Oxyresveratrol-Loaded Electrospun Cellulose Acetate/Poly(ε-caprolactone) Nanofibers with Enhanced Stability and Bioactivity

Abstract

Electrospun fibers serve as a medium for the targeted release of active compounds, facilitating the desired therapeutic effects in drug administration. The point of this study was to find the best conditions for making electrospun fibers from cellulose acetate (CA) and poly(ε-caprolactone) (PCL), mixed with pure oxyresveratrol extract from Artrocarpus lakoocha Roxberg (Moraceae). Additionally, the study focused on evaluating the antioxidant properties, antityrosinase activity, and freeze–thaw stability of the resulting fibers. We incorporated a concentration of oxyresveratrol at 0.1% w/w into various mass ratios of CA/PCL blended fiber sheets (1:0, 3:1, 1:1, 1:3), utilizing mixed solvents of acetone/DMF (2:1% v/v) and chloroform/DMF (9:1% v/v) for preparation. The fiber sheets displayed a continuous and uniform structure, with fiber diameters ranging from 300 to 1000 nanometers. We investigated the release kinetics of oxyresveratrol from the fibrous substrates using the total immersion technique, specifically in phosphate-buffered saline at a pH of 7.4. The results showed that the fiber sheet with a 3:1 w/w ratio of CA to PCL and a 0.1 w/w loading of oxyresveratrol showed the most significant release of oxyresveratrol at the 2 h mark, and it continued to release consistently at this peak value for up to 24 h. The antioxidant and anti-tyrosinase properties of oxyresveratrol in fiber sheets were more stable than those of free oxyresveratrol at the same concentrations. The fiber sheet presents a promising avenue for a user-friendly transdermal patch application.

1. Introduction

Artrocarpus lakoocha Roxberg belongs to the Moraceae family. It has been widely known as Mahat in South and Southeast Asia, particularly in Thailand [1]. It has been used as traditional medicine for treating various diseases, e.g., skin ailments, inflammatory conditions, and diabetes [2]. The dominant bioactive substance in Artrocarpus lakoocha has been reported to be trans-oxyresveratrol (2,3′,4,5′-tetrahydroxystilbene), which is a derivative of resveratrol [3]. Trans-oxyresveratrol is a natural stilbenoid that possesses diverse biological and pharmaceutical activities such as anti-inflammatory [4,5], antioxidant [6], antiviral [7,8], antibacterial [9,10], neuroprotective [11], and anti-tyrosinase effects [12,13,14]. However, it is susceptible to degradation when exposed to oxygen, moisture, and light. Therefore, its applications are limited, especially in the cosmetic industry. The cosmetics sector has utilized trans-oxyresveratrol to reduce skin pigmentation and promote a lighter complexion by inhibiting the activity of the enzyme tyrosinase [15,16].

Various encapsulation techniques have been used to enhance the stability and activity of bioactive compounds through lipid-based nanoemulsions [17], β-cyclodextrin-based inclusions [18], and calcium alginate-based encapsulations [19,20]. However, these encapsulation techniques have several limitations, including difficulty in handling, low drug loading capacity, and scalability [21]. These drawbacks also include kinetic instability, uncontrolled release and difficulty in controlling the particle size. In addition, the high temperature used during the production process could lead to the degradation of bioactive compounds.

Electrospinning, one type of encapsulation, is a promising alternative for solving some drawbacks mentioned above. This technique uses a high electrical force field to generate continuous and charged threads with uniform diameters at the nanometer level from a polymer solution at room temperature. The specific characteristics of electrospun fiber sheets produced display elevated drug loading efficacy and can modulate drug release levels due to their high surface-to-volume ratio [22,23,24]. Furthermore, electrospinning technique applies to a variety of polymeric substances as a medium, such as gelatin [25,26], poly(lactic acid) [27,28], polyvinyl alcohol [29,30], cellulose acetate (CA) [31,32], and polycaprolactone (PCL) [33,34] for the encapsulation of numerous bioactive compounds. From the literature reviews, only a few studies have used the mixture of CA and PCL for encapsulating some bioactive compounds, such as antimicrobial and antioxidant [35,36,37]. However, there have been no reports on the loading of trans-oxyresveratrol with CA mixed with PCL by using the electrospinning technique. Both CA and PCL were chosen for this work due to their biocompatibility, non-toxicity, and natural decomposition [38]. In addition, PCL can also enhance the elongation of nanofiber sheets produced from pure CA.

2. Materials and Methods

2.1. Reagents and Apparatus

Polycaprolactone (PCL, MW = 100,000 Da), cellulose acetate (CA, MW = 30,000 Da), Tyrosinase enzyme, 1,1′-diphenyl-2-picrylhydrazyl (DPPH), and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich. L-3,4-dihydroxyphenylalanine (L-DOPA) was obtained from Tokyo Chemical Industry. Methanol, ethanol, acetone, acetic acid, chloroform, and N,N-dimethylformamide (DMF) were purchased from RCI Labscan (Bangkok, Thailand). All chemicals and reagents were of analytical grade. Artrocarpus lakoocha powder was collected from a local Thai herb trading market in Bangkok, Thailand.

