Blood chemical components analysis of honeysuckle and formulation of xanthan gum/starch-based (PVA-co-AA) hydrogels for controlled release

2.3.1 Preparation of honeysuckle decoction (aqueous extract)

Honeysuckle slices (20 g) were accurately weighed and added 240 mL of purified water in an electric heating sleeve and soaked for 30 min, boiled gently with reflux conditioner for 30 min and then filtered. The extract of Honeysuckle was obtained by adding ultra-pure water to make up the volume, mixed with ultrasonic machine and then filtered using 0.22 µm microporous filter membrane.

2.3.2 Preparation of honeysuckle standard decoction (aqueous extract)

Honeysuckle standard decoction were prepared according to the principles of “Chinese Medicine Granule Management Method (Draft)” issued by the National Pharmacopoeia Committee of China. The honeysuckle decoction pieces (75 g) were accurately weighed, soaked in 12 times as much water for 30 min, gently boiled for 30 min, then filtered while still hot. The residue was decocted with 10 times of water, kept boiling for 20 min on gentle heating, and filtered while hot. The decoctions were combined and then concentrated under pressure to obtain a concentration of 1 g/mL (Xiong and Long 2020).

2.3.3 Preparation of reference solution

The standard substances used, such as neochlorogenic acid, chlorogenic acid, strychnic acid, strychnoside, rutin, isoquercetin, luteoloside, lonicidin, isochlorogenic acid A and isochlorogenic acid C, were accurately weighed and mixed with methanol to obtain the concentrated solution. Then the concentrated solution was diluted with methanol to prepare 80 μg·mL⁻¹ reference solution, which was filtered by 0.22 µm microporous membrane to obtain the in vitro reference solution of Honeysuckle.

2.4.1 Drug administration and serum collection

The SD rats (n = 12, Male) were randomly divided into 4 groups (A, B, C and D) with 3 animals in each group. According to the Chinese Pharmacopoeia (2020 edition) (Leong et al., 2020), the daily clinical dosage of Honeysuckle (Lonicera japonica) is 6–15 g, and the maximum dosage of 15 g was selected (Fang et al., 2021). According to the literature and preliminary experimental results, the intragastric dose of rats was increased to 10 times that of human clinical medication, i.e., raw drug dose of 16 g·kg⁻¹. Rats in groups B, C and D were intra-gastrically gavaged with honeysuckle decoction, and rats in group A received same amount of normal saline. Blood samples were collected from abdominal aorta of rats in groups B, C and D at 60, 80 and 100 min after administration, respectively. The blood samples were kept for 1 h at 4 ℃ and centrifuged at 3500 rpm/min for 15 min to obtain upper serum. Then, stored at −80 ℃ refrigerator for subsequent processing and analysis.

2.4.2 Serum samples preparation

For each group, placed 300 uL serum into a 2 mL EP tube, added 900 µL methanol–acetonitrile (1:1), vortex mixed for 3 min, stand it for 30 s, centrifuged at 12000 rpm at 4 °C for 10 min, taken the supernatant and blow dried with nitrogen at 40 °C. The residue was redissolved with 200 μL methanol, then vortex mixed for 3 min, centrifuged at 12000 rpm at 4 ℃ for 10 min, and then the supernatant was taken for testing.

2.5.1 Chromatographic conditions

The chromatographic separation was performed on an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) with a mobile phase of 0.2 % formic acid aqueous solution (A) and acetonitrile (B) by gradient elution as follows: 0–9 min, 5 %-20 % B; 9–10 min, 20 %-25 % B; 10–20 min, 25 %-80 % B; 20 ∼ 30 min, 80 % ∼ 90 % B; 30 ∼ 30.1 min, 90 % ∼ 5 % B; and 30.1 ∼ 33 min, 5 % B. The column temperature was 40 ℃, the injection volume was 2 μL, and the flow rate was 0.3 mL/min.

