Development and Characterization of Fenugreek Seed Gum-graft-Acrylic Acid Polymer for pH-Sensitive Drug Delivery

Drug delivery systems have progressively evolved from conventional dosage forms toward advanced systems capable of responding to specific physiological conditions. Among these advanced approaches, stimuli-responsive polymers have gained considerable attention because their physicochemical properties can change in response to environmental stimuli such as pH, temperature, ionic strength, and biological signals ^[1]. Among these stimuli, pH is particularly attractive for drug-delivery applications because significant pH variations exist across different physiological and pathological environments ^[2,3].

Natural polysaccharides have attracted considerable interest in pharmaceutical and biomedical applications owing to their hydrophilicity, biodegradability, biocompatibility, and structural versatility ^[4]. However, native natural polymers may exhibit limitations such as inadequate mechanical strength, poor stability, and limited responsiveness to environmental conditions. Chemical modification through graft copolymerization provides an effective approach to improve the functional properties of natural polymers while retaining their inherent advantages. Previous studies have demonstrated that polysaccharide-based polymers grafted with acrylic acid can exhibit pronounced pH-dependent swelling and drug-release behaviour ^[5,6]. For example, Khatoon M., et al.,^[7] developed a glucoxylan-grafted acrylic acid hydrogel and demonstrated that its swelling and drug-release characteristics were influenced by both pH and polymer composition.

Acrylic acid is particularly suitable for the development of pH-responsive polymeric systems because of the presence of ionizable carboxylic acid groups ^[8,9]. Under acidic conditions, these groups remain predominantly protonated, resulting in relatively limited ionization and swelling. At higher pH, ionization of the carboxylic groups to carboxylate ions promotes electrostatic repulsion between polymer chains and increased hydration of the polymer network, leading to enhanced swelling ^[8,10]. Similar pH-dependent behaviour has been reported in various acrylic-acid-containing graft polymer systems, where lower swelling occurs under acidic conditions and greater swelling is observed at higher pH values ^[10,11].

Fenugreek Seed Gum (FSG) is a natural polysaccharide with desirable hydrophilic, swelling, and water-retention properties, making it a potential candidate for pharmaceutical applications ^[12]. However, its native form may possess limited control over environmental responsiveness. Therefore, chemical modification of FSG through graft copolymerization with acrylic acid was explored to develop a semi-synthetic pH-responsive polymer ^[13]. The incorporation of acrylic acid was intended to introduce ionizable carboxylic groups into the polymeric structure, thereby enhancing its pH-dependent swelling behaviour and potential for controlled drug release.

The present study therefore focused on the development of Fenugreek Seed Gum-grafted acrylic acid (FSG-g-AA) using free-radical graft copolymerization ^[14]. The synthesized polymer was systematically optimized using a 3² full factorial design to investigate the influence of formulation variables on pH-responsive swelling behaviour ^[15]. Diclofenac sodium was selected as a model drug because of its pH-dependent solubility and ionization characteristics ^[16,17,18]. The optimized polymer was characterized using Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis–Differential Thermal Analysis (TGA-DTA), and Scanning Electron Microscopy (SEM) ^{[19,20,21,22]}. Furthermore, its pH-responsive behaviour was evaluated through swelling studies at different pH conditions and in vitro drug-release studies to assess its potential application in pH-sensitive drug-delivery systems.

2. MATERIALS

Fenugreek seeds were purchased from Wagdole Ayurvedics, Satara, India. Acrylic acid (AA), diclofenac sodium, N,N′-methylenebisacrylamide (MBA), ammonium persulfate (APS), and ethanol were purchased from Loba Chemie Pvt. Ltd., Mumbai, India. All chemicals and reagents used in the study were of analytical grade and were used as received without further purification. Distilled water was used throughout the experimental work.

3. METHODS:

3.1. Extraction of Fenugreek Seed Gum (FSG)

3.1.1. Defatting of Fenugreek Seeds

Cleaned and dried fenugreek seeds were coarsely powdered and subjected to defatting using petroleum ether at a powder-to-solvent ratio of 1:5 (w/v). The mixture was continuously stirred at room temperature for 24 h using a magnetic stirrer. Subsequently, the defatted mixture was filtered through muslin cloth followed by Whatman filter paper. The obtained residue was air-dried and further dried at 40 °C to ensure the removal of residual petroleum ether. The dried and defatted fenugreek seed powder was stored in an airtight container until further extraction of Fenugreek Seed Gum (FSG) ^[23].

