Topical Tinospora cordifolia BSA Hydrogel Formulation Attenuates UVB‐Induced Skin Inflammation in Golden Hamsters

ABSTRACT

Ultraviolet B (UVB) radiation induces oxidative stress and inflammation, leading to skin damage and related disorders. Tinospora cordifolia, a medicinal plant rich in bioactive phytoconstituents with reported antioxidant and anti‐inflammatory properties, was used in this study to develop bovine serum albumin (BSA)‐based hydrogels encapsulating aqueous and hydroalcoholic extracts of T. cordifolia. The protective effects of these hydrogels were evaluated against UVB‐induced skin inflammation in golden hamsters. The formulations were characterized using rheology, Fourier‐transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) to assess physicochemical properties. Rheological analysis confirmed the hydrogel's viscoelastic nature. FTIR spectra showed successful encapsulation of the extract, indicated by interactions of characteristic functional groups. SEM imaging revealed a porous network structure of the hydrogel, indicating successful formation of the BSA‐based matrix. Biological efficacy was assessed by measuring skin edema, antioxidant activity (SOD and catalase), and pro‐inflammatory cytokines (IL‐1β, IL‐6, TNF‐α). Histology showed reduced epidermal thickening, inflammation, and tissue damage in treated skin. The hydrogels significantly reduced UVB‐induced edema, increased antioxidant enzyme levels, and decreased cytokine levels. Western blot analysis showed downregulation of NF‐κB, COX‐2, and p53, and upregulation of Bcl‐2. These findings suggest that T. cordifolia‐loaded hydrogels offer a promising natural formulation for alleviating UVB‐induced skin inflammation and oxidative damage.

Tinospora cordifolia extract‐loaded BSA hydrogel protects against UVB‐induced skin inflammation.

1. Introduction

UVB radiation encounter our skin daily from the sun and have both beneficial and detrimental effect(s). UVB rays are used in phototherapy to treat various skin disorders like atopic dermatitis, cutaneous T‐cell lymphoma, and vitiligo, and are also used mostly for psoriasis patients [1, 2, 3]. A significant level of UVB radiation reaches the Earth's surface and exposure to it causes erythema, edema, scaling, changes in epidermal thickness, hyperplasia, immunosuppression, and skin aging [4, 5]. UVB rays are potential threat to humans, as they are absorbed by the epidermis and damage DNA by forming cyclo‐butane pyrimidine dimers and pyrimidine (6–4) photoproducts [6]. UVB rays induces the expression of matrix metalloproteinases—MMP‐1, MMP‐3, and MMP‐9—which degrades collagen causing deep wrinkles in the skin [4]. Therefore, effective strategies to prevent UV‐induced skin damage are essential.

Sunscreens of the various are widely used for protection against UV rays and are classified into organic and inorganic type. Organic sunscreens absorb UV light and release it as heat whereas, inorganic sunscreens act as sunblocks by reflecting and scattering UV rays [7]. In vitro studies revealed that oxybenzone present in organic sunscreens has antiandrogenic, pro‐ and antiestrogenic effects. Whereas, oxybenzone along with avobenzone (butyl methoxy dibenzoylmethane), homo salate, octyl dimethyl‐p‐aminobenzoic acid, and octinoxate showed increased cellular proliferation of MCF‐7 breast cancer cells [8]. One of the major concerns of organic sunscreens is coral reef bleaching [7]. Nanoparticles‐based sunscreen, particularly titanium dioxide and zinc oxide, are the most advanced formulations because they effectively block both UVA and UVB radiations [9]. These nano‐sized particles; however, may accumulate in the stratum corneum, thus hampering nanoparticle attenuation properties [10]. Hence, the exploration of plant‐derived bioactive compounds as promising candidates for photoprotection is needed.

Recently, the role of phytocompounds like flavonoids, alkaloids, terpenoids, polyphenols, tannins, anthraquinones, and so forth in preventing skin damage from ultraviolet radiation has received considerable attention. Medicinal plants rich in diverse bioactive phytoconstituents have gained interest as potential sources of natural photoprotective agents. Tinospora cordifolia (Guduchi), is a large, climbing shrub placed under the family Menispermaceae and is used in the Indian traditional medicine because of its various pharmacological activities like antioxidant, antidiabetic, immunomodulatory, and anticancer activity [11, 12]. Tinospora cordifolia preparations are used to treat leprosy, fever, asthma, anorexia, jaundice, gout, skin infections, diabetes, chronic diarrhea, dysentery, and so forth [13]. Tinospora cordifolia extract contains a diverse range of therapeutic compounds like alkaloids, carbohydrates, glycosides, saponins, tannins, flavonoids, steroids, and triterpenoids [14]. Tinospora cordifolia was selected for the present study considering its reported anti‐inflammatory and antioxidant potential, along with the presence of bioactive phytoconstituents which may contribute to the modulation of inflammatory pathways. Phytochemicals may play a critical role in the prevention and/or treatment of several diseases such as cancer and inflammation but these phytochemicals have also certain limitations like poor solubility and low bioavailability [15, 16].