2.2. Preparation of trans-Oxyresveratrol from Artrocarpus Lakoocha Powder Using Microwave-Assisted Extraction (MAE)

1.0 g of Artrocarpus lakoocha powder was extracted with 10 mL of a 2:1 (v/v) ethanol-water mixture using a cooking microwave at 240 W for 1 min. The ethanol-water extract was then filtered through Whatman No. 1 filtering paper. The filtrate was directly subjected to column chromatography over alumina (Al₂O₃) and eluted with a 2:1 (v/v) ethanol-water mixture to yield trans-oxyresveratrol. To purify trans-oxyresveratrol, it was crystallized with ethanol to afford a yellowish solid [39].

2.3. Identification of Trans-Oxyresveratrol

A yellowish solid of trans-oxyresveratrol was characterized using a 400 MHz ¹H NMR spectrometer (Bruker Avance III HD 400 MHz, Switzerland). ¹H-NMR (400 MHz, d-DMSO) δ (ppm): 6.25 (dd, 1H, H-5), 6.32–6.35 (m, 3H, H-3,H-2′, H-6′), 6.77 (d, J = 16 Hz, 1H, H-β), 7.34 (d, J = 16 Hz, 1H, H-α), 9.19 (s, 2H, 3′-OH, 5′-OH), 9.43 (s, 1H, 4-OH), 9.61 (s, 1H, 2-OH) [39]. Qualitative and quantitative analyses of trans-oxyresveratrol were also carried out using the HPLC technique. Solutions of trans-oxyresveratrol standard and sample were prepared in methanol at a concentration of 20 mg/mL. It was then filtered through 20 µm Millipore filters. 20 µL of each filtrate was injected into an Agilent C18 bonded-silica gel column (250 × 4.6 mm, 5 µm) of a Hitachi L-7420 HPLC system connected to a UV-Vis detector. The HPLC analysis was performed using a linear gradient elution of a methanol-water mixture, ranging from 10:90 to 70:30 (v/v), with a flow rate of 1 mL/min and a runtime of 35 min. The wavelength of the UV detector was 320 nm [40]. The content of trans-oxyresveratrol was quantified from the calibration curve of trans-oxyresveratrol standards in a concentration range of 2–50 ppm and was expressed in mg/100 g of dry weight. The analysis was carried out in triplicate. The calibration curve of trans-oxyresveratrol standard with a correlation coefficient (R²) in linear regression greater than 0.99 was used.

2.4. Optimal Fabrication of trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

A 9% (w/w) PCL solution was prepared in a 1:9 (v/v) mixture of dimethylformamide and chloroform and a 17% (w/w) CA solution was prepared in a 2:1 (v/v) mixture of acetone and dimethylformamide. After that, the mixtures of CA and PCL solutions with different mass ratios (0:1, 1:1, 1:3, 3:1, and 1:0) were prepared by stirring at 500 rpm for one hour at room temperature. After being mixed thoroughly, 0.1% (w/w) of trans-oxyresveratrol in acetone was added. The trans-oxyresveratrol-loaded CA-PCL solution was subsequently transferred into a 5 mL syringe and equipped with a metal needle with an inner diameter of 0.22 mm. Its needle was also contacted to a positive electrode (Gamma High Voltage Research, Crystal River, FL, USA), and a negative electrode was attached to the rotary spinning collector under an applied voltage of 15 kV. The distance between the needle tip and the collector was approximately 12 cm. The feed rate of trans-oxyresveratrol-loaded CA-PCL solution was 0.5 mL/h. The nonwoven fibers were collected using a 50 mm rotating drum onto an aluminum foil at a speed of 1200 rpm. The fiber fabrication was conducted at room temperature. The residual solvent in the fiber sheet was removed in a vacuum oven at 70 °C for 12 h [41,42,43].

2.5. The Morphology of trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

The morphology and diameter of CA-PCL fibers were examined using a scanning electron microscope (SEM, Maxim 2000, UK) at an accelerating voltage of 10,000 kV and a magnification of 10,000× Before analysis, all samples of trans-oxyresveratrol-loaded CA-PCL fiber sheets were gold-coated to mitigate charging effects. The fiber diameters, diameter distribution, and uniformity were measured using ImageJ 1.51 K software (n ≥ 100). The thickness of trans-oxyresveratrol-loaded CA-PCL fiber sheets was measured using a JSM-6610 LV (JEOL, USA).

2.6. Determination of Percent Loading of Trans-Oxyresveratrol in trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

A 10 mg sample of a 3:1 w/w CA-PCL fiber sheet loaded with 0.01–0.5% (w/w) trans-oxyresveratrol was sonicated in 10 mL of a 2:1 (v/v) acetone and dimethylformamide mixture for 5 min until a clear solution mixture was obtained. The concentration of trans-oxyresveratrol in the solution mixture was quantified using a standard calibration curve obtained with a UV-Vis spectrometer at the maximum wavelength of 333 nm. The trans-oxyresveratrol content loaded into a CA-PCL fiber sheet was calculated as follows:

$$percent loading of \mathit{trans-oxyresveratrol} (\%)= {\displaystyle \frac{A}{B}}\times 100$$

(1)

where A is the amount of trans-oxyresveratrol experimentally determined from the CA-PCL fiber sheet, and B is the theoretical amount of trans-oxyresveratrol initially introduced into the CA-PCL fiber sheet.