3.1.2. Extraction of Fenugreek Seed Gum (FSG)

Fenugreek Seed Gum (FSG) was extracted by hot-water extraction followed by ethanol precipitation. The defatted seed powder was dispersed in distilled water at a 1:10 (w/v) ratio and heated at 60°C for 3 hr with continuous stirring. The resulting mucilaginous extract was filtered through multilayer muslin cloth to remove insoluble materials. The filtrate was cooled to room temperature, and excess absolute ethanol was added slowly with continuous stirring to precipitate the gum. The precipitated FSG was collected, dried, and stored for further characterization and graft copolymerization studies ^[24,25].

3.2. Synthesis of Fenugreek Seed Gum Grafted Acrylic Acid (FSG-g-AA) Polymer

Fenugreek Seed Gum (FSG) was grafted with acrylic acid (AA) by free-radical graft copolymerization using ammonium persulfate (APS) as the initiator and N,N′-methylenebisacrylamide (MBA) as the crosslinking agent ^[5]. The required quantity of FSG was dispersed in distilled water under continuous magnetic stirring and heated to 60 ± 2 °C. APS was added to initiate free-radical formation on the FSG backbone, followed by the addition of the specified quantity of AA and MBA under continuous stirring. The reaction mixture was maintained at 60 ± 2 °C for 4 h to facilitate graft copolymerization and crosslinking. After completion of the reaction, the product was cooled to room temperature, separated, and repeatedly washed with ethanol to remove unreacted monomer, initiator, and soluble impurities. The purified polymer was dried and stored in an airtight container until further characterization and evaluation ^[26].

Table 1. Composition of Fenugreek Seed Gum-graft-Acrylic Acid Polymer Formulations

3.3. Swelling Studies

The filtration method was used to evaluate the pH-responsive swelling behaviour of the synthesized FSG-g-AA polymer ^[10,27]. The swelling behaviour was evaluated in buffer media of pH 1.2, 6.8, and 7.4. Accurately weighed polymer (50 mg; W₀) was immersed in 100 mL of the respective buffer medium and allowed to swell for 2 h at room temperature. After 2 h, the swollen polymer was separated from the medium by filtration. Excess surface liquid was carefully removed, and the swollen polymer was immediately weighed to determine its swollen weight (W). The experiment was performed in triplicate.

The swelling ratio (SR) was calculated using the following equation:

Swelling Ratio (SR)=W-W0W0

Where, W is the weight of the swollen polymer and W₀ is the initial dry weight of the polymer. The swelling study was performed in triplicate, and the results were expressed as mean ± standard deviation.

3.4. Experimental Design (Design-Expert software)

A 3² full factorial design was employed using Design-Expert® software to systematically investigate the effects of formulation variables on the swelling behavior of the synthesized FSG-g-AA polymer ^[28]. Fenugreek seed gum (FSG) and acrylic acid (AA) were selected as the independent variables, each investigated at three levels. The concentrations of N,N′-methylenebisacrylamide (MBA) and ammonium persulfate (APS) were kept constant throughout all experimental runs. The swelling ratios at pH 1.2, 6.8, and 7.4 were selected as the response variables. The factorial design comprised nine experimental runs, which were prepared and evaluated to determine the effects of FSG and AA concentrations on the pH-responsive swelling behavior of the synthesized polymer.

3.5. Characterization

3.5.1. Fourier Transform Infra-Red Spectroscopy (FTIR)

The Fourier transform infrared (FTIR) spectra of Fenugreek Seed Gum (FSG) and the synthesized FSG-g-AA polymer were recorded using a Bruker ALPHA II (Germany) FT-IR spectrometer equipped with an attenuated total reflectance (ATR) accessory. The spectra were recorded over the wavenumber range of 4000–400 cm⁻¹. The obtained spectra were processed and analyzed using OPUS software (Bruker Optics). The characteristic absorption bands of FSG and FSG-g-AA were compared to identify changes in the functional groups associated with the grafting of acrylic acid onto the FSG backbone. The appearance, disappearance, intensity changes, and shifts of characteristic bands were considered as evidence of structural modification following grafting ^[19,29].

3.5.2. Thermogravimetric Analysis – Differential Thermal Analysis (TGA-DTA)

The thermal behavior of the optimized FSG-g-AA polymer was investigated using a simultaneous thermal analyzer (SDT650 TA Instruments, USA). An accurately weighed sample of the polymer was placed in an alumina sample pan and subjected to controlled heating under a nitrogen atmosphere. The sample was heated over the temperature range of approximately 30–600 °C. The thermogravimetric (TGA) and differential thermal analysis (DTA) curves were recorded simultaneously and analyzed using TA Instruments TRIOS software (Version 5.11.0.31). The obtained thermograms were used to evaluate the thermal degradation behavior and thermal stability of the synthesized FSG-g-AA polymer ^[21,30].