To address these limitations, polymer‐based hydrogels have emerged as an efficient delivery systems that can improve the stability and bioavailability of phytocompounds. Polymer‐based hydrogels can enable sustained drug release for prolonged periods, thus maintaining therapeutic concentrations within a desired range. This property of hydrogel increases drug bioavailability and decreases frequent dosing therefore increasing drug efficiency [17]. Hydrogels are highly crosslinked polymeric networks, formed by hydrophilic macromolecules that can retain large amounts of water [17]. Over the decades, researchers have synthesized both natural and synthetic hydrogels. Naturally occurring polymers like chitosan, hyaluronic acid, alginate, fibrin, and gelatin have been used to fabricate hydrogels [18]. Polymer‐based hydrogels can absorb large amounts of liquids and biological fluids, resembling natural tissues and biological environments [17]. The various biomaterials are being used for hydrogel development, but the serum albumin has gained considerable attention due to its excellent biocompatibility and functional properties. Serum albumin is the most abundant plasma protein in the circulatory system and functions as a carrier because it has the capability to bind reversibly with an wide variety of ligands, such as metabolites, drugs, and organic compounds [19, 20]. Bovine serum albumin (BSA) is a biocompatible, cost‐effective and easily available protein widely used in hydrogel formation due to its ability to undergo crosslinking, forming a stable three‐dimensional network [21, 22]. The incorporation of T. cordifolia extracts into the BSA‐based hydrogel may provide a promising approach for improving their topical delivery and protective efficacy against UVB‐induced skin damage. Therefore, the present study aimed to investigate the photoprotective effects of aqueous and hydro‐alcoholic extracts of T. cordifolia‐loaded BSA hydrogel against UVB‐induced skin inflammation in golden hamsters. The Syrian golden hamsters were selected because they possess immunological similarities with humans, including functionally relevant cytokine‐mediated immune responses, making them suitable for inflammation‐related studies [23].

2. Materials and Methods

Tinospora cordifolia stems were collected in the month of July 2023 from the BHU campus, Varanasi, India. The plant was taxonomically identified by Prof. Nawal Kishore Dubey, Professor, Department of Botany, Banaras Hindu University and the voucher specimen has been deposited in the herbarium (Voucher specimen no. Menisperma: 2026/04). Bovine serum albumin (A2153‐50G) was purchased from Sigma‐Aldrich Chemicals. For all experimental work ultrapure water (12 MΩ‐cm), obtained from the Millipore water purification system was used. ECL kit (Cat. No. WBKLS0100) was purchased from Millipore Corporation, Billerica, USA, and other chemicals used in the study were of analytical grade.

2.1. Animal Model

Golden hamsters, Mesocricetus auratus (adult male weighing 125 ± 5 g) were used and kept in a room with constant temperature 25 ± 2°C and of 12 h light/dark cycle along with water and food, ad libitum. As per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India and Departmental Animal Ethics Committee, Banaras Hindu University (IAEC Approval reference no. BHU/DoZ/IAEC/2018‐19/036) experiments were performed.

2.2. Experimental Design

Animals were divided into the following five groups (n = 6):

1. Control,

2. UVB treated (1.5 J cm⁻² for 10 min daily for 5 days),

3. UVB treated + BSA hydrogel alone,

4. UVB treated + aqueous extract‐loaded BSA hydrogel, and

5. UVB treated + hydro‐alcoholic extract‐loaded BSA hydrogel.

The dorsal hair of hamsters were removed by careful mechanical shaving to minimize potential chemical‐induced alterations in skin inflammatory responses. The UVB exposure was given using a UV source (Philips, India Pvt. Ltd., Gurgaon, India) with a wavelength range of 280–315 nm in a specially designed UV chamber. The UVB irradiance intensity was measured using a UVB radiometer and maintained at approximately 2.5 mW/cm². The animals were exposed at an approximate distance of 17 cm for 10 min daily for 5 consecutive days to achieve a cumulative UVB dose of 1.5 J/cm² [24].

2.3. Synthesis of Plant Extract

Previously published method with slight modifications was used to prepare the T. cordifolia stem extract [25]. To remove physical impurities and to ensure optimal bioactivity, the stems were rinsed with deionized water and dried at room temperature. Approximately 50 g of dried stems were cut into small pieces and then crushed to powder in a mortar and pestle. The powder was then soaked overnight in a solvent at a ratio of 1:4 (w/v) using different solvents (LC/MS‐grade water, 50% water–50% ethanol, and ethanol) with continuous stirring, and heated at 85°C for 15 min next day. The supernatant was filtered through Whatman No. 1 filter paper. The solvent was then evaporated using a rotary evaporator, yielding a concentrated extract (Figure 1).

FIGURE 1.

Stepwise representation of T. cordifolia stem extract preparation (aqueous, hydroalcoholic, and ethanolic), illustrating key steps from initial grinding to the final concentrated extracts.

2.4. UHPLC‐HRMS Analysis of Tinospora cordifolia Stem Extract

Next the UHPLC‐HRMS analysis was conducted following method of Singh et al. (2024) with some minor modifications [26]. Data acquisition was performed using a TSOET (Thermo Scientific Orbitrap Eclipse Tribrid) mass spectrometer (MS) in application H‐ESI mode used for small molecule. To improve the detection of T. cordifolia aqueous extract, UHPLC system was employed with a full approach of MS/ddMS2 (OT HCD), utilizing a Dionex Ultimate 3000 RS Series. The mobile phase having 0.1% formic acid mixed with aqua (A), acetonitrile (B), or methanol (C). The spray voltage was at +3.5 kV for positive mode and −2.5 kV for negative mode. Data analysis was performed using Thermo Fisher Scientific SII's Xcalibur 4.1 software.