2.7. In Vitro trans-Oxyresveratrol Release of trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

The release profiles of trans-oxyresveratrol were obtained using the total immersion method [44]. A 20 mg sample of (1:0, 3:1, 1:1, 1:3, 0:1) w/w CA-PCL fiber sheet loaded with 0.1% (w/w) trans-oxyresveratrol was tested in a 30 mL vial containing 20 mL of 10 mM PBS at pH 7.4 with 0.5% v/v Tween 80 and 10% v/v methanol. The vial was shaken at 100 rpm in an incubator at 37 °C. The accumulative concentrations of trans-oxyresveratrol were assessed by withdrawing 1.5 mL of the release medium from the vial at intervals (0, 1, 2, 3, 6, 9, 12, and 24 h), followed by the addition of an equal volume of fresh medium solution to maintain experimental conditions. The release profiles were conducted using a calibration curve obtained from a UV-Vis spectrophotometer at 333 nm and presented as cumulative release data.

2.8. Chemical Characterization of trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets by Using ATR-FTIR Spectroscopy

The chemical structure of a 3:1 w/w CA-PCL fiber sheet loaded with 10% w/w of trans-oxyresveratrol was characterized by using a Nicolet 6700 FTIR spectroscopy in ATR mode at room temperature in the spectral range of 4000 to 400 cm⁻¹.

2.9. Thermal Stability Analysis by Thermogravimetric Analysis (TGA) of trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

The thermal stability of the fiber mats was assessed using thermogravimetric analysis (TGA). Samples (approximately 4–5 mg) were sectioned into small fragments and positioned in an aluminum pan. Measurements were performed within the temperature range of 50 to 500 °C at a heating rate of 10 °C min⁻¹ under nitrogen flow. Weight loss percentage was measured in relation to temperature.

2.10. Swelling and Weight Loss of trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

Water uptake capability and weight loss percentage of a 0.1% (w/w) trans-oxyresveratrol-loaded (1:0, 3:1, 1:1, 1:3, 0:1) w/w CA-PCL fiber sheet were assessed as follows [45]. Briefly, a trans-oxyresveratrol-loaded CA-PCL fiber sheet of known weight (Mo) was incubated in phosphate-buffered saline (PBS, pH 7.4) at 37 °C for 1 and 7 days. After incubation, the sample was washed with a small amount of distilled water, dried with a filter paper, and weighed immediately (Mw). It was subsequently dried in a vacuum oven at 70 °C until its weight remained constant (Md). Its water uptake capability, swelling ratio (%), and weight loss percentage, weight loss ratio (%), were calculated using the following equations (Equations (2) and (3)).

$$Swelling ratio \left(\%\right)={\displaystyle \frac{\left(M_w-M_o\right)}{ M_o}}\times 100$$

(2)

$$Weight loss ratio (\%)={\displaystyle \frac{\left(M_d-M_o\right)}{ M_o}}\times 100$$

(3)

All experiments were conducted with three replications.

2.11. Tensile Testing of trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

The tensile properties of the electrospun fiber mats were assessed utilizing a universal testing machine (UTM) in accordance with ASTM D638 standards [41]. Specimens were fabricated in a dumbbell shape with dimensions of 115 mm by 25 mm. The thickness was measured at the center and both ends to determine the cross-sectional area (A). Testing was conducted at a crosshead speed of 10 mm/min using a 5000 N load cell. Three specimens were tested for each condition, and the average values of tensile strength and elongation at break were documented.

2.12. Anti-Oxidative Capacity of trans-Oxyresveratrol in trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

The scavenging percentage of DPPH radicals was determined using the DPPH method given by Han et al. (2009) [46]. In summary, 100 µL of methanolic solution of 3:1 (w/w) CA-PCL fiber sheet containing 0.01–0.5% (w/w) trans-oxyresveratrol was mixed with 100 µL of 1 × 10⁻⁴ M 1,1′-diphenyl-2-picrylhydrazyl (DPPH) reagent in methanol. The solution mixture was kept in the dark for 30 min and then analyzed for its DPPH scavenging activity using a microplate reader at an absorbance wavelength of 517 nm on days 1, 3, 7, 14, 21, and 28. Moreover, the anti-oxidative capacity of 0.01–0.5% (w/w) free trans-oxyresveratrol in methanol was also studied for comparison with that of trans-oxyresveratrol loaded into 3:1 (w/w) CA-PCL fiber sheets.

$$DPPH scavenging (\%)={\displaystyle \frac{A_{control}-A_{blank}}{A_{control}}}\times 100$$

(4)

2.13. Anti-Tyrosinase Activity of trans-Oxyresveratrol in trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

The tyrosinase inhibitory effect of trans-oxyresveratrol-loaded CA-PCL fiber sheets and oxyresveratrol-loaded CA/PCL solutions was evaluated using a modified method based on Masuda et al., 2005 [47]. A 20 mg sample of a 3:1 (w/w) CA-PCL fiber sheet containing 0.01–0.5% (w/w) trans-oxyresveratrol. Twenty milligrams of oxyresveratrol were incorporated into a 3:1% w/w CA/PCL matrix, with oxyresveratrol concentrations of 0.01%, 0.05%, 0.1%, and 0.5%% w/w in the fiber mat. The mixture was then dissolved in 10 mL of 20% (v/v) ethanol. The solution was then subjected to a thermal regimen consisting of heating in an oven at 45 °C for 6 h, followed by cooling in a refrigerator at 4 °C for an additional 6 h, completing one cycle. This process was repeated for 28 cycles. The tyrosinase inhibition activity of the remaining trans-oxyresveratrol in the solutions was determined at 0 and 30 min using a microplate reader at an absorbance of 492 nm on days 1, 3, 7, 14, 21, and 28, as described in [47]. Moreover, the anti-tyrosinase activity of 0.01–0.5% (w/w) free trans-oxyresveratrol in methanol was also studied, along with that of trans-oxyresveratrol loaded into 3:1 (w/w) CA-PCL fiber sheets.

$$Tyrosinase inhibition\left(\%\right)={\displaystyle \frac{\left(A-B\right)-\left(C-D\right)}{\left(A-B\right)}}\times 100$$

(5)

where A, B, C and D are the absorbance values of the control, control blank, test sample and test sample blank, respectively.

