in the buffer and stored at -20\u0026deg;C. A working solution with a concentration of 0.1 U/mL was prepared from the stock solution. p-nitrophenyl α-D-glucopyranoside (pNPG) at 2 mM in buffer was used as the substrate, with acarbose as the positive control and the buffer as the negative control. All DES extracts were tested as sample groups, and placebo DES (DES without pomelo extract) was included to confirm the absence of inherent enzyme inhibition. The reaction was incubated and stopped by adding Na₂CO₃. Absorbance was measured at 405 nm, and the percentage inhibition and IC₅₀ values were calculated using the equation: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{P}\\text{e}\\text{r}\\text{c}\\text{e}\\text{n}\\text{t}\\text{a}\\text{g}\\text{e}\\:\\text{I}\\text{n}\\text{h}\\text{i}\\text{b}\\text{i}\\text{t}\\text{i}\\text{o}\\text{n}=[1-\\frac{\\left({OD}_{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\text{}-{OD}_{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\:\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}}\\text{}\\right)}{\\left({OD}_{\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}\\text{}-{OD}_{\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\:\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}}\\right)\\text{}\\text{}}]\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Antiglycation assay\u003c/h2\u003e \u003cp\u003eThe antiglycation assay was performed in a 96-well plate. Each well contained bovine serum albumin (BSA), glucose anhydrous, magnesium oxide, and DES extract. The glycated control contained BSA, glucose, and sodium phosphate buffer with NaN₃, while the blank control consisted of BSA and sodium phosphate buffer. Samples were incubated at 37\u0026deg;C for 14 days. Trichloroacetic acid (TCA, 100%) was added, and the mixture was centrifuged. The pellet was washed with 5% TCA, dissolved in phosphate-buffered saline (PBS), and analyzed for advanced glycation end-products (AGEs) using a spectrofluorometer with excitation at 370 nm and emission at 440 nm. Rutin served as the positive control. All DES extracts were tested as sample groups, and placebo DES (DES without pomelo extract) was included to confirm the absence of inherent enzyme inhibition. The percentage inhibition was calculated using the formula: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{P}\\text{e}\\text{r}\\text{c}\\text{e}\\text{n}\\text{t}\\text{a}\\text{g}\\text{e}\\:\\text{I}\\text{n}\\text{h}\\text{i}\\text{b}\\text{i}\\text{t}\\text{i}\\text{o}\\text{n}=[1-(\\frac{\\text{F}\\text{l}\\text{u}\\text{o}\\text{r}\\text{e}\\text{s}\\text{c}\\text{e}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}{\\text{F}\\text{l}\\text{u}\\text{o}\\text{r}\\text{e}\\text{s}\\text{c}\\text{e}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{e}\\text{x}\\text{t}\\text{r}\\text{a}\\text{c}\\text{t}}\\left)\\text{}\\right]\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3. Ferric reducing antioxidant power (FRAP) assay\u003c/h2\u003e \u003cp\u003eThe antioxidant capacity of the test samples was assessed using the FRAP assay, with Trolox serving as the reference antioxidant standard. A Trolox stock solution (1 mg/mL in methanol) was serially diluted to obtain standard concentrations ranging from 0.47 to 30 \u0026micro;g/mL. The FRAP reagent was freshly prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl, and 20 mM ferric chloride (FeCl₃) in a 10:1:1 (v/v/v) ratio. For each assay, 20 \u0026micro;L of the sample or standard was added to 180 \u0026micro;L of FRAP reagent in a 96-well microplate and incubated at room temperature for 30 minutes. Absorbance was measured at 593 nm using a microplate reader. Appropriate blanks and negative controls were included in each run. All DES extracts were tested as sample groups, and placebo DES (DES without pomelo extract) was included to confirm the absence of inherent enzyme inhibition. Antioxidant activity was calculated based on the Trolox calibration curve and expressed as Trolox equivalent antioxidant capacity (TEAC) per \u0026micro;g of dry sample weight.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Stability study of DES extracts\u003c/h2\u003e \u003cp\u003eThe stability of the extracts was evaluated under different conditions: 30\u0026deg;C/60% RH and 40\u0026deg;C/75% RH for 1, 2, 3, and 6 months. After each period, the extracts were analyzed for naringin content using HPLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were analyzed using SPSS version 28.0 (IBM Corp., USA) and Design-Expert version 13.0 (Stat-Ease Inc., USA). Analysis of variance (ANOVA) was employed to determine the significance of the model and the individual factors. The coefficient of determination (R\u0026sup2;) and the adjusted R\u0026sup2; (Adj-R\u0026sup2;) were used to assess the model's goodness-of-fit and predictive capability. Differences between means were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All experiments were performed in triplicate, and the results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e\u003cem\u003e3.1 Effect of different sources and variety of pomelo on naringin content\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHighly significant differences in peel naringin content (%w/w dry weight, DW) were observed among pomelo varieties and locations, along with a significant variety-by-month interaction (Figure 2A). Interestingly, certain varieties consistently accumulated higher levels of naringin across all locations, and fruits harvested earlier in the season exhibited greater naringin content. Specifically, the KN variety showed the highest mean naringin content (4.28%w/w DW), significantly exceeding that of KY (2.74%w/w DW). Overall, the mean naringin content across varieties followed the order: KN \u0026gt; KP \u0026asymp; KT \u0026gt; KD \u0026asymp; KY \u0026gt; KH. Location also had a modest but statistically significant effect: peels from NP (4.01%w/w DW) contained approximately 2.5 times more naringin than those from PT (1.62%w/w DW), with NS (2.33%w/w DW) yielding intermediate values. NP was the further analysis for location and harvesting period influnce. Notably, NP varitey, a clear decreasing trend in naringin content was observed across the three sampling months (from early to late harvest), with content at the first sampling approximately twice that of the final one. However, this seasonal trend was not evident in the other two locations (Figure 2).