Alginate/Carboxymethyl Cellulose Nanoparticles for Enhanced Delivery of Vitexin: Physicochemical Characterization, Anti-Oxidant, and Multitarget Antidiabetic Potential ^†

Abstract

Vitexin is a potent C-glycosyl flavone from mung bean coats with significant antioxidant properties, constrained by poor solubility and bioavailability. In this study, Nanovitexin (NV) was encapsulated within a biocompatible Alginate/Carboxymethyl Cellulose (Alg/CMC) matrix via a modified solvent evaporation technique assisted by chemical cross-linking. The optimized NV exhibited a mean dry particle size of 50–70 nm, high concentration (0.05–0.25 mg/mL), and stability (Zeta potential >30 mV). FT-IR analysis confirmed the successful entrapment via intermolecular interactions. Notably, NV exhibited enhanced activities compared to free vitexin (FV), showing superior DPPH scavenging (IC₅₀ of 115.38 μg/mL) versus FV (IC₅₀ of 226.06 μg/mL). Furthermore, NV demonstrated significantly enhanced in vitro antidiabetic potential, displayed no cytotoxicity towards HepG2 cells, and effectively protected against H₂O₂-induced oxidative stress. The Alg/CMC nanomatrix effectively improves vitexin bioactivity, suggesting promising potential for pharmaceutical and nutraceutical applications.

1. Introduction

Vitexin (apigenin-8-C-glucoside) is a C-glycosylated flavonoid widely extracted from numerous edible and medicinal plants, prominently including the mung bean seed coat (Vigna radiata L.) [1]. Its chemical structure, featuring a flavonoid core, underpins a broad spectrum of potent pharmacological activities. Extensive preclinical research confirms vitexin’s efficacy as a powerful antioxidant capable of scavenging reactive oxygen species (ROS) and upregulating endogenous defense mechanisms (Nrf2/SOD) [2,3]. Furthermore, it exhibits significant anti-diabetic effects by enhancing glucose uptake via GLUT-4 translocation and modulating key metabolic regulators like PPAR-γ [4,5]. Its utility extends to combating chronic conditions through demonstrated anti-inflammatory properties (e.g., inhibiting NF-κB, MAPKs) [6,7], neuroprotective capacity (e.g., against Alzheimer’s or Parkinson’s disease) [1,8], and cancer cell lines such as hepatocellular carcinoma and lung cancer [1].

Despite its remarkable bioactivities in vitro and promising molecular targets, the clinical and functional application of free vitexin is severely hampered by major physicochemical limitations. Vitexin suffers from extremely low water solubility due to its rigid molecular structure and high hydrophobicity. This results in poor dissolution kinetics [2,9]. Consequently, the oral bioavailability and subsequent therapeutic efficacy in vivo are significantly compromised [2,5]. Several studies confirm the rapid elimination and degradation of unconjugated vitexin in the gastrointestinal tract [2]. Achieving effective plasma concentration often requires a prohibitively high dosage for practical therapeutic use [2]. This limited absorption profile significantly decreases the overall in vivo effects of this otherwise potent phytochemical. The significant pharmacological potential of vitexin is currently challenged by its poor physicochemical profile, necessitating advanced formulation strategies to improve its translational outcome.

Nanotechnology has emerged as the most promising solution, and current strategies largely fall into two categories as nanosuspension technology and polymeric nanoparticles [10]. The drug nanosuspensions is a universal formulation approach for improved drug delivery of hydrophobic drugs and one the most promising approaches for increasing the biopharmaceutical performance of poorly water-soluble drug substances, especially for nature products [11]. Initial research focused on size reduction to enhance dissolution kinetics, a fundamental barrier for this highly hydrophobic flavonoid [9]. Gu et al. (2017) successfully produced vitexin nanocrystals by employing a hybrid technique combining Antisolvent Precipitation (ASP) with High-Pressure Homogenization (HPH) [9]. This dual approach achieved particles with reduced size (approximately 217 nm) and demonstrably improved the in vitro dissolution rate, validating the core principle that particle size reduction is an effective pathway to increase vitexin’s solubility and bioavailability [9].