2.5. Preparation of Composite Hydrogel

The BSA hydrogel was synthesized following previously published method with some modifications [22]. Aqueous solution of BSA was prepared by mixing lyophilized powder of BSA in Millipore water (ultrapure water – 12 MΩ‐cm) at concentration of 100 mg/mL. For composite hydrogel, lyophilized powder of plant extract was mixed along with BSA in 1:2 ratio (plant extract: BSA powder) in ultrapure water. These solutions were then heated at 80⁰C for 30 min to obtain self‐assembled composite hydrogel (Figure 2). Formation of hydrogel was confirmed by vial inversion test. Aqueous extract and hydroalcoholic extract got dissolved evenly in ultrapure water thus forming even hydrogel whereas ethanolic extract was non‐dissolvable thus got precipitated at bottom of vial before hydrogel synthesis. Since the ethanol extract with the protein solution formed an uneven and physically unstable hydrogel, it was not used for biological studies. However, E‐BSA, along with Aq‐BSA and HA‐BSA hydrogels, was freeze‐dried at –80°C and stored at room temperature for preliminary characterization.

FIGURE 2.

Synthesized BSA hydrogels loaded with T. cordifolia extracts. Gelation was confirmed by the vial inversion test, indicating the self‐supporting nature of the hydrogels.

2.6. Characterization

Composite hydrogel was characterized for physiochemical properties. UV–Vis spectroscopy of plant extract, composite hydrogel, and BSA alone was employed to confirm the characteristic absorbance profiles of the individual components and their interaction in the composite hydrogel. FTIR and SEM analysis of lyophilized powder was done to obtain chemical composition and morphological properties of hydrogel, respectively. Rheological analysis of these composite hydrogels was performed to investigate mechanical behavior. The rheological measurements were performed using a parallel‐plate geometry with a 1 mm gap size under isothermal conditions at room temperature (25 ± 2°C). Amplitude sweep measurements were conducted at a constant angular frequency of 1 rad s⁻¹, while frequency sweep measurements were subsequently performed within the linear viscoelastic region (LVR) identified from the amplitude sweep test.

2.7. Skin Edema

A fixed area (5 mm in diameter) from the dorsal skin was collected with the help of a mold. The weight of the skin was measured and average weight of each group was compared with control versus all experimental groups. The results were expressed as skin weight (mg). Skin thickness was measured using a digital micrometer (aerospace screw gauge) of all experimental groups [27]. As a limitation, the retention or degradation of individual bioactive constituents following thermal processing (80°C–85°C) during hydrogel preparation could not be analytically assessed using HPLC or LC–MS.

2.8. Histological Studies

Hamsters were anesthetized at day 5 of UVB exposure. Histological studies of skin were assessed using H&E staining by standard method. The dorsal skin was fixed in neutral buffered formalin and sectioned into 7 µm thickness.

The histopathological changes were observed under a microscope (VX53 OLYMPUS, Tokyo, Japan).

2.9. Biochemical Parameters

2.9.1. Measurement of Antioxidant Enzymes

Measurement of SOD activity was performed with skin tissue along with the help of spectrophotometric method using nitrite formation by SOD radicals [28]. The skin piece then homogenized in ice‐cold PBS (150 mM). Homogenate (10%) was obtained after centrifuged at 12 000× g for 30 min. The supernatant was estimated for protein levels using BSA as the standard. SOD activity was measured by adding 0.05 mL of homogenate to 0.7 mL of a reaction mixture (comprising 50 mM phosphate buffer (pH 7.4), 20 mMol l‐methionine, 1% (v/v) Triton X‐100, 10 mM hydroxylamine hydrochloride, and 50 mMol ethylene diamine tetraacetic acid (EDTA) followed by a brief pre‐incubation at 37°C for 5 min). Then under red light, 50 µM of riboflavin was added to all samples. Only buffer was used as blank instead of sample and exposed to two 20‐W fluorescent lamps in an aluminum foil‐coated wooden chamber. After 10 min of exposure, 0.5 mL of freshly prepared Greiss reagent (1% sulphanilamide, 5% orthophosphoric acid, 0.1% N‐1‐naphthyl ethylenediamine dihydrochloride) was added. Absorbance of the color formed was measured at 543 nm and expressed as units (U)/mg of protein.

An indirect spectrophotometric method was used to measure catalase activity [29]. Briefly, 0.25 mL of PBS mixed with 0.2 mL of H₂O₂ (0.8 mM) and 0.05 mL of enzyme extract was added to it, after 1 min 0.5 mL of this solution was used in a tube and then 0.8 mL of acidic potassium dichromate (K₂Cr₂O₇) solution was added. The mixture was boiled for 10 min and then absorbance of color formed was measured at 570 nm. The CAT activity was expressed as the amount of H₂O₂ depleted/min per mg of protein. The calibration of standard curve was done with different concentrations of 0.2 mM H₂O₂ in PBS.

2.9.2. ELISA

The tissue lysate for ELISA was prepared in phosphate‐buffered saline (PBS) at a concentration of 100 mg/mL. Highly sensitive commercial ELISA kits (Abbkine) were used to measure the tissue TNFα, IL‐6, and IL‐1β as per the manufacturer's instructions.