2.5.2 Mass spectrometry conditions

The mass spectrometry conditions were as follows: Electrospray ion source (ESI); positive and negative ion detection modes, capillary temperature was 350 ℃; flow of sheath gas was 30 L/h; and auxiliary gas flow was 10 L/h. In positive ionization mode the spray voltage was 3 kV, capillary voltage was 35 V, tube lens voltage was 110 V, and in negative ionization mode the spray voltage was −3 kV, capillary voltage was −35 V, and tube lens voltage was −110 V. The sample was scanned in full scan mode with a resolution of 30000, a mass scan range of m/ Z50-1250, and a data-dependent scan of DDA-MS2. Parent ion pil-MS2, and fragment ions were obtained by CID and HCD pyrolysis.

2.6.1 Identification of chemical constituents of honeysuckle

The Xcalibur version 2.1 workstation was used for mass spectrometry data processing. The MS and MS/MS data information in the positive and negative mode of honeysuckle sample solution were compared and analyzed with reference to standards, literature retrieval and database retrieval. The retention time and peak intensity were combined to identify and confirm the chromatographic peaks, so as to identify the chemical components contained in honeysuckle.

2.6.2 Identification of blood prototypic components from honeysuckle

Mass spectrometry data was processed using the Xcalibur workstation. Chromatograms of honeysuckle sample solution, rat serum samples and blank serum samples were compared. MS and MS/MS data information in positive and negative modes were analyzed by reference to standard substances, literature retrieval and database retrieval. Combined with retention time and peak intensity, the chromatographic peaks were identified and confirmed to identify the prototype components of honeysuckle absorbed into the blood.

2.7.1 Drug loading

The honeysuckle was loaded into the hydrogels by preparing a 1 % solution of honeysuckle in pH 7.4 buffer and then the dried hydrogels were weighed and place inside the solution and incubated for 3 days. Then the hydrogels discs were taken out and dried at 40 °C for 1 week. The drug loading was determined by subtracting the initial dry weight of hydrogels before drug loading from the final weight of hydrogels after drug loading, as shown in equation (1).

2.8.1 Fourier transform infrared spectroscopy (FTIR)

FTIR study was conducted to assess the type of bond, structure, and formation of new bonds. FTIR spectra of PVA, xanthan gum, starch, AA, EGDMA, honeysuckle, unloaded and drug-loaded hydrogels were obtained using the attenuated total reflectance FTIR (Spectrum two, Platinum Elmer). FTIR spectra of ground samples were obtained at a wavelength of 4000–400 cm⁻¹.

2.8.2 Thermal analysis (TGA and DSC)

Analyses of pure materials and hydrogels were conducted using a DSC analyzer (Diamond DSC, Perkin Elmer). Heat was applied to the samples at a temperature range between 25 °C and 350 °C at a rate of 10 °C/min, with nitrogen flow remaining constant throughout the study. The experiment was conducted in triplicate, and the data are expressed as means and standard deviations.

Polymers and hydrogels were evaluated for thermal stability with a thermogravimetric analyzer (TG/DTA6300, Seiko, Japan). The sample was accurately weighed (5 mg) and placed in a platinum pan. The analysis was performed at room temperature, and the temperature was maintained at 20 °C per minute until 600 °C of heat was reached under atmospheric nitrogen.

2.8.3 X-ray diffraction (XRD)

The crystalline and amorphous properties of xanthan gum, starch, PVA, and honeysuckle hydrogels were investigated using a TD-3500 X-ray diffractometer (China). A powdered sample was placed in an XRD-sample tray and then XRD was conducted. This test was conducted under the following conditions: irradiation of the target (CuKα), 30 kV of voltage, and 20 mA of current. XRD patterns were acquired by scanning at 2°/minute between 10° and 60°. The data was processed using Jade/MDI software. The crystallinity of the compound was confirmed from peaks, such as sharp intensive peaks in pure materials, whereas diffused peaks in hydrogel samples suggested that the hydrogels are amorphous.

2.8.4 Volume fraction of polymer (V2,s)

Polymer volume fraction (V2,s) indicates the percentage of the polymer in a fully swollen state. Polymer volume fraction was estimated based on equilibrium volume swelling (Veq) data for hydrogel formulations at two different pH values (1.2 and 7.3). This was done by using the following formula.