\u003c/p\u003e\n\u003cp\u003eThese results indicate that naringin accumulation in pomelo peel is influenced both by genetic (varietal) factors and by environmental or phenological factors. The higher naringin content in KN suggests intrinsic genetic differences in flavanone biosynthesis among varieties. This pattern is consistent with other citrus studies showing large cultivar-to-cultivar variation in flavonoid profiles. For instance previous study reported that Thai pomelo cultivars exhibited naringin levels in the pulp ranging from 25.20 to 39.42 mg per 100 g fresh weight, implying corresponding peel levels in the range of 0.8\u0026ndash;3.5%w/w DW when extrapolated [7]. Our observed peel concentrations (approximately 3.28\u0026ndash;4.28%w/w \u0026nbsp;DW) fall within the same order of magnitude as those prior reports. Moreover, the finding that naringin is concentrated in the peel aligns with established citrus chemistry: one study reported that pummelo peel contained up to 3910 \u0026mu;g/mL of naringin, compared to only 220 \u0026mu;g/mL in the juice, reflecting the compound\u0026rsquo;s accumulation in rind tissues [8]. The observed seasonal variation in naringin content is also well-documented in the literature. Immature fruit tends to accumulate higher flavonoid levels, which decline as the fruit matures. Similar seasonal dynamics suggested that naringin content in immature honey pomelo peel decreased from 3.45%w/w \u0026nbsp;DW in April to 1.77%w/w DW in May. Such dramatic reductions are typically due to developmental regulation of flavonoid biosynthesis, wherein juvenile peel tissues accumulate more naringin (imparting bitterness), which is subsequently diluted or metabolized during fruit ripening. This aligns with broader reviews noting that \u0026quot;immature fruit has a higher concentration of naringin\u0026quot; and emphasizing the importance of maturity stage in determining citrus fruit bitterness [9]. Location-based differences in naringin content may reflect microclimatic and edaphic influences, possibly due to cooler night temperatures, different soil properties, or differences in orchard management. Environmental factors such as light intensity, temperature, and water availability are known to influence phenolic and flavonoid metabolism in citrus. Plants exposed to higher UV light often upregulate antioxidant flavonoids like naringin. Conversely, lower sunlight exposure or nutrient-deficient soils can reduce such biosynthetic activity [10]. Although this study did not directly assess climatic variables, the consistent trends across sampling months suggest that environmental and agronomic factors, alongside genetics, influence naringin variation. In summary, naringin content in pomelo peel varies significantly by variety, location, and harvest time, supporting earlier findings in citrus research. Selecting high-naringin cultivars like Khao Nam Phueng and optimizing harvest timing can help maximize yields. Future work should focus on identifying the molecular and environmental mechanisms regulating naringin biosynthesis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2 Preliminary of DES extraction from selected source\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePreliminary screening of three choline chloride-based DES revealed notable differences in their efficiency to extract naringin from the selected pomelo peel source (Table 1). Among the solvents tested, the combination of choline chloride and citric acid yielded the highest naringin content at\u0026nbsp;0.191%w/w dry weight (DW), followed by choline chloride:malic acid (0.183%w/w DW) and choline chloride:oxalic acid (0.126%w/w DW).\u003c/p\u003e\n\u003cp\u003eThese results suggest that the choice of HBD in the DES formulation significantly affects the solubilization and extraction efficiency of flavonoids such as naringin. Citric acid, being a tricarboxylic acid with a relatively higher molecular weight and multiple carboxyl and hydroxyl groups, may form more stable hydrogen bonding networks with choline chloride, enhancing its solvating power toward glycosylated flavonoids [11]. Malic acid, while also effective, exhibited greater variability in extraction efficiency, possibly due to its lower acidity and smaller molecular structure. In contrast, the oxalic acid-based DES demonstrated the lowest extraction capacity. Oxalic acid\u0026rsquo;s strong acidity and smaller molecular size may lead to a more compact solvent network with reduced solubilization capacity for large polar molecules like naringin. The relatively higher extraction efficiency of ChCl:citric acid is consistent with previous studies reporting its favorable performance in extracting flavonoids and polyphenols from plant materials. Furthermore, its biocompatibility and low toxicity make it a promising candidate for food-grade or nutraceutical applications [12]. These findings indicate that ChCl:citric acid is a suitable DES system for further optimization in the green extraction of naringin, potentially offering an eco-friendly alternative to conventional organic solvents such as methanol or ethanol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Naringin content extracted from pomelo peel using different DES components\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eDES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eNaringin content (%w/w DW)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eChCl: citric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003e0.191\u0026nbsp;\u0026plusmn;\u0026nbsp;0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eChCl: oxalic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003e0.126\u0026nbsp;\u0026plusmn;\u0026nbsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003eChCl: malic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 312px;\"\u003e\n \u003cp\u003e0.183\u0026nbsp;\u0026plusmn;\u0026nbsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eNote.