More advanced strategies utilize polymeric matrices to confer protective and targeting functionalities. This approach addresses not only poor solubility but also the rapid degradation and elimination of the compound in the harsh gastrointestinal (GI) tract [2,5]. While these pioneering studies demonstrate successful nano-construction, a critical research gap remains in the optimal utilization of cost-effective, non-toxic, and biocompatible polysaccharide matrices. Specifically, previous works employed Alginate or chemically modified Chitosan systems separately. There is a notable absence of studies exploring the synergistic potential of combining the pH-sensitive and gelling properties of Alginate with the mucoadhesive and protective capabilities of Carboxymethyl Cellulose (CMC). This combination of natural biopolymers holds immense promise as a resilient, single-step fabricated scaffold for nanovitexin, offering simplified production and enhanced stability, which is crucial for scalable pharmaceutical application.

The overall research aims to overcome the critical bioavailability challenges of the potent flavonoid as vitexin. This is achieved by systematically developing and characterizing a novel nanovitexin formulation using a biopolymeric Alginate/CMC scaffold. The study encompasses two specific objectives as Formulation and Characterization: To optimize the fabrication parameters for producing stable nanovitexin particles. We will perform a comprehensive physicochemical characterization of the resulting nanovitexin formulation, including morphology, size distribution, zeta potential, and encapsulation efficiency. Comparative Biological Assessment: To conduct a detailed quantitative comparison of the biological activities between the synthesized nanovitexin and the free vitexin counterpart. This assessment will focus on antioxidant capacity (using DPPH assays), antidiabetic potential (α-glucosidase and α-amylase inhibition) and neuroprotective potential (cholinesterase inhibition, AChE) and cell viability assays and protective in HepG2 cells.

2. Materials and Methods

2.1. Materials

Free vitexin (FV) with a purity of 91.73% was obtained from the isolation and extraction process of mung bean coats (Vigna radiata L.) as described in our previous study [12]. Sodium alginate was purchased from Merck (Rahway, NJ, USA), and Sodium carboxymethyl cellulose (CMC) was obtained from Sigma-Aldrich (Singapore). Activation agents included N-hydroxysuccinimide (NHS) and N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (Sigma-Aldrich, Singapore). Solvents such as Ethanol, Ethyl acetate, Potassium dihydrophosphate, and Acetic acid were supplied by Sigma-Aldrich (Singapore) and were of analytical or HPLC grade. HepG2 cells were kindly provided by Dr. Nguyen Van Tru (Institute of Biology, VAST, Ho Chi Minh City, Vietnam) and cultured in Dulbecco’s minimum essential medium (DMEM)/high glucose supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin under 5% CO₂ at 37 °C. Reagents for biological assays, including DPPH (2,2-diphenyl-1-picrylhydrazyl, 95%), acetylthiocholine iodide (ACTI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), H₂O₂ (34.5–36.5%), and galantamine hydrobromide, were purchased from Sigma-Aldrich (Singapore) or Alfa Aesar (Tokyo, Japan).

2.2. Extraction and Purification of Free Vitexin

Free vitexin (FV) was isolated and purified from mung bean coats (Vigna radiata L.) following established procedures [12]. Briefly, dried mung bean coat powder was subjected to ultrasonic-assisted extraction with 80% ethanol (1:3 w/v ratio). The extract was concentrated, partitioned with ethyl acetate, and purified via recrystallization in hot 80% ethanol. The final FV product was obtained as a yellow powder (purity 91.73%), confirmed by HPLC and NMR spectroscopy (¹H-NMR and ¹³C-NMR) consistent with the vitexin reference standard (>99.5%, Sigma-Aldrich, Singapore).

2.3. Preparation of the Nanovitexin Drug Delivery System Based on Alginate/CMC Carrier

The nanovitexin (NV) system was prepared using a modified solvent evaporation technique assisted by chemical cross-linking. First, sodium alginate solution (1 mg/mL) was activated by adding 3-aminopropyl triethoxysilane (APTES) (10% alcoholic acid solution) and stirring at 400 rpm for 2 h at 80 °C to introduce amino groups (-NH₂). Simultaneously, CMC was activated by slowly adding 25 mg of NHS and 100 mg of EDC to a solution containing 150 mg CMC in 20 mL distilled water (pH adjusted to 8.5 with triethylamine), followed by stirring at 55 °C for 4 h. The activated alginate and CMC were mixed and reacted for 24 h at room temperature to form the Alginate/CMC carrier matrix. Subsequently, 100 mL of an ethanol solution containing 40 mg of FV was added dropwise to the carrier solution under continuous magnetic stirring (350 rpm) for 24 h. The solvent was evaporated, and the mixture was ultrasonicated for 10 min, centrifuged at 5600 rpm for 10 min, and lyophilized to obtain the nanovitexin powder.