2.14. Stability of trans-Oxyresveratrol in trans-Oxyresveratrol-Loaded CA PCL Fiber Sheet

The stability of trans-oxyresveratrol which was loaded into a 3:1 (w/w) CA-PCL fiber sheet containing 0.01–0.5% (w/w) trans-oxyresveratrol was evaluated using an accelerated degradation analysis. A 20 mg sample of the fiber sheet was dissolved in 10 mL of a 2:1 (v/v) acetone and dimethylformamide mixture. The solution was then subjected to a thermal regimen consisting of heating in an oven at 45 °C for 6 h, followed by cooling in a refrigerator at 4 °C for an additional 6 h, completing one cycle. This process was repeated for 28 cycles. The amount of trans-oxyresveratrol lost was measured using a UV-Vis spectrophotometer at 333 nm on days 1, 3, 7, 14, 21, and 28. Moreover, the stability of 0.01–0.5% (w/w) free trans-oxyresveratrol in 10 mL of a 2:1 (v/v) acetone and dimethylformamide mixture was also studied, along with the stability of trans-oxyresveratrol loaded into 3:1 (w/w) CA-PCL fiber sheets.

2.15. Statistical Analysis

All tests were conducted in triplicate. The experimental results are shown as mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) was conducted with three replications (n = 3), testing data with SigmaPlot 10.0.

3. Results and Discussion

3.1. Preparation and Characterization of trans-Oxyresveratrol from Artrocarpus Lakoocha Powder Using Microwave-Assisted Extraction (MAE)

The microwave-assisted extraction (MAE) of Artrocarpus Lakoocha powder yielded a dark brown crude ethanol-water extract with 28% yield. Subsequent purification by column chromatography affords trans-oxyresveratrol (Figure 1) as a yellowish solid with 12% yield. The ¹H-NMR spectrum of trans-oxyresveratrol (Figure S1) was used to confirm its chemical structure, showing six aromatic protons at 6.25–6.35 ppm, two trans-stilbene protons at 6.77 ppm (H-β) and 7.34 ppm (H-α) with a coupling constant J = 16 Hz, and four hydroxyl protons at 9.19 (1-OH and 3-OH), 9.43 (4-OH), 9.61 (2-OH) ppm. Additionally, high-performance liquid chromatography (HPLC) analysis determined the purity of trans-oxyresveratrol to be 95%, confirming its suitability for further experimental applications.

3.2. Optimal Fabrication of trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

The optimal conditions for the fabrication of trans-oxyresveratrol-loaded CA-PCL fiber sheets were determined based on the fiber morphology using different CA:PCL mass ratios with 0.1% (w/w) trans-oxyresveratrol. Figure 2 showed the morphology of fibers fabricated from different CA:PCL mass ratios with 0.1% (w/w) trans-oxyresveratrol by using Scanning Electron Microscopy (SEM). As the PCL content increased, the fiber size and size distribution tended to increase, while the fiber smoothness tended to decrease. However, no beads were observed on all trans-oxyresveratrol-loaded CA-PCL fibers. From the study of efficacy of trans-oxyresveratrol loading in CA-PCL fiber sheets with various CA:PCL mass ratios at 0.1% (w/w) trans-oxyresveratrol (

Table 1. Percent loading of 0.1% w/w trans-oxyresveratrol in various 0:1, 3:1, 1:1, 1:3, 0:1 (w/w) CA-PCL fiber sheets prepared with different trans-oxyresveratrol loading contents.

Formulation	Loading Content (%)
CA:PCL (1:0)	80.53 ± 5.89 a
CA:PCL (3:1)	82.85 ± 0.66 a
CA:PCL (1:1)	80.84 ± 2.67 a
CA:PCL (1:3)	81.36 ± 3.72 a
CA:PCL (0:1)	76.81 ± 1.84 b

^a,b represent a significant difference at p-value < 0.05.

), it was found that the percent loading of trans-oxyresveratrol was found to range from 76.81% to 82.85% with no statistically significant difference at the 95% confidence level. These experimental results lead to use 3:1 w/w CA-PCL fiber sheet loaded with 0.1% (w/w) trans-oxyresveratrol for further study.

3.3. In Vitro trans-Oxyresveratrol Release of trans-Oxyresveratrol-Loaded CA-PCL Fiber Sheets

The release profiles of trans-oxyresveratrol were obtained using the total immersion meth. A 20 mg sample of a 3:1 w/w CA-PCL fiber sheet loaded with 0.1% (w/w) trans-oxyresveratrol was tested in a 30 mL vial containing 20 mL of 10 mM PBS at pH 7.4 with 0.5% v/v Tween 80 and 10% v/v methanol. The vial was shaken at 100 rpm in an incubator at 37 °C. The accumulative concentrations of trans-oxyresveratrol were assessed by withdrawing 1.5 mL of the release medium from the vial at intervals (0, 1, 2, 3, 6, 9, 12, and 24 h), followed by the addition of an equal volume of fresh medium solution to maintain experimental conditions. The release profiles were conducted using a calibration curve obtained from a UV-Vis spectrophotometer at 333 nm and presented as cumulative release data.