\u003c/strong\u003e\u0026nbsp; ChCl = Choline chloride; HBD = hydrogen bond donor; DW = dry weight.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDifferent superscript letters within the same column indicate statistically significant differences (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3 Effect of independent\u0026nbsp;\u003c/em\u003e\u003cem\u003evariables on response variables\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study employed response surface methodology (RSM) based on a Box\u0026ndash;Behnken design to optimize the extraction conditions of naringin from pomelo peel using DES (Figure 3). The independent variables included the molar ratio of malic acid (X₁), citric acid (X₂), and the solvent to powder ratio (X₃), while the responses were naringin content (Y₁), anti-\u0026alpha;-glucosidase activity (Y₂), antioxidant activity by FRAP assay (Y₃), and polarity (Y₄). The relationship between each response and the three independent variables was described using a second-order polynomial regression model. The predicted and experimental values were closely matched, confirming model accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNaringin content (Y\u003csub\u003e1\u003c/sub\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe quadratic regression model for naringin content (Y₁) was found to be highly significant, exhibiting an excellent coefficient of determination (R\u0026sup2;) and adjusted R\u0026sup2;, which together confirm the strong predictive performance of the second-order model. Moreover, the lack-of-fit test was not significant, indicating that the model adequately fits the experimental data. While the linear terms for malic acid ratio (X₁) and citric acid ratio (X₂) did not significantly influence naringin content, the solvent-to-powder ratio (X₃) contributed positively to the model. These findings align with previous DES optimization studies, where the solvent-to-powder ratio has been shown to exert both strong linear and nonlinear effects [13]. As illustrated in Figure 3, the 3D response surface plot demonstrated that naringin content increased with decreasing solvent-to-powder ratios. This trend reflects the combined interaction and quadratic effects, with a slight decline in yield observed at excessively high solvent-to-powder ratios. The quadratic model in actual factor terms was Y₁=0.99\u0026minus;0.07X\u003csub\u003e3\u003c/sub\u003e+0.002X\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnti-\u0026alpha;-glucosidase activity (Y₂)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe linear \u0026nbsp;model for anti-\u0026alpha;-glucosidase activity (Y₂) was also statistically significant, with a high R\u0026sup2;, adjusted R\u0026sup2;, and non-significant lack-of-fit. Significant linear terms, particularly solvent to powder ratio revealed diminishing returns at high activity against \u0026alpha;-glucosidase activity. Since \u0026alpha;-glucosidase inhibition is directly related to naringin concentration, the trends in Y₂ closely mirrored Y₁. The 3D response surface showed that maximum inhibition occurred in the high X₁ and X₂ region, with a broad plateau. Unlike Y₁, inhibition approached 100% without strong decline, though curvature remained evident. X₃ had a moderate positive effect. These results agree with previous reports highlighting naringin as a major contributor to bioactivity [14]. The linear model in actual factor terms was Y₂=31.02\u0026minus;1.16X\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAntioxidant activity (FRAP)(Y₃)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe antioxidant activity, measured using the FRAP assay (Y₃), was effectively modeled using a statistically significant second-order polynomial equation. The model demonstrated strong explanatory power and passed the lack-of-fit test, confirming its adequacy. The quadratic terms for malic acid ratio (X₁) and citric acid ratio (X₂) were significant contributors, with both the interaction term (X₁X₂) and the quadratic terms (X₁\u0026sup2; and X₂\u0026sup2;) showing significant influence. These findings suggest the existence of an optimal concentration range for both acids. While increasing acid content initially enhanced antioxidant activity, excessive levels led to a plateau or slight decline, possibly due to the degradation of acid-sensitive antioxidant compounds. In particular, the negative coefficient of X₂\u0026sup2; indicates that very high citric acid concentrations may be detrimental to antioxidant efficacy.