2.9.3. Western Blotting

The western blotting using the previously published method of Goswami et al. (2014) was adopted for the expression of NF‐κB, COX‐2, p53, and Bcl‐2 protein lysate prepared in ice‐cold RIPA buffer was centrifuged at 12 000× g for 30 min, at 4°C [24]. The protein content was estimated (by the Bradford assay). An equal amount of protein samples was loaded per lane and separated on SDS‐PAGE gel. The proteins were transferred onto a PVDF membrane using electrotransfer (Trans‐blot Turbo, Bio‐RAD) and then blocked for 1 h in blocking buffer containing 5% skimmed milk, 0.1% Tween‐20, and TBS. Membrane was incubated overnight at 4°C with primary antibodies (dilution 1:1000). The membrane after incubation, washed thrice with TBST for 15–20 min. The secondary antibody (conjugated with horse‐radish peroxidase‐HRP) was used at a specified dilution (1:1500). With the secondary antibody it was incubated 2 h at 37°C. Loading control was β‐actin. The immunoreactive signal was detected by using Imobilon Western chemiluminescent (Millipore) with the ChemiDoc system (Amersham Imager 680). The density of specific bands was quantified using the Alpha Imager 2200 software (Alpha Innotech). The details of the antibodies used are NF‐κB (cell signaling technology), β‐actin (cell signaling technology), Bcl‐2 (Santa Cruz Biotechnology, USA), COX‐2 (cell signaling technology), p53 (Santa Cruz Biotechnology, USA). The target proteins and β‐actin loading controls were analyzed on separate membranes. Values were expressed as the relative density of the target protein to β‐actin.

2.10. Statistical Analysis

Data were analyzed using GraphPad Prism 8. For Western blot analysis, densitometric values obtained from independent biological replicates (n = 3 animals/group) were used for statistical analysis. One‐way ANOVA was performed followed by Tukey's post hoc test. Results were expressed as mean ± standard deviation (SD), and p < 0.05 was considered statistically significant.

3. Results

3.1. Tinospora cordifolia Stem Extract: An UHPLC‐HRMS Analysis

A total of 2393 and 2397 molecular features were detected in aqueous and hydro‐alcoholic extracts of T. cordifolia stem, respectively. These features were putatively annotated using mzCloud, ChemSpider, and massList databases and were classified into major phytochemical categories including organic acids, phenolics, flavonoids, terpenes, fatty acids, coumarins, and sugars. Some major putatively annotated phytoconstituents detected in the HRMS analysis, along with their retention time, molecular mass, and reported anti‐inflammatory activity, are provided in Supplementary Table S1. The whole chromatogram metabolites present in T. cordifolia stem extract with relative abundance has been presented in (Supplementary Figure S1).

3.2. UV–Visible Spectroscopy

The UV–vis spectrum of the plant extract displayed a sharp absorbance peak at approximately 270 nm, indicating the presence of UV‐absorbing phytochemicals.

The BSA showed a characteristic absorbance at approximately 280 nm, which corresponds to the aromatic amino acid residues present in the protein.

In the case of the composite hydrogel, a broad peak was observed around 275–285 nm, with slight shifts in wavelength and absorbance intensity compared to the individual components (Figure 3).

FIGURE 3.

UV–Vis absorption spectra of Tinospora cordifolia plant extract, BSA, and plant extract‐loaded composite BSA hydrogel.

3.3. Rheological Properties

Amplitude and frequency sweep tests were conducted to analyze the viscoelastic properties of composite hydrogels. An amplitude sweep at a constant frequency of 1 rad s^{− 1} was performed to determine the deformation behavior, and linear viscoelastic range (LVR) in the non‐destructive deformation region [30, 31]. The LVR limit, expressed as strain percentage, was identified based on the storage modulus (G′), as G′ and the loss modulus (G′′) remain independent of applied shear strain within this range. The abrupt decrease in G′ indicated the LVR limit (Figure 4A).

FIGURE 4.

Rheological analysis of BSA‐based hydrogels. (A) Amplitude sweep and (B) frequency sweep of Aq‐BSA, HA‐BSA, and BSA‐alone hydrogels, demonstrating their viscoelastic properties. Filled and unfilled symbols represent G' and G'', respectively.

Compared to BSA hydrogel alone, the composite hydrogel exhibited higher G′ and G′′ values, indicating an enhanced energy storage capacity. As strain increased, G′ gradually decreased, reflecting the hydrogel's smooth texture and its potential applicability as an ointment. The incorporation of plant extract extended the plateau region, suggesting a more robust and stable three‐dimensional crosslinked hydrogel network. Additionally, all hydrogels displayed elastic behavior (G′ > G′′) at low shear rates. With increasing strain, G′ gradually decreased, followed by a crossover point (G′ = G′′) and transition toward viscous behavior (G′′ > G′) at higher deformation.

To further investigate the structure and viscoelastic behavior of the hydrogels, frequency sweep tests were performed within the LVR range (Figure 4B). At a constant strain of 1%, G′ and G′′ were plotted against frequency. Across the tested frequency range, all hydrogels exhibited G′ > G′′, confirming their predominantly elastic nature and structural stability over extended storage. Moreover, both G′ and G′′ remained nearly constant and independent of frequency within the range of 0.1–10 rad s^{− 1}, indicating a well‐maintained network.

3.4. FTIR Analysis

FTIR spectroscopy was employed to analyze the chemical interactions and functional groups present within the hydrogels, providing insights into their molecular structure and cross‐linking mechanisms. As depicted in Figure 5, a broad and intense peak near 3500 cm^{− 1} was observed across all samples, including BSA and the composite hydrogels. This peak corresponds to the stretching vibrations of hydroxyl (─OH) groups, indicating the presence of hydrogen bonding interactions. The amide I (1650 cm^{− 1}) and amide II (1520 cm^{− 1}) bands observed in the pure BSA hydrogel confirm that the protein's intrinsic backbone remains largely intact during gelation. These peaks, attributed to C═O stretching and N–H bending, respectively, suggest that BSA retains its secondary structure. However, the composite hydrogel exhibited a reduction in the intensity of these amide bands, along with a decrease in C–O stretching (1650 cm^{− 1}) and N–H bending. Additionally, a peak around 1107 cm^{− 1} was observed in the composite hydrogel, which was not prominent in pure BSA hydrogel. The FTIR spectra of E‐BSA hydrogel were presented in Supplementary File S2.