The release efficacy of oxyresveratrol from CA-PCL polymer blend fibers at ratios of 1:0, 3:1, 1:1, 1:3, and 0:1 (CA:PCL) mixed with 0.1% w/w oxyresveratrol was evaluated under simulated skin conditions in a phosphate-buffer solution (PBS, pH 7.4) [48,49,50]. Oxyresveratrol, as a phenolic compound, has limited water solubility. To enhance its solubility, the medium was adjusted following the methodology of Han et al. [46], using 1% Tween 80 and 10% methanol in a 10 mM PBS solution (pH 7.4). Fibers of different CA:PCL ratio (20 mg each) was immersed in a 20 mL phosphate-buffer solution (pH 7.4). At specific time intervals (0, 0.5, 1, 2, 3, 6, 9, 12, and 24 h), 1.5 mL of the solution was collected to determine the amount of oxyresveratrol released, comparing it to a standard oxyresveratrol solution (2–10 ppm). Measurements were conducted using a UV-visible spectrophotometer at 333 nm, and a cumulative release percentage graph was plotted over time.

The release profiles of oxyresveratrol (Oxy) from different CA:PCL:Oxy formulations are presented in Figure 3. For the CA:PCL:Oxy ratio of 1:0:0.1% w/w, the initial release at 0 h was 2.71% ± 2.27, followed by a sharp increase that reached 95.29% ± 4.78 at 2 h and remained stable up to 24 h. In contrast, the 0:1:0.1% w/w (PCL-only) formulation exhibited a lower initial release of 1.70% ± 1.05, with a maximum release of 78.74% ± 1.67 at 2 h, which was sustained throughout 24 h. The intermediate formulations with CA:PCL ratios of 3:1, 1:1, and 1:3 showed comparable release profiles, characterized by a rapid increase with peak release at 2 h, followed by a plateau maintained until 24 h. Overall, the data confirm that the presence of citric acid enhances Oxy release efficiency, with all formulations displaying an initial burst release within 2 h and stable release thereafter. The electrospinning technique offers a significant advantage in effectively controlling the release mechanisms of drugs. Increasing the PCL polymer ratio resulted in a decreased percentage of oxyresveratrol release, due to PCL’s larger fiber size and low hydrophilicity, which restricted oxyresveratrol diffusion. In contrast, higher CA content enhanced Oxyresveratrol release. CA fibers exhibited smaller dimensions and higher homogeneity, resulting in greater surface area, which improved drug release efficiency. Based on these findings, the optimal fiber formation for face mask preparation was determined to be a CA:PCL polymer blend at a 3:1% w/w ratio. These fibers exhibited ideal release behavior, a smooth surface with high homogeneity, and an average fiber diameter of 452 nm, resulting in a high surface area and increased water absorption capacity, which facilitates the effective release of oxyresveratrol.

3.4. Proving the Structural Identity of Oxyresveratrol from CA-PCL Nanofiber Sheet Using the FT-IR Technique

The presence of oxyresveratrol in fiber blends of cellulose acetate and polycaprolactone (3:1% w/w) was investigated. To ensure measurable detection, oxyresveratrol was incorporated at a concentration of 10% w/w. The IR spectrum (Figure 4) showed significant peaks from the CA and PCL polymers, with essential peaks being the –OH group of CA at 3269 cm⁻¹, the CH₂ group, and the carbonyl group of PCL at 2928 cm⁻¹ and 1699 cm⁻¹, respectively. The analysis revealed that the incorporation of oxyresveratrol into the fibers resulted in the emergence of a broad O–H stretching peak at a frequency of 3269 cm⁻¹. The shift in the –OH peak in oxyresveratrol is noteworthy, likely resulting from significant hydrogen bonding interactions with cellulose acetate [21]. A C–H stretching peak of oxyresveratrol was identified at a frequency of 1591 cm⁻¹. The process of preparing fibers that incorporated oxyresveratrol within a polymer blend of CA and PCL yielded positive results, demonstrating that oxyresveratrol was retained in the fibers following their formation. Nonetheless, the FTIR data can be considered preliminary findings, indicating that further research on the quantity of oxyresveratrol incorporated into the fibers may be necessary.

3.5. Study of the Thermal Properties of CA-PCL Nanofibers Using Thermogravimetric Analysis (TGA) with Encapsulated Resveratrol

The thermogram allows for the analysis of the decomposition temperature (Td) of each material. The TGA thermogram (Table S2) of PCL indicates significant thermal stability at elevated temperatures, with a decomposition temperature of 389.06 °C and a weight loss of 97.01% upon complete disintegration, leaving 0.52% ash residue. The decomposition temperature for CA was 337.18 °C, with a corresponding weight loss of 81.47%, resulting in 16.33% ash after complete breakdown (Figure 5). The breakdown of oxyresveratrol occurred in two temperature ranges: at 80.65 °C, it exhibited a weight loss of 5.88%, and at 223.16 °C, the residual components disintegrated, resulting in a weight loss of 50.00% and a post-decomposition ash content of 43.49%. The thermogram of the polymer fibers composed of CA:PCL in a 3:1% w/w ratio exhibited decomposition at two temperature ranges: 331.10 °C, corresponding to the decomposition of CA with a weight loss of 53.33%, 391.73 °C, associated with the decomposition of PCL, resulting in a weight loss of 35.52%.