\u003c/p\u003e\n\u003cp\u003eThe 3D response surface plot for Y₃ displayed a characteristic hill-shape, with peak antioxidant activity occurring at moderate-to-high values of X₁ and X₂. Increasing the solvent to powder ratio (X₃) also improved antioxidant activity, likely by enhancing mass transfer; however, the benefit diminished at higher levels. Notably, the highest FRAP values did not coincide with maximum acid concentrations, but rather overlapped with the optimal regions for naringin content (Y₁) and extraction yield (Y₂), indicating a balance between these variables. Interestingly, these results diverge from those observed for \u0026alpha;-glucosidase inhibitory activity, suggesting that the antioxidant effects may be attributable to other bioactive flavonoids beyond naringin, such as hesperidin, narirutin, and eriocitrin [15]. The quadratic model in actual factor terms was\u0026nbsp;Y\u003csub\u003e3\u003c/sub\u003e=456.46\u0026minus;180.00X\u003csub\u003e1\u003c/sub\u003e\u0026minus;58.97X\u003csub\u003e2\u003c/sub\u003e\u0026minus;9.52X\u003csub\u003e3\u003c/sub\u003e+1.25X\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e\u0026minus;2.04X\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e+39.43X\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e+20.64X\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e+0.32X\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePolarity (Y₄)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe quadratic regression model developed to predict solvent polarity based on malic acid, citric acid, and solvent-to-powder ratio was found to be statistically\u0026nbsp;non-significant\u0026nbsp;, indicating that the model lacks predictive strength for this particular response. Additionally,\u0026nbsp;none of the linear, interaction, or quadratic terms\u0026nbsp;reached statistical significance, although the quadratic term for citric acid approached borderline significance, suggesting a potential nonlinear relationship worth further exploration.\u003c/p\u003e\n\u003cp\u003eWhile the model\u0026apos;s predictive utility is limited, the equation offers insight into the general directionality of the effects. For example, the\u0026nbsp;positive coefficients\u0026nbsp;for the linear terms of malic acid, citric acid, and solvent-to-powder ratio suggest that increases in these factors may correspond to slight increases in polarity. However, the\u0026nbsp;negative quadratic term for citric acid (\u0026ndash;0.42)\u0026nbsp;implies that extremely high concentrations may reduce solvent polarity, which is chemically plausible due to changes in hydrogen bonding and ionic strength at elevated acid levels. These results suggest that\u0026nbsp;solvent polarity, as measured or defined in this context, is not strongly influenced by the selected formulation variables within the tested range. It is possible that the measurement method for polarity was either too insensitive or not directly affected by these components. Alternatively, the design space may have been too narrow to observe meaningful variation in this response.\u003c/p\u003e\n\u003cp\u003eOverall, the response surface models developed in this study effectively captured the relationships between extraction variables and key response outcomes. The solvent to powder ratio consistently emerged as a critical factor across bioactivity responses, while citric and malic acid ratios exhibited more complex, nonlinear effects. In contrast, the model for polarity was not statistically significant, suggesting that polarity, as measured here, may not be strongly influenced by the studied formulation parameters or that its measurement method lacked sensitivity. These findings underscore the potential of DES optimization for enhancing functional compound extraction, while also highlighting the need for further investigation into physicochemical descriptors like polarity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e The ANOVA results of response surface model for naringin content, anti-a-glucosidase activity, antioxidant, and Polarity of pomelo peel DES extract\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003eDf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003enaringin content (%w/w DW)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eanti-a-glucosidase activity (IC\u003csub\u003e50\u003c/sub\u003e,\u0026nbsp;mg/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eantioxidant (FRAP) (FEAC/mg DW)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003ePolarity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eQuadratic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eLinear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eQuadratic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eLinear\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e -value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e49.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e26.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt; 0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e393.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4708\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e-Malic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.2312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.1346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.7206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e72.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5107\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e-Citric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.1630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5729\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e217.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.8319\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eX\u003csub\u003e3\u003c/sub\u003e- C-Solvent to powder ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e395.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e79.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e151.