2.4. Physicochemical Characterization

The structural interactions were evaluated using Fourier-Transform Infrared (FT-IR) spectroscopy based on the absorption of specific functional groups [13]. Morphology was examined using Field-Emission Scanning Electron Microscopy (FE-SEM). The vitexin content and encapsulation efficiency (EE) were determined using High-Performance Liquid Chromatography (HPLC) (Agilent 1100 Series, Santa Clara, CA, USA, Eclipse XDB C18 column, 4.6 × 250 mm, 5 μm). The mobile phase consisted of 0.5% ortho-phosphoric acid (A) and acetonitrile (B) with a gradient program, detected at 336 nm. The encapsulation efficiency (EE) was calculated using Equation (1):

$$EE (\%) = {\displaystyle \frac{Total Vitexin Encapsulated}{Total Initial Vitexin}} \times 100\%$$

(1)

2.5. In Vitro Biological Activity Evaluation

2.5.1. DPPH Radical Scavenging Assay

The antioxidant activity was determined using the DPPH method. Samples (FV or NV, 20–200 μg/mL) were mixed with DPPH (0.2 μM) and incubated in the dark for 30 min. Absorbance was measured at 517 nm. Ascorbic acid served as the positive control. The IC₅₀ value was calculated from the linear regression curve.

2.5.2. α-Glucosidase and α-Amylase Inhibition Assays

α-Glucosidase inhibitory activity was evaluated colorimetrically using p-nitrophenyl-α-D-glucopyranoside (pNPG) as the substrate [14]. Absorbance was measured at 405 nm. For α-amylase inhibition, the DNSA method was employed using starch as the substrate, measuring absorbance at 540 nm [15]. Acarbose was used as the positive control for both assays.

2.5.3. AChE Inhibitory Activity Assay

Acetylcholinesterase (AChE) inhibitory activity was determined using Ellman’s method [16]. The reaction mixture containing sample, phosphate buffer, and AChE was incubated, followed by the addition of DTNB and ACTI. Absorbance was measured at 412 nm with Galantamine as the positive control.

2.6. Cytotoxicity and Cell Protection Assays

Cell viability was assessed using the MTT assay HepG2 cells (0.5 × 10⁵ cells/mL) were treated with FV or NV (0.04–100 μg/mL) for 24 h. For oxidative stress protection, cells were pre-incubated with samples (50 μg/mL) or ascorbic acid (20 μg/mL) for 24 h prior to exposure to 10 mM H₂O₂ for 1 h. Cell survival was calculated based on absorbance at 570 nm [17].

2.7. Statistical Analysis

Data were analyzed using Microsoft Excel 2013 and presented as Mean ± SD. One-way ANOVA was utilized to determine statistical differences, with p < 0.05 considered significant.

3. Results

3.1. Fabrication and Physicochemical Characterization of Nanovitexin

The nanovitexin system was successfully fabricated via the solvent evaporation method using a chemically cross-linked Alginate/CMC carrier. The schematic structure is illustrated in Figure 1 and

Table 1. Characteristic peaks on the FT-IR spectrum (cm⁻¹).

Functional	Peaks (cm−1)
	Alginate	Carboxymetyl Cellulose	Alginate/Carboxymetyl Cellulose Carrier
Si-O			659
C-O-C	1014	1080	1018
-C-O		1340	1323
-COO-		1440	1413
N-H			1588 and 600
-C=O	1631	1650
C-H sp3	2923	2940	2909
The -OH group in the trans isomer			3303
-OH	3421	3470

.