3.5.3. Differential Scanning Calorimetry (DSC)

The thermal transitions of the optimized FSG-g-AA polymer were investigated using a differential scanning calorimeter (DSC25 TA Instruments, USA) under a nitrogen atmosphere. An accurately weighed sample of the polymer was placed in an aluminium sample pan, with an empty aluminium pan used as the reference. The sample was subjected to controlled heating over the selected temperature range, and the DSC thermogram was recorded. The obtained thermogram was processed using TA Instruments TRIOS software (Version 5.11.0.31) and evaluated to determine the characteristic thermal transitions and thermal behavior of the synthesized FSG-g-AA polymer ^[20,30].

3.5.4. Scanning Electron Microscope (SEM)

The surface morphology of the synthesized FSG-g-AA polymer was examined using scanning electron microscopy (SEM). The dried polymer sample was mounted onto an aluminium stub using conductive adhesive and sputter-coated with a thin layer of gold prior to analysis. The coated sample was examined using a Carl Zeiss SUPRA 55 (Germany) scanning electron microscope (SEM), equipped with a secondary electron detector (SED). The analysis was performed at an accelerating voltage of 20 kV and a working distance (WD) of 12.3 mm. SEM micrographs were acquired at different magnifications to evaluate the surface morphology, surface characteristics, and structural features of the synthesized FSG-g-AA polymer at the microscopic level ^[22].

3.7.5. In Vitro Drug Release

In vitro drug release studies were performed using Diclofenac Sodium as a model drug, incorporated into tablets prepared with the optimized FSG-g-AA polymer. The study was carried out using a USP dissolution apparatus II (Paddle) at 50 rpm, 900 mL dissolution medium and maintained at 37 ± 0.5 °C to evaluate the pH-responsive drug-release behaviour of the optimized polymer system ^[31].

Table 2. Composition of Diclofenac loaded FSG-g-AA Polymer Tablet

The in vitro drug release study was conducted for 8 hr to evaluate the pH-responsive drug release behavior of the optimized polymer system. The tablets were initially placed in simulated gastric fluid (SGF, pH 1.2), followed by simulated intestinal fluid (SIF, pH 6.8 and 7.4). Samples (2 mL) were withdrawn at predetermined time intervals of 1, 2, 3, 4, 5, 6, 7, and 8 hr and immediately replaced with an equal volume (2 mL) of fresh dissolution medium to maintain sink conditions throughout the study ^[32]. The experiment was performed in triplicate.

The percentage cumulative drug release was calculated using the following formula:

%Cumulative Drug Release=Amount of drug releasedTotal drug content×100

4. RESULTS AND DISCUSSION

4.1. Extraction of Fenugreek Seed Gum (FSG)

Fenugreek Seed Gum (FSG) was successfully extracted using the hot-water extraction method, yielding a mucilaginous polysaccharide fraction. The obtained gum was separated from insoluble seed materials, dried, and recovered as a stable powder. The successful extraction confirmed the suitability of the method for obtaining FSG as a natural polymeric backbone for subsequent graft copolymerization and development of the pH-responsive FSG-g-AA polymer.

Figure 1. Wet Fenugreek Seed Gum (FSG)

4.2. Synthesis of FSG-g-AA Polymer

The FSG-g-AA polymer was successfully synthesized by free-radical graft copolymerization of acrylic acid (AA) onto the Fenugreek Seed Gum (FSG) backbone using ammonium persulfate (APS) as the initiator and N,N′-methylenebisacrylamide (MBA) as the crosslinking agent. The synthesis yielded a stable polymeric product, which was purified, dried, and subsequently used for further characterization and evaluation of its pH-responsive properties.