2.8.5 Scanning electron microscopy (SEM)

SEM (Quanta 250, FEI, Czech) was used to investigate the microstructure, porosity, and morphology of the hydrogels. During this experiment, vacuum-dried samples were fixed to aluminum stubs and sputter-coated with gold, and the surface morphology was observed under 15 kV accelerated current under a cross-sectional view.

2.8.6 Sol–gel study

The sol–gel analysis of all developed formulations was performed in order to determine the soluble and insoluble crosslinking components of the hydrogels. Gel is the insoluble fraction of hydrogels, whereas sol is the soluble fraction. The sol–gel analysis was conducted using a Soxhlet extraction technique. A specific volume of deionized distilled water was added to a round bottom flask containing the hydrogel disc and connected with the condenser. Extraction continued at 85 °C for 14 h. Following extraction, the hydrogel disc was dehydrated in a vacuum oven until completely dry, and then weighed again. The following equations were used to determine the sol–gel analysis.

The weight shown in Y1 is the initial weight of the hydrogels (prior to extraction), whereas the weight shown in Y2 is the final weight of the dried hydrogels (after extraction).

2.8.7 Porosity study

The porosity of all the hydrogels was evaluated and analyzed using a solvent replacement technique. The hydrogel discs (L1) of all the formulations were immersed in absolute ethanol (purity > 99.9 %) for three days. Hydrogel discs were removed after three days, blotted with filter paper to remove excess solvent, and weighed again (L2). Disc thickness and diameter were also measured. Porosity was determined using the given equation.

ρ represents the density of absolute ethanol, while V represents the swelling volume of the hydrogel.

2.8.8 Equilibrium swelling ratio

The equilibrium-swelling ratio (ESR) of hydrogels was measured at pH 1.2 and pH 7.4. Hydrogels were weighed accurately and regularly in order to determine how much their weight had changed over time. Hydrogel weights were recorded until equilibrium was reached. The percentage swelling was calculated by using the following formula.

Where, Wf is the final weight and Wi represents initial weight of hydrogel.

2.8.9 In-vitro drug release and data modeling

Drug release from developed hydrogels was studied in vitro at two pH values, namely pH 1.2 and pH 7.4. Dry discs of hydrogels containing the drug were immersed individually in 900 mL of phosphate buffer solutions at pH 1.2 and 7.4 in a USP dissolution apparatus type II at 37 ± 0.5 °C and 50 revolutions per minute. A 5-ml aliquot was taken at specified intervals and replenished with a fresh medium of the same volume to maintain sink conditions. After the samples were collected, they were filtered and analyzed using a UV–vis spectrophotometer (T6 New Century; Beijing GM) at λ^max of 324 nm (standard chlorogenic acid) in triplicate.

Release of drugs from a hydrogel matrix is affected by a number of factors, including polymer chain relaxation and swellability, matrix composition, type of drug, and pH of the release medium. Because hydrogels exhibit controlled swelling, solvent diffusion is necessary in order to cause hydrogel swelling. Models of zero order, first order, Higuchi and Korsmeyer Peppas were used to predict drug release patterns.

2.8.10 Statistical analysis

The data was analyzed using ANOVA method to determine the significant differences. A (p-value of less than 0.05) was considered significant. The data is presented as mean ± standard deviation.

3.3.1 Flavonoids

Flavonoids undergo a series of cleavage, loss and rearrangement reactions centered on the C ring. Most of the free flavonoids have molecular ion peaks [M] + as base peaks, [M−H]+, [M−CO] + and RDA. Fragment ion peaks generated by fragmentation are important peaks, which can determine the type of aglycone and the type and number of substituents on the A, B, and C rings. Flavonoid glycosides are classified into flavonoid C glycosides and flavonoid O-glycosides due to the difference in the glycoside bond atoms connecting to the sugar group being C atom and O atom. The flavonoid C-glycosides are mainly cleaved by the cross-ring excision of the sugar ring, and almost no aglycone ions are produced; the cleavage of the flavonoid O-glycosides is mainly based on the cleavage of the glycosidic bond, the cleavage of the sugar-sugar bond and the cross-ring excision and cleavage of the sugar ring [21].