The ash content following complete decomposition was 9.55%. The polymer fibers composed of CA:PCL in a 3:1% w/w ratio, with the incorporation of 10% w/w of oxyresveratrol to enhance the detection signal, exhibited no variation in weight loss compared to the polymer fibers of the same CA:PCL ratio without oxyresveratrol at the same temperature. The material decomposed at three distinct temperature ranges: 228.01 °C, corresponding to oxyresveratrol decomposition, with a weight loss of 20.92%, 324.46 °C, associated with CA decomposition, resulting in a weight loss of 17.55% and 390.33 °C, linked to PCL decomposition, exhibiting a weight loss of 23.42%. Upon complete decomposition, the ash content measured 33.50%. The experiment demonstrates that a polymer blend of CA:PCL in a 3:1% w/w ratio, used for fiber preparation with oxyresveratrol, exhibits favorable thermal stability. After being processed into fibers, oxyresveratrol retains its decomposition temperature and maintains suitable properties for use as a skin adhesive material.

3.6. Study of Swelling Properties in Water and Percentage of Weight Loss

The investigation of swelling behavior through water immersion involved the use of prepared fibers with a specified weight of 10 mg, which were submerged in a phosphate-buffer solution (PBS, pH 7.4) containing 1% Tween 80 and 10% Methanol in 10 mM PBS for 1 day and 7 days. This study aimed to determine the fiber formulation with superior swelling capacity in water and its potential impact on the release efficiency of resveratrol from the fiber [51]. The results are presented in Figure 6. After one day of immersion, the swelling percentage exceeded 100%, ranging from 256.57 ± 33.85% to 363.23 ± 50.14%. The bar chart indicates that including CA in the fibers enhances swelling capacity in water due to its hydrophilic properties, which facilitate efficient water absorption. Conversely, incorporating PCL into the fibers reduces swelling behavior in water due to the hydrophobic nature of PCL [52,53]. Following a 7-day assessment, all fibers exhibited increased swelling characteristics. The findings indicated that a higher CA content in the fibers corresponded to improved swelling properties. In comparison, higher PCL content reduced the swelling potential, thereby limiting the release capacity of substances from PCL-containing fibers.

Weight loss is contingent upon the stability and degradation of the material. Prepared fibers with a known weight were immersed in a phosphate-buffer solution (pH 7.4) for 1 day and 7 days to assess weight loss. After 1 day of immersion, the observed weight loss ranged from 0.65 ± 0.51% to 1.33 ± 0.53%. The fibers exhibited minimal weight loss, with the most significant loss occurring in the CA:PCL 3:1 condition. The presence of CA in the fibers may have caused partial dissolution in water, resulting in weight loss after immersion in the medium solution (Figure 7). The percentage of weight loss increased after 7 days of immersion, ranging from 1.65 ± 0.51% to 4.30 ± 1.33%. The weight loss observed after 7 days was comparable to the weight loss observed after 1 day, suggesting that higher CA content in the fibers leads to greater weight loss. This is attributed to the polar characteristics of CA, which facilitate the detachment of specific components from the fiber sheet.

3.7. Study of the Mechanical Properties of Nanofiber Mats of Oxyresveratrol from Polymer Blends of CA:PCL in Various Ratios

The analysis of the elastic modulus for each fiber type (Figure 8), which reflects the material’s response to stress, reveals that CA-oxy fibers exhibit low resistance to external forces, categorizing them as brittle materials. PCL-oxy fibers, on the other hand, show a higher elastic modulus, allowing for greater resistance to external forces compared to CA-oxy fibers. The investigation of mixed polymer fibers (CA:PCL:oxy) at different ratios revealed that the polymer combinations enhanced the elastic modulus, thereby increasing the strength and ductility of the fibers. The experiments indicate that the CA-oxy polymer fibers are brittle, characterized by a limited strain range and low elongation at break, leading to diminished mechanical properties. Conversely, PCL-oxy fibers exhibit superior mechanical properties, characterized by high flexibility and strength, classifying them as a malleable material. Incorporating PCL into CA fibers enhances the strength, resulting in increased resistance to separation. This research identifies CA:PCL polymer blend fibers in a 3:1 w/w ratio as suitable candidates for developing a removable skin adhesive material. The tensile strength tests of these fibers demonstrate favorable mechanical characteristics, specifically sufficient strength and flexibility, making them appropriate for further applications.

3.8. The DPPH Radical Scavenging Activity of Oxyresveratrol Encapsulated in CA-PCL Nanofibers Compared to Free Oxyresveratrol

The antioxidant capacity of free oxyresveratrol and resveratrol after fiber formation was examined. Fibers were synthesized by incorporating oxyresveratrol at varying concentrations of 0.01, 0.05, 0.1, and 0.5% w/w into a polymer blend of CA:PCL in a 3:1% w/w ratio. A 50 mg sample of each fiber formation was dissolved in 5 mL of methanol. The DPPH radical scavenging capacity was then assessed using a microplate reader at a wavelength of 517 nm. Figure 9 illustrates the concentrations of free oxyresveratrol and oxyresveratrol encapsulated in the nanofibers at 0.01, 0.05, 0.1, and 0.5% w/w. The oxyresveratrol encapsulated in the fibers exhibited DPPH radical scavenging activity like that of free oxyresveratrol at equivalent concentrations. An increase in oxyresveratrol concentration correlates with enhanced radical scavenging activity. The radical scavenging percentages at c of 0.1% and 0.5% w/w were 90.67% ± 2.54 and 102.01% ± 0.69, respectively, with no statistically significant difference observed at the 95% confidence level. The experiment indicates that the optimal ratio of oxyresveratrol for encapsulation in fibers is 0.1% w/w, as this concentration achieves nearly 100% radical scavenging efficiency without unnecessary use of the substance. Consequently, the 0.1% w/w oxyresveratrol encapsulated in fibers, along with free oxyresveratrol, was further analyzed for its radical scavenging efficiency under accelerated conditions.