FIGURE 5.

FTIR spectra of A‐BSA (aqueous BSA), HA‐BSA (hydroalcoholic BSA), E‐BSA (ethanolic BSA), and BSA‐alone hydrogels, highlighting characteristic functional group vibrations.

3.5. Morphology

The SEM analysis of lyophilized hydrogel samples revealed a highly porous morphology across all composition, and the voids with several micron‐sized as depicted in Figure 6. BSA hydrogel exhibited a smooth, porous morphology, while the plant extract was seen to express over amyloid fibrils. A reduced degree of porosity was observed in the HA‐BSA and Aq‐BSA hydrogels, with the lowest porosity observed in the E‐BSA extract. During the synthesis process, the ethanolic plant extract did not dissolve in water, resulting in an uneven gel. Consequently, the HA‐BSA, Aq‐BSA, and E‐BSA hydrogels displayed a well‐organized porous morphology, in contrast to the less structured appearance of the E‐BSA hydrogel. Additionally, by adjusting the concentration of the extract, the porosity of the hydrogel could be tuned to meet specific requirements. The SEM images of E‐BSA were presented in Supplementary File S3.

FIGURE 6.

Scanning electron microscopy (SEM) images of Aq‐BSA, HA‐BSA, and BSA‐alone hydrogels, illustrating their surface morphology.

3.6. Macroscopic/Morphological Evaluation

The macroscopic examination of hamster skin following UVB exposure revealed marked photodamage, including erythema, redness, edema, and scaling, being most prominent on Day 3 and Day 5, indicating severe skin inflammation. In contrast, treatment groups receiving BSA hydrogel alone, as well as BSA hydrogel loaded with aqueous or ethanolic extracts of T. cordifolia, exhibited a visible reduction in these signs of damage. The ethanolic extract‐treated group showed comparatively better improvement, with reduced redness and a smoother skin surface. Representative images of each experimental group are presented in Figure 7, illustrating these morphological changes.

FIGURE 7.

Macroscopic evaluation of skin in the Control, BSA, Aq‐BSA, and HA‐BSA treatment groups on Days 1, 3, and 5, showing progressive visible changes. Green arrows indicate erythema, red arrows indicate edema, and blue arrows indicate scaling.

3.7. Skin Edema

Skin thickness and weight were significantly increased in the UVB‐treated group (p < 0.001) compared to the control. However, treatment with BSA (p < 0.01), aqueous extract‐loaded BSA hydrogel (p < 0.01), and hydroalcoholic extract‐loaded BSA hydrogel (p < 0.05) significantly reduced these parameters (Figure 8).

FIGURE 8.

Effect of UVB and T. cordifolia stem extracts loaded BSA hydrogel on (A) skin weight and (B) skin thickness, indicators of edema, in control, UVB, BSA, Aq‐BSA, and HA‐BSA, treatment groups measured at the end of the experiment (Day 5). Statistical significance is indicated as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Data are presented as mean ± standard deviation (one‐way ANOVA, post hoc test).

3.8. Histology

The UVB experimental group showed a significant increase in epidermal thickness (p < 0.001) and edema. In contrast, BSA hydrogel treatment led to a mild reduction in both epidermal thickness (p < 0.001) and edema. Aqueous extract‐loaded BSA hydrogel also resulted in a reduction in epidermal thickness (p <0.01) and edema, with hydroalcoholic extract‐loaded BSA hydrogel showing a more pronounced effect (p < 0.05) (Figure 9).

FIGURE 9.

Effect of UVB and T. cordifolia stem extract‐loaded BSA hydrogel on the histoarchitecture of skin in (A) Control, (B) UVB, (C) BSA, (D) Aq‐BSA, and (E) HA‐BSA treatment groups. (F) Measurement of epidermal thickness. Yellow arrow indicate edema, red arrow indicates hair follicle, ‘Ep’ indicates epidermis, ‘D’ indicates dermis, ‘Ad’ indicates adipose, ‘M’ indicates muscle, and ‘HEp’ indicates hyperplasia of epidermis. Statistical significance is indicated as, p < 0.05 (*), p < 0.01 (**), and p < 0.001(***). Data are presented as mean ± standard deviation (one‐way ANOVA, post hoc test).

3.9. Antioxidant Status

A significant decrease in the activities of SOD and CAT enzymes was observed in UVB‐treated groups (p < 0.001). However, the BSA‐treated group showed a slight increase in enzyme activity (p < 0.001), while the aqueous extract‐loaded BSA hydrogel‐treated groups (Aq‐BSA) exhibited a more significant increase (p < 0.01). HA‐BSA showed the most restored SOD and catalase activity (p < 0.05) compared to the control groups (Figure 10).

FIGURE 10.

Effect of UVB and T. cordifolia stem extract‐loaded BSA hydrogel on antioxidant enzyme activity in control, UVB, BSA, Aq‐BSA, and HA‐BSA treatment groups. (A) Superoxide dismutase (SOD) activity and (B) catalase activity. Statistical significance is indicated as, p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Data are presented as mean ± standard deviation (one‐way ANOVA, post hoc test).

