Biodegradable gelatin-based antioxidant film composition

Abstract

A biodegradable gelatin-based antioxidant film composition comprising: a) Gelatin as polymer matrix, b) a β-AKBA analogue incorporated into the matrix to improve the antioxidant properties, c) a plasticizer to improve the flexibility of the film and d) optional excipients to modulate the optical, mechanical and hydrophobic properties, wherein the β-AKBA analogue is dispersed in the gelatin matrix to achieve improved antioxidant activity and modified physical properties.

Description

[0001] The present invention relates to a biodegradable biopolymer film composition developed for sustainable packaging and biomedical applications. In particular, the invention comprises an antioxidant gelatin-based film to which a β-AKBA analogue is added, which improves the functional properties of the film. The composition comprises a gelatin matrix, a β-AKBA analogue for enhanced antioxidant activity, and optional plasticizers or crosslinking agents to improve mechanical stability and hydrophobicity.

[0002] The global plastic pollution crisis has reached alarming proportions: billions of tons of plastic waste are accumulating in landfills and natural ecosystems. Conventional petroleum-based plastics are non-biodegradable, last for centuries, and break down into microplastics that enter the food chain, posing environmental and health risks. The urgent need for sustainable alternatives has spurred research into biodegradable biopolymers, which offer promising solutions due to their natural degradability and environmentally friendly properties. However, many biopolymers, including gelatin, require functional enhancements to match the performance of synthetic plastics in packaging and biomedical applications.

[0003] Gelatin, a widely studied biodegradable biopolymer, possesses excellent film-forming ability, biocompatibility, and biodegradability, making it a good candidate for sustainable materials. However, gelatin-based films often exhibit limitations in mechanical strength, water resistance, and functional properties, limiting their practical applications. To overcome these issues, bioactive compounds can be incorporated to enhance film properties and provide additional functionalities such as antioxidant, antimicrobial, and therapeutic effects. One such compound, acetyl-11-keto-β-boswellic acid (β-AKBA), derived from Boswellia serrata resin, has demonstrated significant pharmacological benefits, including anti-inflammatory, cytotoxic, and anticancer properties.Despite their potential, the integration of β-AKBA analogues into biodegradable polymer matrices is still underexplored, requiring further investigations into their compatibility and effectiveness in improving film performance.

[0004] By incorporating β-AKBA analogues into gelatin films, this invention aims to develop a multifunctional material that not only addresses environmental concerns but also provides therapeutic benefits. The antioxidant properties of β-AKBA can protect packaged food products from oxidative degradation, thus extending shelf life, while its bioactive nature can support biomedical applications such as wound dressings or drug delivery. This research aims to overcome the limitations of conventional gelatin films by improving their mechanical, chemical, and surface properties to provide a sustainable and functional alternative to conventional plastics.

[0005] An object of the present disclosure is to provide an environmentally friendly, biodegradable film with improved functional properties for sustainable applications.

[0006] Another object of the present disclosure is to improve the tensile strength and flexibility of the film while maintaining structural integrity.

[0007] Another aim of the present disclosure is to incorporate β-AKBA analogues into the film matrix to significantly increase the antioxidant activity.

[0008] Another object of the present disclosure is to modify the transparency and haze of the film and to optimize light scattering for specific applications.

[0009] Another objective of the present disclosure is to improve water resistance, reduce moisture absorption, and extend the shelf life of the film.

[0010] Another objective of the present disclosure is to ensure the stability and bioactivity of β-AKBA analogues within the film matrix.

[0011] Another object of the present disclosure is to enable use in the biomedical, pharmaceutical and food packaging industries.

[0012] The present invention relates to a biodegradable gelatin-based film composition containing β-AKBA analogues to improve its functional and structural properties.

[0013] Another embodiment of the present invention is the integration of β-AKBA analogues into a gelatin matrix to improve the mechanical strength, hydrophobicity and optical properties of the film.

[0014] Another embodiment of the present invention is the synthesis of β-AKBA analogues through a two-step chemical modification process to ensure their stability and compatibility within the film matrix.