Figure 2a shows the FT-IR spectra of Alginate, Carboxymethyl cellulose (CMC), and the Alginate/CMC carrier system. Figure 2b illustrates the FT-IR spectra of the Alginate/CMC carrier, free vitexin, and the synthesized vitexin nanoparticles. Successful nano-encapsulation is evidenced by distinct shifts in characteristic absorption bands, indicating strong intermolecular interactions between the drug and the polymeric matrix. Specifically, the O-H stretching vibrations of the carrier (3303 cm⁻¹) and vitexin (3207 cm⁻¹) shifted to 3222 cm⁻¹ in the nanoparticle spectrum. Additionally, the C=O peak of vitexin (1649 cm⁻¹) and the N-H band of the carrier (1588 cm⁻¹) merged and shifted to 1563 cm⁻¹. Further structural confirmation is provided by the shift of the C-OH band (1180–1174 cm⁻¹) and the presence of Si-O symmetric stretching at 653 cm⁻¹. The slight variation in C-O-C vibration (1035 cm⁻¹) compared to the starting materials also corroborates the formation of a stable nanostructure. These spectral shifts confirm the successful encapsulation and strong intermolecular interactions (hydrogen bonding) between vitexin and the polymeric matrix.

Morphological analysis via FE-SEM (Figure 3) revealed that the nanoparticles are spherical and uniform, with a dry diameter ranging from 50 to 70 nm. However, Dynamic Light Scattering (DLS) measurements indicated a larger average hydrodynamic diameter of 178.6 nm in aqueous solution (Figure 4). The nanovitexin system demonstrated excellent stability, evidenced by a zeta potential of −33.5 mV (Figure 5).

3.2. Nanoencapsulation Efficiency (EE)

The encapsulation efficiency was determined using HPLC-MS. The chromatograms showed a single peak for nanovitexin at a retention time of 20.473 min (m/z 431.500), consistent with the vitexin standard. Based on the linear regression equation (y = 52.22756x + 40.08303, R² = 0.9986), the encapsulation efficiency was calculated to be 93.67%, as detailed in

Table 2. Vitexin content in the nanovitexin system.

Sample	Peak Area (mAu·s)	Vitexin Concentration in Diluted Sample (µg/mL)	Vitexin Content in Nanovitexin Powder (g)	Average Content (g)
1	3886	73.636	368.18	374.67 ± 5.65
2	3982	75.474	377.37
3	3993	75.696	378.48

.

3.3. Enhanced Antioxidant Activity

The DPPH radical scavenging assay results in Figure 6 showed that the Alginate/CMC blank carrier had negligible antioxidant activity (IC₅₀ > 500 μg/mL). In contrast, NV exhibited significantly superior antioxidant capacity compared to FV. The IC₅₀ value of NV was 115.38 μg/mL, nearly half that of FV (226.06 μg/mL), representing a 51.04% improvement in activity (p < 0.05).

3.4. In Vitro Antidiabetic Potential

• α-Glucosidase Inhibition

As shown in Figure 7, NV demonstrated remarkable potency with an IC₅₀ of 2.03±0.14 μg/mL. This represents a 57.71 fold enhancement compared to FV (117.15 ± 5.40 μg/mL) and is significantly more potent than the positive control, Acarbose (123.81 ± 3.58 μg/mL).

• α-Amylase Inhibition

The results in Figure 8 indicate that NV inhibited α-amylase with an IC₅₀ of 60.06 ± 0.42 μg/mL, which was 1.57 fold more effective than FV (95.02 ± 4.60 μg/mL) and superior to Acarbose (245.85 ± 7.71 μg/mL).

3.5. Cytotoxicity and Hepatoprotective Effects

In the cytotoxicity assay (Figure 9a), both FV and NV showed no significant toxicity towards HepG2 cells at concentrations up to 100 μg/mL, maintaining cell viability above 86%. Under H₂O₂-induced oxidative stress (Figure 9b), the model group viability dropped to 62.67%. Treatment with NV (50 μg/mL) effectively restored cell viability to 90.24 ± 2.45%, which was significantly higher than both the FV group (74.29 ± 3.33%) and the Ascorbic acid positive control (78.42 ± 4.20%) (p < 0.05).