3.10. ELISA

UVB exposure significantly elevated pro‐inflammatory cytokine levels (IL‐1β, IL‐6, and TNF‐α), compared to the control group (p < 0.001). The BSA hydrogel significantly reduced these levels (p < 0.001), with Aq‐BSA showing greater reduction (p < 0.01), while HA‐BSA exhibited the most significant reduction in the cytokines (p < 0.05) (Figure 11).

FIGURE 11.

Levels of tissue pro‐inflammatory cytokines (A) IL‐1β, (B) IL‐6, and (C) TNF‐α, in control, UVB, BSA, Aq‐BSA, and HA‐BSA, treatment groups. Statistical significance is indicated as, p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Data are presented as mean ± standard deviation (one‐way ANOVA, post hoc test).

3.11. Western Blotting

UVB exposure significantly upregulated pro‐inflammatory markers (NF‐κB, COX‐2) and p53 (p < 0.001), while significantly downregulating the anti‐apoptotic marker Bcl‐2 (p < 0.001). BSA treatment significantly reduced NF‐κB, COX‐2, and p53 (p < 0.001) and significantly upregulated Bcl‐2 (p < 0.001). The Aq‐BSA hydrogel further significantly suppressed NF‐κB, COX‐2, and p53 and significantly restored Bcl‐2 levels (p < 0.01). The HA‐BSA hydrogel demonstrated the most significant reduction in NF‐κB, COX‐2, and p53 (p < 0.05) and significantly upregulated Bcl‐2 (p < 0.05) compared to the control (Figure 12).

FIGURE 12.

Western blot analysis of key inflammatory and apoptotic markers in control, UVB, BSA, Aq‐BSA, and HA‐BSA, treatment groups. (A) Representative immunoblot images. (B–E) Densitometric analysis of Bcl‐2, NF‐κB, COX‐2, and p53 expression. Densitometric analysis was performed from independent biological replicates (n = 3) and normalized against β‐actin. Statistical significance is indicated as, p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Data are presented as mean ± standard deviation (one‐way ANOVA, post hoc test).

4. Discussion

The absorbance at 270 nm in the plant extract is due to conjugated aromatic compounds, commonly found in polyphenols and flavonoids. The 280 nm peak in BSA arises from the presence of tryptophan and tyrosine residues. The slight shift in the composite hydrogel indicates possible physical or chemical interaction between the protein and phytochemicals, reflecting successful integration of the extract within the BSA matrix.

The rheological properties of hydrogels are crucial in determining their mechanical stability and potential applications. The amplitude sweep analysis revealed that composite hydrogels possess a stronger network than BSA hydrogel alone, as evidenced by their higher G′ and G′′ values. This suggests that the addition of plant extract enhances the gel structure, increasing its resistance to deformation and improving its ability to store elastic energy. The gradual decrease in G′ with increasing strain indicates the deformation response of the hydrogel network under applied stress, suggesting its flexible nature for topical applications [32]. The extended plateau region in the composite hydrogel indicates a more stable and interconnected crosslinked network, which could contribute to better mechanical integrity and prolonged usability. Additionally, the transition from elastic (G′ > G′′) to viscous (G′′ > G′) behavior with increasing shear strain demonstrates the hydrogel's ability to withstand stress while maintaining its functional properties [33].

The frequency sweep analysis supports these findings, as all hydrogels maintained G′ > G′′ over the tested range, confirming their predominantly elastic nature [34, 35]. The frequency independence of G′ and G′′ suggests that the hydrogel network remains intact under varying external forces, ensuring long‐term stability [36]. This stability is particularly beneficial for biomedical applications, where consistent mechanical properties are essential for sustained performance [37]. The combination of enhanced viscoelasticity, increased network strength, and shear‐dependent viscoelastic behavior makes the composite hydrogel a promising candidate for topical formulations.

The spectral changes observed in the composite hydrogel indicate molecular interactions between BSA and the additional hydrogel components. The reduction in amide band intensity suggests that these components alter BSA's native structure to some extent, potentially disrupting its secondary structure and leading to modified hydrogen bonding interactions [38]. The presence of a characteristic peak around 1107 cm^{− 1}, attributed to C−O−C stretching vibrations, indicates chemical cross‐linking within the polymeric network, which enhances the hydrogel's mechanical stability [39, 40]. In contrast, the retention of the amide bands from BSA suggests that non‐covalent interactions, such as hydrogen bonding, electrostatic forces, and van der Waals interactions, contribute to hydrogel formation and stabilization [38]. These interactions help to maintain the protein's structure while reinforcing the hydrogel network, ensuring an optimal balance between flexibility and robustness. FTIR analysis further reveals that the composite hydrogel's stability is governed by a combination of covalent cross‐linking within the polymer matrix and weaker intermolecular forces between BSA and the additional components. These molecular interactions not only influence the hydrogel's structural integrity but also regulate its functional properties, including hydration capacity, mechanical strength, and biodegradability, making it a promising candidate for topical biomedical applications [39, 41].

The SEM images reveal notable differences in the porosity and morphology among various hydrogel compositions. The BSA hydrogel alone exhibits a highly porous, honeycomb‐like structure with irregular shapes and interconnected macropores, characteristic of protein‐based gelation [42]. However, the incorporation of plant extracts appears to influence the protein structure, leading to enhanced network formation. This is evidenced by increased roughness and density in the hydrogel network, suggesting that bioactive compounds within the plant extracts facilitate additional cross‐linking, possibly through intermolecular interactions with protein fibrils [43]. Furthermore, the observed variations in pore sizes across different formulations underscore the tunability of the hydrogel's physical properties, which is crucial for applications requiring controlled permeability and sustained release of bioactive compounds.