         

(A)                                                                   (B)

Figure 2. FSG-g-AA Graft Copolymer: (A) Before Drying and (B) After Drying

Figure 3. Comparative Swelling Ratios of FSG-g-AA Polymer Batches (F1-F9)

Figure 4. Contour Plot and 3D Response Surface Plot for Swelling Ratio at pH 1.2

Response 2: Swelling Ratio at pH 6.8

Table 6. ANOVA for Swelling Ratio at pH 6.8

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	11.17	5	2.23	26.95	0.0107	significant
A-FSG	6.20	1	6.20	74.84	0.0032
B-AA	0.1667	1	0.1667	2.01	0.2512
AB	2.72	1	2.72	32.85	0.0105
A2	2.07	1	2.07	24.95	0.0154
B2	0.0089	1	0.0089	0.1073	0.7648
Residual	0.2486	3	0.0829
Cor Total	11.42	8

Figure 5. Contour Plot and 3D Response Surface Plot for Swelling Ratio at pH 6.8

Response 3: Swelling Ratio at pH 7.4

Table 7. ANOVA for Swelling Ratio at pH 7.8

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	22.32	5	4.46	11.92	0.0341	significant
A-FSG	13.20	1	13.20	35.26	0.0095
B-AA	0.1350	1	0.1350	0.3605	0.5905
AB	5.29	1	5.29	14.13	0.0329
A2	3.65	1	3.65	9.73	0.0525
B2	0.0450	1	0.0450	0.1202	0.7517
Residual	1.12	3	0.3744
Cor Total	23.44	8

Figure 6. Contour Plot and 3D Response Surface Plot for Swelling Ratio at pH 7.4

Figure 7. FTIR Spectrum of FSG, and FSG-g-AA

Figure 8. TGA-DTA thermogram of FSG-g-AA

Figure 9. DSC thermogram of FSG-g-AA

Figure 10. (A, B, C, D) SEM microphotographs of FSG-g-AA

Table 9. In Vitro Drug Release of Diclofenac Sodium at Different pH Conditions (Mean ± SD)

Time (hr)	% CDR (pH 1.2)	% CDR (pH 6.8)	% CDR (pH 7.4)
0	0 ± 0.00	0 ± 0.00	0 ± 0.00
1	4.29 ± 0.30	5.45 ± 0.41	11.42 ± 0.54
2	6.20 ± 0.48	8.91 ± 0.57	18.07 ± 0.86
3	7.16 ± 0.50	15.86 ± 1.08	26.38 ± 1.38
4	8.92 ± 0.57	24.55 ± 1.00	37.98 ± 2.32
5	10.84 ± 0.62	31.50 ± 1.42	51.28 ± 2.95
6	14.51 ± 0.67	38.45 ± 3.03	62.90 ± 3.60
7	18.97 ± 0.88	45.40 ± 3.33	74.52 ± 3.85
8	23.60 ± 1.00	50.62 ± 2.45	86.15 ± 4.50

Figure 11. In Vitro Drug Release of Diclofenac Sodium from FSG-g-AA

After 8 hr, drug release was only 23.60% at pH 1.2, whereas 50.62% and 86.15% release occurred at pH 6.8 and 7.4, respectively. The substantially greater release at higher pH corresponds with the increased swelling of the FSG-g-AA matrix.

At acidic pH, protonation of carboxylic groups limits polymer expansion and consequently restricts drug diffusion. At higher pH, ionization of carboxylic groups increases electrostatic repulsion, hydration and swelling, facilitating drug diffusion from the polymer matrix. This mechanism is consistent with the pH-responsive behaviour reported for related acrylic-acid-based graft polymers.

The progressive increase in drug release from pH 1.2 to pH 7.4 therefore demonstrates that the synthesized FSG-g-AA polymer can provide a distinctly pH-dependent release environment.

CONCLUSION

A smart semi-synthetic pH-sensitive polymer was successfully developed by grafting acrylic acid onto Fenugreek Seed Gum through free-radical graft copolymerization and characterized using FTIR, DSC, TGA-DTA, and SEM techniques. A 3² full factorial design was successfully employed to evaluate the effects of FSG and acrylic acid concentrations on the swelling behaviour of the polymer. Among the nine formulations, F7 containing 750 mg FSG and 2 mL acrylic acid exhibited the highest swelling ratios of 2.1, 6.2, and 9.8 at pH 1.2, 6.8, and 7.4, respectively, and was selected as the optimized formulation. The optimized FSG-g-AA exhibited acceptable micromeritic properties, characteristic FTIR bands, thermal stability, and a rough, porous morphology. Furthermore, diclofenac sodium release was distinctly pH-dependent, with cumulative drug release of 23.60%, 50.62%, and 86.15% at pH 1.2, 6.8, and 7.4, respectively, after 8 h. Overall, the developed FSG-g-AA demonstrated promising potential as a natural polysaccharide-based smart polymer for pH-sensitive drug delivery, particularly for enhancing drug release under higher-pH conditions. Further investigations involving release kinetics, stability, safety, reproducibility, and in vivo evaluation are warranted to establish its pharmaceutical applicability.

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