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.3920\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5587\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e X\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5941\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5072\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4465\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5336\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e X\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.2999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e64.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5476\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4926\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e X\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5941\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e171.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.2915\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.7701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2370.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0553\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.8234\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.9532\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e649.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e6.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0559\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eX\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e40.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e100.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.7761\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4187\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eLack of fit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e7.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.1164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e3.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.2373\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.1279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.9354\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eAdj R\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.9688\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.8461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.9961\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.7602\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Significant at p\u0026lt;0.05, Df = degree of freedom.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4. Verification of predictive model\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the reliability and predictive accuracy of the developed quadratic models, an experimental formulation was prepared using the optimized conditions: Malic acid molar ratio at 1, citric acid molar ratio at 1, and solvent to powder ratio at 12.24. The experimental values obtained for all three response variables; naringin content, anti-\u0026alpha;-glucosidase activity, and antioxidant activity (FRAP); were compared against the model-predicted values and their 95% prediction intervals (Table 3). The observed naringin content (0.28% w/w DW) was closely aligned with the predicted value (0.30% w/w DW) and fell within the prediction interval of 0.26\u0026ndash;0.34, indicating good model accuracy. Similarly, the observed anti-\u0026alpha;-glucosidase activity (IC₅₀= 9.99 \u0026micro;g/mL) showed acceptable agreement with the predicted value (16.10 \u0026micro;g/mL), although it was near the upper bound of the prediction interval (12.74\u0026ndash;19.46 \u0026micro;g/mL). This slight deviation could be attributed to biological variability or matrix effects during formulation. In contrast, the antioxidant activity (FRAP assay) showed excellent correlation between predicted (199.50 FEAC/\u0026micro;g DW) and observed (192.31 FEAC/\u0026micro;g DW) values, though the observed mean slightly undershot the lower bound of the prediction interval, possibly due to degradation of labile antioxidants during extraction. Overall, the results confirm the robustness and validity of the optimization models in predicting key bioactive properties under practical formulation conditions. Minor discrepancies highlight the complexity of physicochemical interactions in DES but remain within acceptable experimental margins. The present model reliably predicts extraction outcomes for naringin and related activities from pomelo peel. Its adequacy is supported by both the internal validation and consistency with literature reports of RSM-optimized DES extractions [16]. These findings reinforce the value of combining DES and RSM for green extraction: not only are DES themselves sustainable solvents, but the optimized process yields high concentrations of target compounds while minimizing trial-and-error. Future work could further leverage this model to explore other DES formulations or scale-up extraction, confident in the predictive power demonstrated here. In conclusion, the Box\u0026ndash;Behnken RSM model proved to be a robust tool for maximizing naringin recovery from pomelo peel, aligning with contemporary green extraction strategies and prior successful validations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Confirmation of model predictions at malic acid molar ratio=1, citric acid molar ratio=1, solvent to powder ratio=12.24 (n=3)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eResponse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003ePredicted mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003eObserved mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 236px;\"\u003e\n \u003cp\u003e95% prediction interval\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003enaringin content (%w/w DW)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e0.26-0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eanti-a-glucosidase activity (IC\u003csub\u003e50\u003c/sub\u003e,\u0026nbsp;mg/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e16.