3.9. Study of the DPPH Radical Scavenging Activity of Oxyresveratrol Encapsulated in Fibers Under Accelerated Conditions Compared to Free Oxyresveratrol

The study investigates the DPPH radical scavenging ability of free oxyresveratrol and oxyresveratrol incorporated into fibers. Fibers were prepared by incorporating oxyresveratrol at a concentration of 0.1% w/w into a polymer blend of CA:PCL in a 3:1% w/w ratio. A total of 50 mg of the blend was dissolved in 5 mL of methanol and subjected to a thermal cycling process. It was then stored in a hot air oven at 45 °C for 8 h, followed by storage in a refrigerator at 4 °C for 8 h. The absorbance of DPPH at 517 nm was measured weekly for one month using a microplate reader, following the method established by Neo et al. [52]. Figure 10 illustrates the percentage of DPPH radical scavenging activity. On day 1, free oxyresveratrol exhibited an antioxidant efficiency against DPPH of 90.55% ± 2.22, which progressively declined to 55.34% ± 4.33 by day 28. In contrast, oxyresveratrol incorporated into fibers at a concentration of 0.1% w/w showed an antioxidant efficiency against DPPH of 90.67% ± 2.54 on day 1, which declined to 75.14% ± 1.11 by day 28. Free oxyresveratrol demonstrated lower stability under accelerated conditions than oxyresveratrol encapsulated in fibers at the same concentration, with a statistically significant difference at a 95% confidence level, leading to a reduction in DPPH antioxidant efficiency. The experiment demonstrated that the electrospinning technique effectively preserves the stability of oxyresveratrol and maintains its antioxidant efficiency against free radicals, making it a promising method for enhancing the stability of oxyresveratrol in practical applications.

3.10. Study of the Tyrosinase Inhibitory Activity of Fiber-Encapsulated Oxyresveratrol Compared to Free Oxyresveratrol at the Same Concentration

This study examines the tyrosinase inhibitory activity to assess the effectiveness of the prepared product in skin whitening. Tyrosinase is an enzyme responsible for skin pigmentation, promoting the darkening of skin pigment. However, substances that inhibit tyrosinase activity can result in skin whitening. This experiment compared the efficacy of free oxyresveratrol to oxyresveratrol in fiber form at identical concentrations. Fibers combining oxyresveratrol at various concentrations were obtained from a CA:PCL polymer blend at a 3:1% w/w ratio. A 20 mg fiber sample was dissolved in 5 mL of 20% v/v ethanol, and the absorbance of L-dopachrome at 492 nm was measured using a microplate reader, following the method of Masuda et al. [44]. The percentage of tyrosinase inhibition is illustrated in Figure 11.

The experiment found that increasing the concentration of oxyresveratrol enhanced tyrosinase inhibition. The tyrosinase inhibitory activity of free oxyresveratrol at concentrations of 0.01, 0.05, 0.1, and 0.5% w/w was 52.01% ± 1.68, 84.62% ± 3.30, 94.88% ± 2.30, and 99.27% ± 1.68, respectively. Meanwhile, the tyrosinase inhibitory activity of oxyresveratrol encapsulated in fibers at the same concentrations was 50.92% ± 1.68, 86.81% ± 2.91, 95.24% ± 3.36, and 98.17% ± 2.77, respectively. Both free oxyresveratrol and oxyresveratrol encapsulated in fibers at the same concentrations exhibited comparable tyrosinase inhibitory activity. This demonstrates the effectiveness of the encapsulation technique for delivering oxyresveratrol via nanofibers. At 0.1% and 0.5% w/w concentrations, the percentage inhibition of tyrosinase was 95.24% ± 3.36 and 98.17% ± 2.77, respectively, with no statistically significant difference at the 95% confidence level. Therefore, 0.1% w/w oxyresveratrol is the optimal concentration for fiber encapsulation, as it achieves tyrosinase inhibition comparable to 0.5% w/w without unnecessary material usage. Based on this finding, 0.1% w/w oxyresveratrol encapsulated in nanofibers and free resveratrol will be further studied for tyrosinase inhibitory effects under accelerated conditions.