[0015] Another embodiment of the present invention is the preparation of the film using a controlled drying process to achieve a uniform structure and optimal dispersion of the active compounds.

[0016] Another embodiment of the present invention is the use of a plasticizer to increase the flexibility of the gelatin-based film without compromising its structural integrity.

[0017] Another embodiment of the present invention is the development of a scalable and reproducible process for the production of β-AKBA-loaded films suitable for industrial applications.

[0018] Another embodiment of the present invention is the application of the developed film composition in biomedical, pharmaceutical and food packaging fields due to its improved functional properties.

[0019] Another embodiment of the present invention is the ability of the β-AKBA analogues to interact with the gelatin matrix, resulting in improved material properties compared to conventional gelatin films.

[0020] The present invention relates to the development of a novel biodegradable film combination containing β-AKBA analogues to improve their functional properties. The invention involves a two-step synthesis process in which β-AKBA, a natural compound, is chemically modified to form its analogues, which are then incorporated into a gelatin-based film matrix. This preparation method ensures uniform dispersion of the active ingredient, resulting in a structurally stable film. This innovative approach not only utilizes environmentally friendly materials but also optimizes the physicochemical properties of the film for various applications.

[0021] Furthermore, the invention investigates the effects of incorporating β-AKBA analogues on key properties such as mechanical strength, surface hydrophobicity, and optical properties. The modified films exhibit significant advantages over conventional gelatin films and are therefore suitable for various applications in the biomedical, pharmaceutical, and food packaging industries. The invention also presents an efficient and scalable manufacturing process that ensures consistency and reproducibility for industrial production. EXAMPLE 1; COMPOSITION

[0022] A biodegradable, antioxidant gelatin-based film composition comprising: a) Gelatin as polymer matrix, b) a β-AKBA analogue incorporated into the matrix to improve the antioxidant properties, c) a plasticizer to improve the flexibility of the film and d) optional excipients for modulating the optical, mechanical and hydrophobic properties, wherein the β-AKBA analogue is dispersed in the gelatin matrix to achieve improved antioxidant activity and modified physical properties.

[0023] For the synthesis of the 1H-1,2,3-triazole analogue of β-AKBA (4), the natural product β-AKBA (1), isolated from the gum resin of Boswellia sacra, was used as the starting material in a first step. It has a free acid group at position C-24 and was reacted with propargylamine in the presence of HATU coupling agent to obtain the corresponding compound 2 in high yield (90%). In the second step, the desired product, 1H-1,2,3-triazole analogue of β-AKBA (4), was prepared in high yield (92%) by reacting compound 2 with aromatic 4-trifluoromethyl azide (3a) in the presence of copper iodide (CuI), triethylamine as the base, and acetonitrile as the solvent at room temperature for 3 hours. The detailed synthesis procedure is as follows: EXAMPLE 2: Synthesis of (Prop-2-yn-1-ylcarbamoyl) 3a-Acetyloxy-11-oxo-urs-12-en-24-oate (2)