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e19.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e12.74-19.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eantioxidant (FRAP) (FEAC/mg DW)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e199.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e192.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e195.08-203.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: All observed values represent the average of three replicates.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5. FT-IR spectrum analysis of DES extract\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe FTIR spectra presented in Figure 4 compare (A) naringin extracted with DES, (B) the DES solvent alone (choline chloride, malic acid, and citric acid), (C) pure naringin, and (D\u0026ndash;F) individual components of the DES: choline chloride, malic acid, and citric acid, respectively. The spectrum of pure naringin (Figure 4C) exhibits characteristic peaks, including a broad O\u0026ndash;H stretching vibration around 3416 cm⁻\u0026sup1;, attributed to phenolic and hydroxyl groups, and C\u0026ndash;H stretching bands in the region of 2957\u0026ndash;2859 cm⁻\u0026sup1;. A distinct carbonyl stretching band appears near 1630 cm⁻\u0026sup1;, corresponding to the flavanone structure of naringin, while the peaks at approximately 1470 cm⁻\u0026sup1; and 1361 cm⁻\u0026sup1; are related to aromatic C=C stretching and phenolic ring vibrations, respectively. The region between 1240\u0026ndash;1139 cm⁻\u0026sup1; displays C\u0026ndash;O stretching, including contributions from ether and glycosidic linkages in the naringin molecule [17]. Importantly, the spectrum of the naringin extract in DES (Figure 4A) retains all major characteristic peaks of pure naringin with no observable shifts in wavenumber, indicating that the molecular structure of naringin remains intact after extraction. The O\u0026ndash;H stretch, C=O vibration, and aromatic peaks are all clearly visible and match those of the reference compound (Figure 4C), confirming that the DES system effectively extracted naringin without causing structural modification. The lack of any new peaks or significant shifts further supports the absence of chemical interaction between naringin and DES components at the molecular level. In contrast, the FTIR spectrum of the DES mixture without naringin (Figure 4B) shows a broad O\u0026ndash;H stretching band around 3360 cm⁻\u0026sup1;, attributed to strong hydrogen bonding among citric acid, malic acid, and choline chloride. Carbonyl stretching bands from the carboxylic acids appear around 1743 and 1691 cm⁻\u0026sup1;, and C\u0026ndash;O stretching bands are observed in the 1240\u0026ndash;1139 cm⁻\u0026sup1; range [18]. These peaks are distinct from those of naringin and do not interfere with the identification of naringin in the extract. This comparison confirms that the functional groups of naringin in the extract (Figure 4A) originate from the flavonoid itself and not from the DES matrix.\u003c/p\u003e\n\u003cp\u003eThe individual spectra of choline chloride (D), malic acid (E), and citric acid (F) also exhibit their respective characteristic peaks, such as broad O\u0026ndash;H stretching, C\u0026ndash;H bending, and C=O stretching, yet none show the distinct aromatic or flavanone-related bands present in pure naringin [18]. Thus, the FTIR results clearly demonstrate the successful extraction of naringin by the DES system, with the compound retaining its structural integrity. These findings align with previous studies where unchanged spectral positions indicated the absence of chemical transformation post-extraction.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.6. Antiglycation activity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe BSA\u0026ndash;glucose assay revealed that pure naringin exhibited potent antiglycation activity with an IC₅₀ value of 36.77 \u0026micro;g/mL. In contrast, the naringin formulated in DES showed significantly reduced activity, with an IC₅₀ of 104.75 \u0026micro;g/mL. The DES placebo, which contained no naringin, exhibited negligible antiglycation activity (IC₅₀\u0026gt;1000 \u0026micro;g/mL). The differences between pure naringin and its DES formulation were statistically significant and both were significantly different from the placebo group. The positive control, aminoguanidine, effectively inhibited protein glycation at low concentrations, consistent with its established use as a standard glycation inhibitor. Flavonoids, including naringin, are known to inhibit the formation of advanced glycation end-products (AGEs) primarily through their antioxidant and free-radical scavenging activities. Certain flavonoids, such as luteolin, quercetin, and rutin, can inhibit AGE formation more effectively than aminoguanidine at 10 mM concentration, suggesting that natural polyphenols can be superior antiglycation agents [19]. The IC₅₀ value obtained for pure naringin in the current study compares favorably with previous findings, highlighting its strong potential as an inhibitor of protein glycation. However, the reduced antiglycation activity observed in the naringin\u0026ndash;DES formulation may be attributed to physicochemical interactions within the solvent matrix. DES are widely recognized as green solvents capable of enhancing the solubility, stability, and even bioavailability of poorly water-soluble compounds. Despite these benefits, in the context of the in vitro BSA\u0026ndash;glucose assay, the DES matrix may have limited naringin\u0026rsquo;s accessibility or reactivity. This could occur through molecular sequestration via hydrogen bonding, or by modifying key microenvironmental factors such as pH or viscosity. Another