Taking compound No. 31 as an example, the quasi-molecular ion peak was detected as m/z 285.0393[M-H]- in the negative ion mode. The software Xcalibur 2.1 was used to analyze its elemental composition and deduce its possible molecular formula as C₁₅H₁₀O₆. In mass spectrometry, quasi-molecular ions are more likely to lose H₂O and CO to generate fragment ions m/z 267.07288 ([C₁₅H₈O₅]-) and m/z 256.95416 ([C₁₄H₁₀O₅]-), respectively. As a result of fragment ions 151.03949 ([C₇H₄O₄]-), and 133.01379 ([C₈H₆O₂]-) combined with relevant references [22], compound No. 31 was identified as luteolin. Its MS spectrum and its possible cleavage pathway is shown in Fig. 2.

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Fig. 2

MS spectrum (A) and the possible cleavage pathway (B) of luteolin.

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3.3.2 Organic acid components

The basic structures of these compounds are aromatic rings substituted with phenolic hydroxyl groups, fatty acids, etc., which are easily broken at the carbonyl group to form fragment ions [23]. Among them, chlorogenic acid compounds have ester groups in their structures. In mass spectrometry, the ester groups are easily broken to form fragment ions with a smaller mass-to-nucleus ratio and higher abundance than the parent ion. Fragment ions of [M−H₂O]- and [M−CO₂]- appeared in secondary mass spectrometry [24].

Taking compound No. 11 as an example, the quasi-molecular ion peak was detected as m/z 353.0876 [M−H]- in the negative ion mode, and its possible molecular formula was deduced to be C₁₆H₁₈O₉. In its secondary mass spectrometry, the quasi-molecular ion removed 1 molecule of caffeoyl group and 1 molecule of quinacyl group respectively and generated fragment ions like quinic acid ion (m/z1 91.05562) and caffeic acid ion (m/z 179.03438), and after comparing with the reference substances, it is inferred that compound No. 11 is chlorogenic acid. Its secondary mass spectrum and its possible cleavage pathway are shown in Fig. 3.

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Fig. 3

Secondary mass spectrum (A) of Chlorogenic acid and its proposed fragmentation pathway (B).

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Taking compound No. 10 as an example, the quasi-molecular ion peak was detected as m/z 181.05 [M + H] + in positive ion mode, and its possible molecular formula was deduced to be C₉H₈O₄. In the secondary mass spectrometry, the fragment ion was m/z 163.03963, and it is speculated that the quasi-molecular ion should be obtained by removing [M + H-COOH] +. The m/z 135.04451 indicates that [M + H-H₂O-CO] + is removed from the parent ion, and based on a comparison of its spectrum to that of the reference substance, it has been deduced that compound No. 10 is caffeic acid, and its MS spectrum and possible cleavage pathway are depicted in Fig. 4.

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Fig. 4

Secondary mass spectrum (A) of caffeic acid and its proposed fragmentation pathway (B).

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3.3.3 Iridoid components

The primary spectrum of iridoids in positive ion mode commonly contains [M + H]+, [2 M + H]+, [M + Na]+, and [2 M + Na] + ions, as well as sometimes [M + K]+, and [2 M + K] + ion peaks. The negative ion mode is characterized by [M−H]- and its dimer [2 M−H]-. An iridoid glycoside quasimolecular ion or its adducted molecular ion is used as a parent ion in the positive or negative ion mode. The MS/MS cleavage is characterized by the loss of neutral molecules, such as CO₂, H₂O, Glc, etc., and some substituents, with the sequence of cleavage relating to the parental nucleus or side chains [25].

Taking compound No. 36 as an example, the [2 M + H]+, [2 M + Na]+, and [M + H] + in positive ion mass spectrometry were analyzed, and the quasi-molecular ion peak was detected at m/z 405.1379 [M + H]+, and its possible molecular formula is deduced to be C₁₇H₂₄O₁₁. Based on the secondary mass spectrometry results of the fragment ion at m/z 243.94196, it is speculated that the quasi-molecular ion is formed by the removal of [M + H-Glc]+, based on the reference [17], so a comparison with the reference substance indicates that compound No. 36 is deoxyloganin. The results show that UPLC-LTQ-Orbitrap-MS can quickly and systematically analyze the components of honeysuckle into the blood, and it suggests that the components directly absorbed into the blood may be the medicinal substances of honeysuckle, which lays the foundation for the subsequent determination of honeysuckle quality markers. basis, so as to realize the quality control of honeysuckle.