[0024] A representative protocol for the HATU-mediated coupling agent reaction of the carboxylic acid of β-AKBA (1) and propargylamine to provide the desired product (2) is as follows. β-AKBA (1) (1.0 equiv.) and [1-bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (1.2 equiv.) were added to propargylamine (1.1 equiv.) in anhydrous DMF in the presence of N,N-diisopropylethylamine (DIPEA) (2.5 equiv.), and the reaction mixture was stirred at room temperature for 10–12 hours until the reaction was complete (monitored by TLC analysis). Upon completion of the reaction, the product was extracted with EtOAc (3 × 30 mL).The combined organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure on a rotary evaporator to obtain the crude product, which was purified by column chromatography on silica gel using n-hexane / ethyl acetate (85:15 v / v) as eluent to afford the desired pure compound 2 (90%). Light green solid (light green crystals from MeOH solvent); Yield = 90%; mp 196-198 °C, 1H NMR (600 MHz, chloroform-d) δ 5.68 (t, J = 4.5 Hz, 1H), 5.51 (s, 1H), 5.27 (s, 1H), 4.03 - 3. 95 (m, 2H), 2.51 (d, J = 13.2 Hz, 1H), 2.37 (s, 1H), 2.25 (t, J= 14.0 Hz, 1H), 2.18 (s, 1H), 2.04 (s, 4H), 1. 85 (dd, J = 12.6, 8.1 Hz, 1H), 1.75 (dd, J = 22.8, 12.7 Hz, 2H), 1.68 (dd, J = 17.6, 7.6 Hz, 1H), 1.58 (dd, J = 15. 2, 2.5 Hz, 1H), 1.51 - 1.38 (m, 5H), 1.36 (d, J = 10.9 Hz, 2H), 1.30 (s, 3H), 1.26 (d, J = 11.3 Hz, 1H), 1. 21 - 1.15 (m, 6H), 1.11 (s, 3H), 1.08 (s, 3H), 0.98 (d, J = 11.3 Hz, 1H), 0.90 (s, 3H), 0.78 (s, 3H), 0.76 (d, J = 6.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 199.0, 174.8, 170.1, 164.7, 130.5, 79.3, 73.3, 71.6, 60.3, 58.9, 50. 3, 46.6, 45.0, 43.7, 40.8, 39.3, 39.2, 37.3, 34.8, 33.9, 33.1, 30.8, 29.1, 28.8, 27.4, 27.2, 24.4, 23.8, 21.3, 21.1, 20.4, 19.4, 18.2, 17.3, 13.3; HRMS (ESI+): found [M+H]+: 550.39124 (calculated for C35H52NO4, 550.39127). EXAMPLE 3: Synthesis of 1(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)3a-acetyloxy-11-oxo-urs-12-en-24-oate (4)

[0025] CuI (2.0 equivalents) and Et3N (3.0 equivalents) were added to a solution of compound 2 (1.0 equivalent) and aromatic 4-trifluoromethyl azide 3a (1.2 equivalents) in acetonitrile (10 mL), and the mixture was stirred at room temperature for 3 hours until the reaction was complete (monitored by TLC analysis). After the reaction was complete, the reaction mixture was diluted with EtOAc (30 mL), and 15 mL of saturated aqueous NH4Cl solution was added. NH4Cl solution was added, and the aqueous layer was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with brine (1 × 20 mL), dried over anhydrous MgSO4, filtered, and the filtrate was concentrated in vacuo. The crude residue was further purified by flash column chromatography (silica gel, n-hexane / EtOAc, 75:25) to obtain the desired pure 1H-1,2,3-triazole analogue of β-AKBA (4) (92%). A gummy pale yellow solid; yield = 92%; 1H NMR (600 MHz, chloroform-d) δ 8.06 (s, 1H), 7.83 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 8. 6 Hz, 2H), 6.37 (t, J = 5.6 Hz, 1H), 5.49 (s, 1H), 5.32 (s, 1H), 4.56 (qd, J = 15.1, 5.7 Hz, 2H), 2.46 (dd, J = 10.0, 3.2 Hz, 1H), 2. 36 (s, 1H), 2.31 - 2.25 (m, 1H), 2.08 - 2.04 (m, 4H), 1.88 - 1.83 (m, 1H), 1.72 (ddd, J = 19.9, 10.8, 4.7 Hz, 5H), 1.58 (dd, J = 15. 1, 3.1 Hz, 1H), 1.46 (ddd, J = 31.7, 14.4, 8.1 Hz, 4H), 1.38 - 1.33 (m, 2H), 1.31 (d, J = 12.1 Hz, 4H), 1.23 (d, J = 7.2 Hz, 1H), 1. 20 - 1.16 (m, 2H), 1.12 (s, 3H), 1.10 (s, 3H), 0.99 (dd, J = 11.4, 2.4 Hz, 1H), 0.91 (s, 3H), 0.87 (s, 3H), 0.77 (s, 3H), 0.76 (d, J = 6.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 199.0, 175.7, 170.2, 164.9, 145.5, 139.3, 130.5, 127.1, 120.8, 120.5, 73.4, 60.3, 59. 0, 50.3, 46.6, 45.0, 43.7, 40.9, 39.3, 39.2, 37.4, 34.8, 34.0, 33.0, 30.9, 28.8, 27.5, 27.2, 24.5, 23.9, 21.3, 21.1, 20.5, 19.5, 18. 3, 17.4, 13.0; 19F NMR (564 MHz, Chloroform-d) δ -62.6; HRMS (ESI+): gefunden [M+H]+: 737.42445 (berechnet für C42H56N404, 737.42432). EXAMPLE 4: Production of the films