possible explanation is that the DES formulation diluted the effective concentration of naringin available to exhibit antiglycation activity [20]. Notably, the placebo DES (without naringin) showed no antiglycation effect, confirming that the solvent components themselves were biologically inert in this context. While DES can enhance solubility and protect bioactive compounds from degradation, the current findings highlight a potential drawback: they may also interfere with the interaction of active compounds and biological targets \u003cem\u003ein vitro\u003c/em\u003e. Therefore, the selection of an appropriate solvent system is critical when formulating plant-derived compounds for biological assays or therapeutic use. Future research should focus on evaluating the pharmacokinetics and \u003cem\u003ein vivo\u003c/em\u003e bioavailability of DES-formulated flavonoids to better understand their true therapeutic potential.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.7. Stability study of DES containing naringin\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe average naringin content in DES extract stored at 30\u0026deg;C/75%RH and 40\u0026deg;C/75%RH over a 6-month period was presented in Figure 5. At time 0, the naringin content was 0.28%w/w DW. A progressive decline in naringin content was observed under both storage conditions. After 6 months, the content had decreased to 0.25%w/w DW at 30\u0026deg;C/75%RH and 0.24%w/w DW at 40\u0026deg;C/75%RH. Each storage condition indicated a significant effect of time on naringin content. At 6-month, naringin content values were significantly lower than the baseline and 1-month values under both storage conditions. This trend demonstrates that both temperature and duration significantly influenced the chemical stability of the DES extract. The bar graph (Figure 4) further illustrates this trend, showing a steeper decline under the 40\u0026deg;C/75%RH condition. The pattern of naringin degradation appeared consistent with first-order kinetics, as supported by a linear fit of ln\u003csub\u003e(concentration)\u003c/sub\u003e versus time (data not shown). This is in agreement with prior studies indicating that flavonoid degradation, including that of naringin and related glycosides, follows first-order kinetics under accelerated stability conditions [21]. The estimated half-lives under 30\u0026deg;C and 40\u0026deg;C conditions were approximately 8\u0026ndash;10 months and 3\u0026ndash;4 months, respectively, based on extrapolation. While DES are known to enhance the stability of phenolic compounds due to strong hydrogen bonding and reduced water activity, the observed degradation over 6 months was still substantial, particularly under accelerated conditions. These findings align with previous study which reported degradation of phenolic compounds in DES-based orange peel extracts over time, at a slower rate than in conventional solvents [22]. It is important to note that under the ICH Q1A(R2) guideline, a decrease greater than 5% in the assay value is considered a significant change during stability testing [23]. In this study, the extract lost more than 5% of its original naringin content by 3 months at 40\u0026deg;C and by 6 months at 30\u0026deg;C, indicating potential challenges in long-term storage stability. Therefore, product formulations using this DES extract would require stability-enhancing strategies, such as inclusion of antioxidants, encapsulation, or packaging under inert conditions to prolong shelf life. The practical implications are critical, particularly for botanical ingredients intended for pharmaceutical, nutraceutical, or cosmeceutical applications, which must comply with regulatory standards for shelf-life and active compound stability. Further kinetic modeling using Arrhenius equations and real-time stability testing would be valuable in determining accurate expiration dates.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study introduces a novel green extraction method using DES to recover naringin from pomelo peel, offering an environmentally safe alternative to conventional solvents. The approach efficiently converts pomelo peel; an agro-industrial byproduct; into a stable, multifunctional extract suitable for nutraceutical applications. Significant variation in naringin content was observed across different pomelo cultivars, growing regions, and harvest periods. The Khao Nam Phueng variety consistently exhibited the highest levels, while location and early-season harvesting were also critical factors influencing yield. These findings highlight the importance of cultivar selection and harvest timing in maximizing bioactive compound recovery. The optimized DES extract demonstrated strong in vitro bioactivities, including enzyme inhibition, antioxidant capacity, and antiglycation potential, while maintaining compound integrity and extract stability. The use of green solvents enables direct application of the extract in functional health products without the need for solvent removal. This work not only establishes a sustainable, scalable extraction method but also underscores the value of integrating agricultural biodiversity and harvest strategy into raw material standardization. Future research should focus on \u003cem\u003ein vivo\u003c/em\u003e efficacy, formulation optimization, and regulatory assessment, as well as extending this green extraction platform to other phytochemical-rich plant residues.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research project was also supported in part by the Faculty of Pharmacy, Mahidol University, Bangkok, Thailand.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not contain any human or animal study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Data will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eM.M. 