3.4.1 FTIR analysis

The preparation of xanthan gum/starch based-(PVA-co-AA) hydrogels was confirmed by evaluating the FTIR spectra of xanthan gum, starch, PVA, AA, honeysuckle and developed hydrogels, as shown in Fig. 5A. FTIR spectrum of xanthan gum showed stretching vibration of C — O of primary alcohols and asymmetrical stretching of carboxylate ions by peaks at 1011 and 1598 cm⁻¹, respectively. Starch showed peaks at 3270, 2917 and 987 cm^−1°indicating stretching vibration of the O—H, C—H, and C—O groups, respectively (Sethi et al., 2020). The characteristic peaks of PVA appeared at 3281, 2907, 1709 and 1234 cm⁻¹, which were assigned to stretching vibration of O—H, CH₂ asymmetric stretching, C⚌O carbonyl stretching, and C—H deformation, respectively (Kharazmi et al., 2015). Similarly, FTIR spectra of AA showed peaks at 2983, 1690, and 1427 cm⁻¹, which were assigned to stretching vibration of C—H, C—O, C⚌O, and deformation of C—O—H (Liew et al., 2016). FTIR spectra of EGDMA showed peaks at 1722, 937, and 809 cm⁻¹, which were assigned to the stretching vibration of C—O, and waging and twisting bands of = CH2, respectively (Barkat et al., 2021). The crosslinking of polymers and monomers and the chemical interaction among the contents of hydrogel led to changes in the peak distribution of certain polymers and monomers. The prominent peaks of xanthan gum were changed from 1011 and 1598 cm⁻¹ to 1152 and 1690 cm⁻¹ peaks in the synthesized hydrogels. Similarly, a few peaks of AA and starch were moved from 2983 and 1235 cm⁻¹ and 987 to the 2915 and 1132 cm⁻¹ and 1152 cm⁻¹ peaks in the developed hydrogel network. Some of the peaks of xanthan gum, PVA, AA, and starch disappeared, while certain new peaks appeared. Thus, shifting peaks, disappearances, and the formation of new peaks indicated that AA, starch, and PVA were successfully grafted onto the backbone of xanthan gum, producing a new polymeric network. The FTIR spectral analysis of honeysuckle revealed stretching vibration of O—H and N—H, C—H, C⚌O, C⚌C, and C—N by peaks at 3289, 2905, 1683, 1585, and 1034 cm − 1, respectively (Zhang et al., 2022). Drug-loaded hydrogels showed a fluctuation in certain peaks of honeysuckle. The peaks at 2905 and 1585 cm⁻¹ were moved to 2916 and 1570 cm⁻¹ peaks of the drug-loaded hydrogel. Due to the successful loading of the drug into the hydrogel, there was no chemical interaction observed between honeysuckle peaks and hydrogel components.

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Fig. 5

FTIR (A), DSC (B), TGA (C) and XRD (D) of the pure materials and developed hydrogels.

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3.4.2 Thermal analysis

Polymers and hydrogels were tested for thermal stability using a DSC analysis, as shown in Fig. 5B. Xanthan gum shows an endothermic peak at 100 °C (melting) and an exothermic peak at 297 °C (degradation). Starch shows an endothermic peak at 82 ℃ (initial moisture loss) and exothermic peak at 258 ℃ (melting and decomposition of the polymer). The endothermic peak of PVA occurs at 91 °C (glass transition and moisture loss) and a sharp peak at 191 °C (melting of the polymer), while the endothermic peak of the unloaded hydrogels occurs at 100 °C (evaporation of water) and the second endothermic peak occurs at 239 °C (onset of degradation of the gel network). This is in line with previous studies which demonstrate a highly stable polymeric gel network (Shalviri et al., 2010).