4. Discussion

The clinical application of vitexin is currently limited by its poor solubility and rapid metabolic elimination. In this study, we successfully developed a nanovitexin delivery system using a novel Alginate/CMC matrix. The physicochemical characterization revealed a discrepancy between the dry size observed in SEM (50–70 nm) and the hydrodynamic size in DLS (178.6 nm). This swelling behavior is advantageous for hydrophilic polymer systems, as the hydrated matrix facilitates bioadhesion and prolongs retention time [18]. Furthermore, the DLS size of <200 nm is optimal for avoiding rapid renal clearance while enabling effective tissue permeation [18]. FT-IR analysis provided molecular evidence of encapsulation. The shift in the O–H stretching vibration to 3222 cm⁻¹ suggests strong intermolecular hydrogen bonding between vitexin and the carrier, stabilizing the drug in an amorphous state [2]. This difference is attributed to the swelling capability of the hydrophilic Alg/CMC matrix in the aqueous environment, creating a hydration shell around the particles.

Importantly, the validity of the nano-formulation is further confirmed by the particle size distribution analysis (Figure 4). The DLS profile displays a single, narrow peak, indicating a low polydispersity index (PDI value of 0.22). This unimodal distribution suggests that the synthesized nanovitexin is highly uniform and monodisperse, devoid of significant aggregation. Furthermore, the high absolute Zeta potential value of −33.5 mV corroborates the physical stability of the system, preventing particle coalescence through electrostatic repulsion. Therefore, combining the morphological evidence from FESEM (50–70 nm) with the uniformity data from DLS, we can confirm the successful fabrication of vitexin nanoparticles with a consistent size range suitable for biomedical applications.

A remarkable finding of this study is the high encapsulation efficiency of 93.67%, which surpasses previous reports for Zein/Pectin systems (67%) [18] or Alginate-Chitosan microspheres (68.92%) [19]. This efficiency likely results from the high compatibility between the polar functional groups of vitexin and the hydrophilic Alg/CMC network. Pharmacologically, the nano-formulation drastically improved the therapeutic potential of vitexin. The antioxidant activity of NV was nearly double that of FV. This aligns with Li et al. [2], who noted that nanostructuring increases the specific surface area available for redox reactions.

Regarding antidiabetic potential, NV exhibited a 57.71-fold increase in α-glucosidase inhibition compared to FV. While vitexin is known to bind to the hydrophobic pocket of α-glucosidase, its bulk form has limited affinity due to steric hindrance [20]. The reduction of particle size to the nanoscale likely facilitates rapid dissolution and molecular interaction according to the Ostwald–Freundlich principle, thereby enhancing inhibitory efficacy [3]. Interestingly, NV showed a strong but balanced inhibition of α-amylase (IC₅₀ of 60.06 μg/mL). This profile—strong α-glucosidase inhibition coupled with moderate α-amylase inhibition—is clinically favorable for minimizing the gastrointestinal side effects often associated with synthetic inhibitors like acarbose. Finally, the study demonstrated the superior hepatoprotective effect of NV. While oxidative stress is a key driver of metabolic disorders, NV maintained HepG2 cell viability at 90.24 ± 2.45% under H₂O₂ challenge, outperforming even ascorbic acid. This enhanced protection is attributed to the improved intracellular uptake of the nanoparticles compared to the crystalline free drug [9,21].

5. Conclusions

This study presents the first successful fabrication of vitexin-loaded nanoparticles using a biocompatible Alginate/Carboxymethyl Cellulose matrix via chemical cross-linking and solvent evaporation. The optimized nanovitexin system exhibited uniform particle size, high stability, and exceptional encapsulation efficiency (>93%). Compared to free vitexin, the nano-formulation demonstrated significantly enhanced antioxidant activity, superior inhibition of diabetes-related enzymes (α-glucosidase and α-amylase), and remarkable cytoprotective effects against oxidative stress in HepG2 cells. These results confirm that the Alg/CMC nanocarrier effectively overcomes the biopharmaceutical limitations of vitexin. Consequently, nanovitexin emerges as a promising candidate for the development of novel functional foods or pharmaceuticals aimed at managing diabetes and oxidative stress-related disorders. Future in vivo pharmacokinetic and pharmacodynamic studies are warranted to fully translate these findings into clinical applications.