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Nicorescu, et al., The influence of solid-to-solvent ratio and extraction method on total phenolic content, flavonoid content and antioxidant properties of some ethanolic plant extracts, Revista de Chimie 67 (2016) 1922-1927.\u003c/li\u003e\n \u003cli\u003eK. Zhang, Z. Ding, W. Duan, et al., Optimized preparation process for naringenin and evaluation of its antioxidant and \u0026alpha;‐glucosidase inhibitory activities, Journal of Food Processing and Preservation 44 (2020) e14931 https://doi.org/https://doi.org/10.1111/jfpp.14931.\u003c/li\u003e\n \u003cli\u003eR. Tocmo, J. Pena‐Fronteras, K.F. Calumba, et al., Valorization of pomelo (\u003cem\u003eCitrus grandis\u003c/em\u003e Osbeck) peel: A review of current utilization, phytochemistry, bioactivities, and mechanisms of action, Compr. Rev. Food Sci. Food Saf 19 (2020) 1969-2012 https://doi.org/ https://doi.org/10.1111/1541-4337.12561.\u003c/li\u003e\n \u003cli\u003eI. Gupta, S.N. Adin, M. Aqil, M. 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Zhan, et al., Antiglycating effects of citrus flavonoids and associated mechanisms, Food Science and Human Wellness 13 (2024) 2363-2372 https://doi.org/https://doi.org/10.26599/FSHW.2022.9250247.\u003c/li\u003e\n \u003cli\u003eG. Zengin, M.d.l.L. C\u0026aacute;diz-Gurrea, \u0026Aacute;. Fern\u0026aacute;ndez-Ochoa, et al., Selectivity tuning by natural deep eutectic solvents (NADESs) for extraction of bioactive compounds from \u003cem\u003eCytinus hypocistis\u003c/em\u003e\u0026mdash;studies of antioxidative, enzyme-inhibitive properties and LC-MS profiles, Molecules 27 (2022) 5788 https://doi.org/https://doi.org/10.3390/molecules27185788.\u003c/li\u003e\n \u003cli\u003eL.M. Cordenonsi, N.G. Bromberger, R.P. Raffin, E.E. Scherman, Simultaneous separation and sensitive detection of naringin and naringenin in nanoparticles by chromatographic method indicating stability and photodegradation kinetics, Biomedical chromatography 30 (2016) 155-162 https://doi.org/https://doi.org/10.1002/bmc.3531.\u003c/li\u003e\n \u003cli\u003eM. Panić, M. Andlar, M. Ti\u0026scaron;ma, et al., Natural deep eutectic solvent as a unique solvent for valorisation of orange peel waste by the integrated biorefinery approach, Waste management 120 (2021) 340-350 https://doi.org/https://doi.org/10.1016/j.wasman.2020.11.052.\u003c/li\u003e\n \u003cli\u003eD.J. Mazzo, The ICH stability guideline. International Stability Testing, CRC Press, 2020, pp. 1-13.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anti-α-glucosidase activity assay, Antiglycation assay, DES, Naringin, pomelo peel","lastPublishedDoi":"10.21203/rs.3.rs-6892292/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6892292/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiabetes remains a critical public health issue, intensifying the demand for safe, affordable, and eco-friendly therapeutic alternatives. Pomelo peel, rich in the flavonoid naringin, offers promising antidiabetic potential. This study introduces a novel green extraction approach using natural deep eutectic solvents (DES), offering a safer alternative to organic solvents. Among six Thai pomelo cultivars, Khao Nam Phueng exhibited the highest naringin content (4.28% w/w dry weight), significantly surpassing Khao Yai (2.74% w/w DW). Regional variation was also observed: peels from Nakhon Pathom yielded 4.01%, compared to only 1.62% from Pathum Thani. Seasonal decline was evident, with early-harvest fruit showing nearly double the naringin content of late-season samples. Initial screening identified choline chloride:citric acid (1:1) as the most effective DES, extracting 0.19%w/w DW naringin, followed by malic acid (0.18% w/w DW) and oxalic acid (0.13%w/w DW). Optimization using Box–Behnken design improved yield to 0.28% w/w. The extract displayed potent α-glucosidase inhibition (IC₅₀=9.99 µg/mL) and strong antioxidant activity (FRAP=192.3 FEAC/µg DW). Antiglycation activity was moderate (IC₅₀=104.75 µg/mL), lower than pure naringin (IC₅₀=36.77 µg/mL), likely due to solvent-matrix interactions. Predictive response surface models showed high accuracy, and the extract remained chemically stable over six months with only slight degradation (0.24–0.25% w/w). This work demonstrates a novel, scalable method to valorize agro-industrial pomelo peel waste into standardized, high-quality antidiabetic extracts using sustainable DES technology. These findings support broader applications of DES in green phytopharmaceutical development and highlight cultivar and harvest timing as key factors in maximizing bioactive compound recovery.\u003c/p\u003e","manuscriptTitle":"Development of Naringin Extract from Pomelo Peel Using Natural Deep Eutectic Solvent System as Green Technology for Antidiabetic Purpose: Box-Behnken design approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 11:11:23","doi":"10.21203/rs.3.rs-6892292/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b5140469-e961-4460-8572-e5d73d9ce11d","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:11:57+00:00","versionOfRecord":{"articleIdentity":"rs-6892292","link":"https://doi.org/10.1007/s12247-025-10084-7","journal":{"identity":"journal-of-pharmaceutical-innovation","isVorOnly":false,"title":"Journal of Pharmaceutical Innovation"},"publishedOn":"2025-09-30 15:58:07","publishedOnDateReadable":"September 30th, 2025"},"versionCreatedAt":"2025-06-23 11:11:23","video":"","vorDoi":"10.1007/s12247-025-10084-7","vorDoiUrl":"https://doi.org/10.1007/s12247-025-10084-7","workflowStages":[]},"version":"v1","identity":"rs-6892292","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6892292","identity":"rs-6892292","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}