The thermogravimetric analysis of xanthan gum, starch, PVA, and XG/starch-based (PVA-co-AA) hydrogels was carried out to determine the thermal stability of the developed hydrogels. The initial weight loss of starch at 97 °C was found to be 10 % as a result of the release of moisture and volatile matter absorbed by the starch. Weight loss was observed in two steps, and was found to be 60 % and 81 % at 335 °C and 551 °C, respectively, indicating that the initial weight loss was much greater than the final weight loss of starch. The initial weight loss of xanthan gum was estimated to be approximately 14 % due to the release of moisture and volatile matter at 178 °C. Afterwards, a two-step decomposition process occurred, with a first-stage weight loss of 60 % occurring in the temperature range of 214–297 °C and a second-stage weight loss of 74 % occurring in the range of 326–550 °C. When PVA was heated to 288.5 °C, it lost 10.3 % of its weight, which indicates that the bound water had been lost. As the temperature increased, the weight of the PVA decreased further, and when the temperature reached 363.8 °C, the PVA weight was reduced by 50.2 %, demonstrating that the PVA had been continuously degraded with increased temperature. The honeysuckle loaded XG/starch-based (PVA-co-AA) hydrogels showed an initial weight loss of approximately 4 % at 203 °C. This may be explained by the loss of hydroxyl groups and the removal of absorbed and bonded moisture. Approximately 18 % of the weight was lost during the first stage of decomposition at 316 °C. Afterwards, there was a further decomposition of approximately 54 % weight loss at 553 °C, which resulted from fragmentation of side chains and the polymer infrastructure, producing carbonaceous residues. As a result of chemical and physical interactions between different polymer constituents, hydrogel shows a lower thermal decomposition rate than starch, xanthan gum, and PVA. As a result of the formation of a dense cross-linked structure within the hydrogel, its resistance to thermal decomposition was enhanced (Lemos et al., 2021). Decomposition of the hydrogel generally occurred at a higher temperature than the decomposition of its basic components. As a result of the above discussion, it was concluded that the cross-linked hydrogel is more thermally stable than pure polymers (Fig. 5C). We found that XG/starch-based hydrogels (PVA-co-AA) were comparable in thermal stability to xanthan gum-based hydrogels previously reported in the literature (Sethi et al., 2020).

3.4.3 X-ray diffraction analysis

XRD analysis was used to evaluate the physical properties of xanthan gum, starch, PVA, and the developed hydrogel, as shown in Fig. 5D. Compounds that exhibit sharp and highly intense peaks are usually described as having a crystalline nature, while compounds exhibiting diffuse peaks are described as having an amorphous nature. Xanthan gum exhibited a broad and amorphous peak at 2θ = 20.50°. Due to its amorphous nature, xanthan gum aggregates with other side chains, making packaging polymers difficult. The XRD analysis of starch revealed both crystalline and amorphous peaks at 2θ = 20.48°, and 24.1°, indicating that the starch possesses a semi-crystalline structure. PVA is a semicrystalline material with hydrogen bonds between its OH groups that enable it to interlink its chains. XRD analysis of PVA revealed prominent peaks at 2θ = 20° and 39.3°. XRD analysis of honeysuckle revealed prominent peaks at 2θ = 23.5° and 26.02°. XRD analysis of the developed hydrogel matrix indicated that the crystalline and intense peaks of xanthan gum, PVA and starch had been reduced or disappeared as a result of crosslinking and grafting. The results demonstrate that crosslinking, grafting, and polymerization of hydrogel contents reduced crystallinity of pure reagents and replaced sharp peaks with low-intensity peaks, showing successful crosslinking, grafting, and polymerization. Our findings are consistent with those reported in the literature, which also demonstrated that fabricated hydrogels have a decreased crystallinity (Luo et al., 2021).

3.4.4 Polymer volume fraction

Table 1 shows the polymer volume fraction determined at pH 7.4 for the developed hydrogels. A significant effect was observed in the polymer volume fraction due to the contents of the hydrogel, such as xanthan gum, starch, PVA, and EGDMA. The volume fraction of polymers decreased with increasing polymer composition. I.e., xanthan gum, PVA, and starch. Due to high concentrations of monomers and polymers, the hydrogel swells significantly, which is the main cause of this phenomenon. In contrast to the polymer and monomers, with the increase in composition of EGDMA, the polymer volume fraction increased due to low swelling in hydrogels. The high swelling index of the hydrogel was indicated by low and high polymer volumes.