[0026] Gelatin (1.5%) was dissolved in distilled water with continuous stirring at 50 °C for two hours until a homogeneous solution was obtained. Glycerol (1%) was added as a plasticizer to increase flexibility. The film-forming gelatin solution was divided into two labeled beakers, including GA1 (control) and GA2, which was loaded with an analogue of β-AKBA. After complete dispersion of the β-AKBA analogue, 20 ml of solution was removed from each beaker and poured into pre-leveled Petri dishes, where they were allowed to dry at room temperature under controlled conditions. After complete drying, the films were carefully peeled off and stored in a desiccator until further analysis.

[0027] Visual assessment of the films revealed significant differences between GA1 (control) and GA2 (with β-AKBA analogue). The GA1 film appeared more transparent, while GA2 exhibited higher opacity. This difference can be attributed to the incorporation of the β-AKBA analogue, which likely disrupted the uniform polymer network of the gelatin, resulting in increased light scattering. EXAMPLE 5: Light transmission and haze

[0028] To evaluate the transparency and light scattering properties of the GA1 and GA2 films, their transmission and haze values ​​were measured using a haze meter. The film samples were carefully placed in the measuring device, ensuring correct alignment to avoid measurement errors.

[0029] The results show that the transmittance of GA2 was significantly lower and the haze significantly higher compared to GA1. The control film (GA1) exhibited a transmittance of 92.06 ± 0.015%, while GA2 had a lower transmittance of 90.65 ± 0.156%. Such a decrease suggests that the β-AKBA analogue impairs the optical transparency of the film.

[0030] The results show that the turbidity values ​​further support these observations: GA1 had a low turbidity value (3.167 ± 0.46%), while GA2 had a significantly increased turbidity value (28.05 ± 0.78%). The β-AKBA analogue was not homogeneous in the gelatin matrix, resulting in strong diffraction and scattering of light. EXAMPLE 6: Hydrophobicity of the surface

[0031] The hydrophobicity of the film surface was assessed using water contact angle measurements. A 2 × 2 cm film sample was placed on a flat analysis surface, and a 1 µl water droplet was carefully deposited in the center using a precision micropipette. The contact angle was then recorded with a goniometer and analyzed using dpiMAX software.

[0032] Contact angle measurements showed that the addition of β-AKBA analogue affected the surface wettability of the films. The contact angle of the GA1 film was lower (66.57°), indicating that the surface of the GA1 film was more hydrophilic than that of GA2 (83.32°). This may be related to the interaction between the β-AKBA analogue and the gelatin matrix and the lower abundance of hydrophilic functional groups on the surface. EXAMPLE 7: Physical and mechanical properties of the filmsFilm thickness

[0033] The thickness of the produced films GA1 and GA2 was determined using a digital micrometer. Five random points per sample were measured, and the average thickness (mm) was recorded.

[0034] The results show a significant increase in film thickness for the β-AKBA analogue, indicating that its presence leads to thicker films. The GA1 film (control) had an average thickness of 0.082 ± 0.040 mm, and GA2 (with β-AKBA) had a thickness of 0.13 ± 0.007 mm. This increase in thickness is most likely due to the β-AKBA analogue due to the fixed amount of film matrix, which increased the patterning of the films. Mechanical strength

[0035] The mechanical properties of the films were tested using a universal testing machine with a 5 kg load cell. Prior to testing, the films were preconditioned for 40 hours at 25 °C and 50% relative humidity. They were then cut into 7 × 60 mm strips and subjected to a tensile test at a speed of 30 mm / min to determine the tensile strength (TS, MPa) and elongation at break (EAB, %). The results were processed and analyzed using the Exponent Connect software platform.