The decrease in porosity observed in HA‐BSA and Aq‐BSA hydrogels compared to the BSA hydrogel could be attributed to interactions between the plant extracts and the BSA matrix, leading to a denser network and reduced void formation. Studies have shown that incorporating plant extracts into protein‐based hydrogels can significantly alter their structural properties. For instance, the addition of phenolic compounds from plant extracts can induce cross‐linking with proteins through hydrogen bonding and covalent interactions, resulting in a more compact and less porous network [44].

The E‐BSA hydrogel, which exhibited the lowest porosity, might be affected by the incompatibility of the ethanolic extract with the aqueous medium, leading to poor dissolution and distribution of the extract during gelation. This inconsistency can result in a less organized hydrogel structure. Research indicates that the choice of solvent for plant extracts can significantly impact the properties of the resulting hydrogel. For example, the incorporation of ethanolic extracts into hydrogels has been shown to influence the gelation process and the structural integrity of the hydrogel network [45].

The observation that the porosity of the hydrogel can be tuned by adjusting the concentration of the plant extract is significant for tailoring the hydrogel's properties for specific applications, such as controlled drug release or tissue engineering. Porosity is a key factor that influences the mechanical properties and drug‐loading capacity of hydrogels, and this feature can be optimized by adjusting the extract concentration during synthesis [46]. In summary, the interactions between plant extracts and the BSA matrix, as well as the compatibility of the extract solvent with the hydrogel system, play crucial roles in determining the porosity and overall structure of the resulting hydrogels.

Moreover, these findings suggest that HA‐BSA and Aq‐BSA, hydrogels could be more advantageous for applications requiring well‐structured, organized pores, compared to the less organized E‐BSA hydrogel.

The skin is the organ most susceptible to damage by UV light due to its direct exposure [47]. UVB radiation is known to cause significant damage to the skin, including erythema, redness, and edema and scaling. It also causes oxidative stress, and DNA damage, which can lead to premature aging and increased skin cancer risk [6]. Protective formulations with antioxidant and anti‐inflammatory properties are being actively investigated to counteract the harmful effects of oxidative stress and inflammation caused by environmental factors such as UV radiation [48].

The observed reduction in redness and swelling in the T. cordifolia extract‐loaded BSA hydrogels suggests a potential anti‐inflammatory effect, with HA‐BSA treatment group showing greater efficacy. These macroscopic observations align with the skin edema measurements, histological analysis, and Western blot findings, further supporting the role of T. cordifolia extract loaded‐BSA hydrogels in mitigating UVB‐induced inflammation. UVB exposure caused significant skin edema, as evidenced by increased skin thickness and weight. Treatment with BSA and both aqueous and hydroalcoholic extract‐loaded BSA hydrogels effectively reduced these parameters, suggesting their potential to alleviate UVB‐induced skin inflammation.

Histological observation suggest that UVB treatment significantly increased epidermal thickness and edema, consistent with well‐established findings on UVB‐induced skin inflammation [4]. Treatment with BSA hydrogel led to a mild reduction in both parameters, suggesting some anti‐inflammatory effects. The aqueous extract‐loaded BSA hydrogel further reduced epidermal thickness and edema, indicating that the incorporation of T. cordifolia extract enhances the hydrogel's protective potential. Notably, the hydroalcoholic extract‐loaded BSA hydrogel showed the most pronounced effect, likely due to the enhanced bioactivity of the hydroalcoholic extract, which has been shown to possess stronger anti‐inflammatory and antioxidant properties [49]. These findings support the use of T. cordifolia‐loaded hydrogels in alleviating UVB‐induced skin damage. UVB also increases reactive oxygen species (ROS) and decreases antioxidant enzymes, hence leading to oxidative stress in the epidermis [50]. Similarly, extracts of Dipteracanthus prostratus have been reported to exhibit antioxidant activity and sun protection potential, suggesting their potential role in reducing UV‐induced oxidative damage [51]. UVB exposure significantly decreased the activities of SOD and CAT enzymes, highlighting the increased oxidative stress. Treatment with BSA hydrogel resulted in a slight increase in enzyme activity, suggesting some antioxidant potential. The aqueous extract‐loaded BSA hydrogel (Aq‐BSA) demonstrated a more significant improvement in SOD and CAT activity, supporting the antioxidant properties of T. cordifolia extract. Notably, the hydroalcoholic extract‐loaded BSA hydrogel (HA‐BSA) showed the greatest restoration of enzyme activity, indicating enhanced antioxidant effects, likely due to the synergistic action of the hydroalcoholic extract. Natural products have also been reported to exert antiphotoaging effects by regulating oxidative stress, inflammation, and extracellular matrix degradation [52].

UVB induces expression of pro‐inflammatory cytokines, such as interleukin (IL‐1, IL‐6) and tumor necrosis factor‐α (TNF‐α) via the activation of NF‐κB [52]. Treatment with BSA hydrogel significantly reduced these cytokine levels, suggesting its potential to modulate inflammation. The aqueous extract‐loaded BSA hydrogel (Aq‐BSA) showed a greater reduction, aligning with findings from earlier research indicating that plant extracts like T. cordifolia can enhance anti‐inflammatory responses. The hydroalcoholic extract‐loaded BSA hydrogel (HA‐BSA) exhibited the most pronounced decrease in cytokine levels, supporting reports that hydroalcoholic extracts tend to have stronger anti‐inflammatory effects compared to aqueous formulations. UVB exposure significantly upregulated pro‐inflammatory markers (NF‐κB, COX‐2) and p53 while downregulating the antiapoptotic marker Bcl‐2, in line with previous reports linking UVB‐induced inflammation and apoptosis [53, 54, 55, 56]. The BSA hydrogel treatment led to moderate reductions in NF‐κB, COX‐2, and p53, along with a slight increase in Bcl‐2. These effects might be attributed more to the protective role of BSA, potentially providing a physical barrier that mitigates further skin damage, rather than direct anti‐inflammatory modulation.