3.11. Study of the Inhibitory Effect on Tyrosinase Enzyme of Oxyresveratrol Encapsulated in Fibers Under Accelerated Conditions Compared to Free Oxyresveratrol at the Same Concentration

This experiment investigated the efficacy of free oxyresveratrol and oxyresveratrol in fiber form at identical concentrations under accelerated conditions. The fibers were prepared by incorporating 0.1% w/w oxyresveratrol into a CA:PCL polymer blend at a 3:1% w/w ratio, totaling 20 mg. This mixture was dissolved in 5 mL of 20% v/v ethanol and subjected to a thermal cycling process, stored in a hot air oven at 45 °C for 8 h, followed by 8 h in a refrigerator at 4 °C. The absorbance of L-dopachrome at 492 nm was measured weekly over one month using a microplate reader. Figure 12 illustrates the percentage of tyrosinase inhibition. Initially, free oxyresveratrol and oxyresveratrol encapsulated in a mixed polymer fiber at 0.1% w/w demonstrated tyrosinase inhibition percentages of 94.88% ± 2.30 and 95.24% ± 3.36, respectively. Under accelerated conditions, tyrosinase inhibition progressively diminished, with values decreasing to 67.38% ± 0.62 for free oxyresveratrol and 75.63% ± 1.24 for fiber-encapsulated oxyresveratrol by day 28. Free oxyresveratrol exhibits reduced stability, corresponding with a further decline in tyrosinase inhibition capacity. The mixed polymer fiber demonstrated superior efficacy in preserving oxyresveratrol activity compared to free oxyresveratrol. The experiment confirmed that an increased oxyresveratrol concentration significantly enhanced the tyrosinase inhibition capacity, with 0.1% w/w oxyresveratrol identified as the optimal concentration for encapsulation in fibers intended for skin-whitening applications.

3.12. Study of the Stability of Oxyresveratrol Encapsulated in CA-PCL Nanofibers Under Accelerated Conditions Compared to Free Oxyresveratrol at the Same Concentration

This study investigates the stability of oxyresveratrol, specifically its temperature resistance during fiber formation. It investigates the residual quantity of oxyresveratrol after integration into a polymer fiber blend of cellulose acetate (CA) and polycaprolactone (PCL) at a 3:1 w/w ratio, with oxyresveratrol concentrations ranging from 0.01% to 0.5% w/w, compared to free oxyresveratrol at equivalent concentrations. The samples were prepared at a concentration of 10 ppm in 10 mL of acetone and subjected to a storage regimen of 45 °C in a hot air oven for 8 h, followed by refrigeration at 4 °C for 8 h, constituting one cycle. A total of 28 cycles were conducted. The absorbance at 333 nm was measured weekly over one month using a UV-visible spectrophotometer. The residual quantity of oxyresveratrol was determined by comparison with a standard oxyresveratrol solution at concentrations ranging from 2 to 10 ppm. Initially, it was observed that fibers infused with oxyresveratrol at various concentrations and free oxyresveratrol at a concentration of 10 ppm exhibited similar oxyresveratrol levels exceeding 97%.

However, this concentration progressively declined, reaching 65.42% ± 0.78 by day 28 for the free oxyresveratrol. In fibers with oxyresveratrol concentrations ranging from 0.01% to 0.5% w/w, it was noted that the residual oxyresveratrol after 28 days was marginally higher in fibers with a concentration of 0.01% w/w than in those with higher concentrations. At 0.05% w/w, the remaining amount of oxyresveratrol was 76.63% ± 1.42 and 74.42% ± 2.48, respectively. In the fibers with 0.1% and 0.5% w/w oxyresveratrol, the remaining amounts on day 1 were 100.03% ± 1.42% and 97.71% ± 2.10%, respectively, and by day 28, they were 77.47% ± 1.60% and 76.54% ± 3.22%, respectively (Figure 13). All four concentrations exhibit comparable retention levels of oxyresveratrol. The encapsulation of oxyresveratrol within polymer fibers demonstrates superior stability compared to free oxyresveratrol at equivalent concentrations, even under accelerated conditions across all 28 cycles.

4. Conclusions

Nanofiber sheets containing oxyresveratrol were successfully produced using electrospinning techniques with environmentally friendly and biocompatible polymers, namely cellulose acetate (CA) and polycaprolactone (PCL). The crude extract from the heartwood of the Artrocarpus lakoocha yielded 28%, and the oxyresveratrol was isolated via column chromatography, achieving a 12% yield as a yellow solid. The isolated oxyresveratrol achieved a purity of 95%, as determined by HPLC analysis. To prepare polymer fiber sheets, cellulose acetate and polycaprolactone were blended at various ratios, incorporating 0.1% w/w oxyresveratrol. SEM analysis revealed fiber sizes ranging from 270.49 nm to 854.05 nm. The release efficacy of oxyresveratrol from fibers was evaluated through immersion in a simulated skin environment (pH 7.4), with fibers containing a CA:PCL (3:1) polymer blend exhibiting optimal oxyresveratrol release efficacy. The DPPH radical scavenging capacity of free oxyresveratrol and fiber-encapsulated oxyresveratrol was assessed under accelerated conditions over 28 days. The fibers exhibited comparable properties to free oxyresveratrol, with fiber-encapsulated oxyresveratrol demonstrating superior DPPH radical scavenging activity. Additionally, the tyrosinase enzyme inhibitory activity of free oxyresveratrol and oxyresveratrol-loaded fibers was examined, with fiber-encapsulated oxyresveratrol exhibiting superior inhibition. The stability of free oxyresveratrol was compared to that of fiber-encapsulated oxyresveratrol under accelerated conditions, demonstrating greater stability in the fiber form. Notably, the electrospun CA-PCL nanofiber system stabilizes oxyresveratrol under accelerated storage conditions. The findings suggest that electrospun nanofibers were well-suited for drug delivery applications, providing enhanced stability and the controlled release of oxyresveratrol.