[0036] The results show that the tensile strength (TS) of the GA1 film was 0.026 ± 0.002 MPa, while GA2 exhibited a lower TS value of 0.02 ± 0.001 MPa. The decrease in tensile strength after incorporation of β-AKBA suggests that the active ingredient may disrupt the gelatin polymer network, resulting in reduced intermolecular interaction. Regarding the elongation at break (EAB), the GA1 film exhibited an elongation of 210.45 ± 11.88%, while GA2 exhibited a slightly lower EAB of 202.4 ± 9.36%. This slight reduction suggests that the incorporation of β-AKBA did not significantly alter the flexibility of the film. Antioxidant activity

[0037] The antioxidant potential of the GA1 and GA2 films was determined using the DPPH radical scavenging assay. The reaction mixture was analyzed using a Vis spectrophotometer at a wavelength of 517 nm. The percentage inhibition was calculated from the average of three independent measurements per sample.

[0038] The results show that the β-AKBA-loaded film exhibits significantly higher DPPH inhibitory activity (41.55±0.91%) compared to GA1 (13.85±0.91%), confirming the successful enhancement of antioxidant properties. The remarkable increase in DPPH inhibition in GA2 suggests that the active ingredient effectively retains its functional properties in the gelatin matrix and contributes to the improved antioxidant performance of the film. Statistical evaluation

[0039] All results are expressed as mean ± standard deviation (SD) based on triplicate measurements. A one-way ANOVA followed by Fisher's post hoc test was performed to determine statistical significance between the different samples. Examples

[0040] 1. A biodegradable gelatin-based antioxidant film composition comprising: a) Gelatin as polymer matrix, b) a β-AKBA analogue incorporated into the matrix to improve the antioxidant properties, c) a plasticizer to improve the flexibility of the film and d) optional excipients for modulating the optical, mechanical and hydrophobic properties, wherein the β-AKBA analogue is dispersed in the gelatin matrix to achieve improved antioxidant activity and modified physical properties.

Claims

[1] Biodegradable gelatin-based antioxidant film composition comprising: a) Gelatin as polymer matrix, b) a β-AKBA analogue incorporated into the matrix to improve the antioxidant properties, c) a plasticizer to improve the flexibility of the film and d) optional excipients to modulate the optical, mechanical and hydrophobic properties, wherein the β-AKBA analogue is dispersed in the gelatin matrix to achieve improved antioxidant activity and modified physical properties. [2] A biodegradable gelatin-based antioxidant film composition according to claim 1, wherein the β-AKBA analogue is selected from (prop-2-yn-1-ylcarbamoyl) 3α-acetyloxy-11-oxo-urs-12-en-24-oate or 1(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl) 3α-acetyloxy-11-oxo-urs-12-en-24-oate. [3] The biodegradable gelatin-based antioxidant film composition according to claim 1, wherein the plasticizer is glycerin added at a concentration of 1 wt%. [4] The biodegradable gelatin-based antioxidant film composition according to claim 1, wherein the β-AKBA analog increases the film thickness to a range of 0.13 ± 0.007 mm. [5] A biodegradable gelatin-based antioxidant film composition according to claim 1, wherein the incorporation of the β-AKBA analogue results in a reduction in transmittance to 90.65 ± 0.156% and an increase in haze value to 28.05 ± 0.78%. [6] The biodegradable gelatin-based antioxidant film composition according to claim 1, wherein the β-AKBA analogue increases the water contact angle of the film surface to 83.32°, thereby enhancing hydrophobicity. [7] The biodegradable gelatin-based antioxidant film composition of claim 1, wherein the β-AKBA analogue reduces the tensile strength of the film to 0.02 ± 0.001 MPa while maintaining flexibility with an elongation at break of about 202.4 ± 9.36%. [8] The biodegradable gelatin-based antioxidant film composition according to claim 1, wherein the β-AKBA analogue increases the antioxidant activity and causes a DPPH inhibition of 41.55 ± 0.91%.