In contrast, the aqueous extract‐loaded BSA hydrogel (Aq‐BSA) further suppressed NF‐κB, COX‐2, and p53, while restoring Bcl‐2 levels, suggesting that T. cordifolia extract enhances the hydrogel's anti‐inflammatory and antiapoptotic properties. The hydroalcoholic extract‐loaded BSA hydrogel (HA‐BSA) exhibited the most significant reductions in inflammatory markers and a notable increase in Bcl‐2, further corroborating findings that hydroalcoholic extracts tend to exert stronger bioactivity due to better solubility and stability of active compounds. The protective effect of phytochemicals present in T. cordifolia against UVB‐induced skin inflammation may be due to their antioxidant and modulatory activities. Tinospora cordifolia contains various bioactive phytoconstituents, and antioxidant‐rich plant extracts have been reported to reduce oxidative stress by scavenging free radicals [14]. Therefore, the antioxidant potential of the formulation may contribute to protection against UVB‐induced oxidative damage. Reduction of oxidative stress and ROS accumulation by antioxidant phytoconstituents may help regulate oxidative stress‐associated inflammatory pathways, including NF‐κB, and may affect downstream inflammatory mediators such as COX‐2 and cytokines [57]. Excessive oxidative stress can cause cellular damage by affecting mitochondrial function and cellular balance. Oxidative stress‐mediated damage may also affect apoptosis‐related pathways by altering apoptotic regulators [58]. Therefore, the observed modulation of Bcl‐2 and p53 expression in the present study suggests that the hydrogel formulation may help regulate UVB‐induced apoptotic responses. An un‐irradiated hydrogel‐only control group was not included in this study, which is a limitation. Future studies may incorporate an unirradiated hydrogel control group to further confirm the safety of the formulation and assess any baseline skin response due to the hydrogel or plant extract. The T. cordifolia extract‐loaded BSA hydrogel demonstrated significant UVB protective and anti‐inflammatory effects, as evidenced by reduced skin edema, enhanced antioxidant enzyme activity, and suppressed pro‐inflammatory cytokine levels. Western blot analysis further confirmed the modulation of NF‐κB and COX‐2 pathways, supporting its role in mitigating UVB‐induced inflammation. These findings align with previous studies on herbal‐based photoprotective agents and suggest that T. cordifolia‐based formulations could serve as effective candidates for managing UVB‐induced skin damage. This study did not include an unirradiated composite hydrogel control group, limiting the baseline skin compatibility and contact toxicity assessment. Further investigations, including long‐term efficacy assessments and mechanistic studies, are needed to fully understand its therapeutic potential.

5. Conclusion

Our study highlights the potential of T. cordifolia extract‐loaded BSA hydrogels as effective formulations for mitigating UVB‐induced skin damage. The results demonstrate that both aqueous and hydroalcoholic extract‐loaded BSA hydrogels not only reduced UVB‐induced oxidative stress and inflammation, as evidenced by improved antioxidant enzyme activities, modulation of inflammatory markers, and restoration of skin structural alterations., suggesting their dual protective and therapeutic effects. The protective effect of the hydrogel formulation may be due to its antioxidant potential, which helps reduce oxidative stress and regulate UVB‐induced inflammatory and apoptotic responses. While BSA alone provides a protective barrier to reduce skin damage, the incorporation of T. cordifolia extracts enhances the anti‐inflammatory, antioxidant, and antiapoptotic properties of the hydrogel. Further studies are required to explore the detailed mechanism of action, optimize the formulation, and evaluate long‐term safety. These findings support the use of T. cordifolia‐based formulations as promising candidates for UVB protection and skin inflammation management, together contributing toward the development of novel, plant‐based dermatological applications.

Author Contributions

Nitesh Kumar Mishra: writing – original draft, conceptualization, validation, methodology, formal analysis, data curation. Shikha Tripathi: validation, methodology, data curation, writing. Soumya Ranjan Mohanty: methodology, data curation, writing. Khushi Gusain: methodology, data curation, writing. Avanish Singh Parmar: writing – review and editing, Sanjeev Kumar Yadav: supervision, resources, funding acquisition, writing – review and editing. Chandana Haldar: writing – review and editing.

Funding

The authors acknowledge the Institute of Eminence (IoE) Research Grant for Faculty and the IoE Bridge Grant awarded to Dr. Sanjeev Kumar Yadav. Additionally, support from the Indian Council of Medical Research (ICMR) through the Extramural Grant (P‐14/0797) to Dr. Sanjeev Kumar Yadav is duly acknowledged. Mr. Nitesh Kumar Mishra gratefully acknowledges the University Grants Commission (UGC) for the award of a NET‐JRF fellowship.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71599‐sup‐0001‐SuppMat.tif.

Supporting File 2: cbdv71599‐sup‐0002‐SuppMat.tif.

Supporting File 3: cbdv71599‐sup‐0003‐SuppMat.tif.

Supporting File 4: cbdv71599‐sup‐0004‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.