3.4.5 Scanning electron microscopy

SEM was used to examine the morphology and surface properties of the synthesized hydrogels, as illustrated in Fig. 6 (A, B, and C). The SEM analysis of the hydrogel formulation revealed an irregular porous surface with a hard texture. This is due to the partial collapse of gel during the process of dehydration, which is evident from the presence of large cracks and wrinkles on the surface. Hydrogels with hard and porous surfaces demonstrate strong interactions between their components after polymerization.

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Fig. 6

Scanning electron microscopic images of the developed hydrogels at 200 X (A), 400 X (B), and 800 X(C). The effect of the concentration of the feeding components on the porosity and sol–gel fraction, such as PVA (D), Starch (E) and crosslinker EGDMA (F).

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3.4.6 Sol gel analysis

Sol represents the uncrosslinked soluble fraction of hydrogel, while gel represents the crosslinked insoluble fraction. Since the gel and sol fractions have an inverse relationship, an increase in one causes a decrease in the other, as shown in Fig. 6 D–F. The concentrations of different hydrogel contents, such as PVA, starch, and EGDMA, had a significant impact on both the sol and gel fractions. By increasing the feed ratio of PVA, there was an increase in the gel fraction. This result could be attributed to the increase in the number of free radicals generated by increasing PVA feed ratios, which were highly reactive. As a result, a higher concentration of PVA will result in more free radicals being generated for polymerization and cross-linking of hydrogel contents, resulting in a greater content of gel. In contrast to PVA, the gel fraction decreased with the increase in starch content and had an unfavorable effect on gelation. Hydrogel bulk and crosslinking densities increased as the EGDMA feed ratio increased. As a result of the high crosslinking of the hydrogel, the pore size decreased, and the gel fraction increased. A higher polymer, monomer, and crosslinker feed ratio was associated with an increase in gel fraction, as stated by Khanum and her colleagues (Khanum et al., 2018). This finding supports our hypothesis. The sol fraction decreased with an increase in PVA feed ratio, unlike the gel fraction and vice versa for EGDMA.

3.4.7 Porosity study

The porosity of all formulations of Xanthan gum/starch-based (PVA-co-AA) hydrogels was evaluated. In the present study, the primary objective was to assess the ability of fluids to penetrate the pores of hydrogel networks. The porosity of the reaction mixture was affected by the various combinations of hydrogel contents. Fig. 6D and 6F indicate that porosity increased with increasing PVA composition. This may be caused by the highly viscous nature of the reaction mixture formed after the hydrogel materials were polymerized. Increasing the concentration of PVA in the mixture resulted in the formation of a highly viscous mixture, which prevented bubbles from evaporating. As a result, interconnected channels were formed, increasing the porosity of the hydrogel. Porosity decreased as the feed ratio of EGDMA and starch was increased, contrary to what was observed for the PVA. As the concentration of EGDMA increased, porosity decreased as a result of the formation of high crosslinked bulk networks of hydrogel, which reduced the size of the pores in the developed hydrogel. Thus, there were fewer channels, resulting in lower penetration and porosity. In conclusion, higher porosity results in greater swelling and higher drug loading.

3.4.8 Swelling study

As shown in Fig. 7 (A and C), swelling studies were conducted on xanthan gum/starch-based (PVA-co-AA) hydrogels to determine their response to pH levels of 1.2, and 7.4. Swelling was almost negligible at pH 1.2 in comparison with pH 7, which indicates the hydrogel's pH sensitivity. Protonation and deprotonation of the –COOH groups of the polymers and monomers are responsible for the low and high swelling of the prepared hydrogel. When –COOH groups were protonated, conjugates were generated with counterions by strong hydrogen bonding, causing the charge density of the –COOH groups to decrease, resulting in a reduction in pore size and low swelling at pH 1.2. When the pH was increased from 1.2 to 7.4, there was a noticeable difference in the swelling of the synthesized hydrogel. During alkaline conditions, carboxyl groups ionize, which results in electrostatic attraction between –COO- groups, thus forming loose network structures that are able to accommodate a large volume of water. Moreover, polysaccharides contain hydroxyl groups that may also deprotonate in alkaline conditions, resulting in greater swelling (Hua et al., 2020). Therefore, fabricated hydrogels showed swelling in the order of pH 7.4 > 1.2. Different combinations of xanthan gum, starch, PVA, and EGDMA affected the swelling of formulated hydrogels. An increase in the polymer and crosslinker content resulted in an increase in swelling. Initial increases in xanthan gum concentration resulted in an increase in swelling ratio due to its hydrophilicity and anionic properties, but the swelling ratio decreased as xanthan gum concentration was increased further. As the concentration of xanthan gum is increased, the amount of network space that is available for water is reduced, thus decreasing the swelling capacity of the formulation. A reduction in swelling of hydrogel was observed with the increasing concentration of EGDMA, contrary to what was observed for the polymers (Thombare et al., 2018). The polymeric hydrogel's pore size decreased as the crosslinking and bulk density were increased, resulting in a decrease in the penetration of water into the formulated hydrogel. With an increase in composition, EGDMA developed a harder network and had a decrease in water penetration, which led to a decrease in swelling as the composition increased. Other polysaccharide hydrogels showed similar results in alkaline media (Gholamali 2021).

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Fig. 7

PXS hydrogels swelling at pH 1.2 (A), drug release at pH 1.2 (B), swelling at pH 7.4 (C), and drug release at pH 7.4 (D).

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3.4.9 Drug release and kinetics modelling

In vitro drug release studies were conducted using a xanthan gum/starch-based (PVA-co-AA) hydrogel at two pH values, namely pH 1.2 and pH 7.4 (Fig. 7B and D). The maximum amount of drug was released at pH 7.4 compared to pH 1.2, which was attributed to the pH-sensitive swelling behavior described above. There was also protonation and deprotonation of COOH groups of monomers, resulting in low and high levels of drug release from the fabricated hydrogel. At pH 7.4, deprotonation increased the charge density of COOH groups substantially (Cortes et al., 2020). At pH 7,4 due to the high charge density, the attractive force between COOH groups decreased and the repulsive force increased, resulting in increased swelling and drug release. The protonation of monomers at pH 1.2, however, led to the formation of conjugates with counterions, leading to the strengthening of the polymeric structure of the hydrogel through hydrogen bonding. The strong hydrogen bonding resulted in a low amount of swelling, resulting in a low amount of drug release. In a similar manner to swelling, drug release was also affected by the combination of polymers and crosslinkers. The incorporation of xanthan gum, starch, and PVA increased the rate of drug release. The release of drugs decreased as the composition of EGDMA increased. The reason for this is the crosslinking and hardness of the hydrogel, which both inhibit water penetration through the small pores, leading to a decrease in the release of the drug (Akhtar et al., 2015). Our results demonstrate that hydrogel remains protonated/unionized at low pH values of 1.2 as a result of the pKa values of its reagents, resulting in low swelling and drug release. Deprotonation and ionization of the polymer and monomer functional groups occurred as the pH increased from 1.2 to 7.4, causing the hydrogel networks to expand, leading to an increase in swelling and drug release.

The best fit release kinetic model was determined based on in vitro drug release data for all formulations by modeling zero order, first order, Higuchi, and Korsmeyer-Peppas models (Table 3). According to the r² and n values, the best-fit model and drug release mechanism are confirmed. The “r²″ value indicates the regression coefficient. The ”r²″ values of Korsmeyer-Peppas model for all formulations of the fabricated hydrogel are closer to 1 compared with other kinetic models, which indicates that this was the best fit model. Furthermore, the type of diffusion is determined by the value of “n”. Whenever “n” > 0.45, the diffusion is non-Fickian, while when “n” ≤ 0.45, the diffusion is Fickian. The Fickian diffusion was observed in all formulations with n values ranging from 0.084 to 0.222 (Emam and Shaheen 2022).