Original Article

Preparation of Sterculia foetida-pullulan-Based Semi-interpenetrating Polymer Network Gastroretentive Microspheres of Amoxicillin Trihydrate and Optimization by Response Surface Methodology


  • Jayshri HADKE
  • Shagufta KHAN

Received Date: 17.11.2019 Accepted Date: 14.01.2020 Turk J Pharm Sci 2021;18(4):388-397 PMID: 34496478


In this study, a novel Sterculia foetida and pullulan-based semi-interpenetrating polymer network gastroretentive microsphere formulation was prepared using the emulsion crosslinking method and optimized by central composite design.

Materials and Methods:

The effects of the amounts of glutaraldehyde, S. foetida, and pullulan on the percent drug entrapment efficiency (EE), percent mucoadhesion at 12 h, and percent in vitro drug release at 12 h were optimized. The microspheres were characterized using scanning electron microscopy, fourier transform infrared spectroscopy, and differential scanning calorimetry.


The formulation containing 4% v/v glutaraldehyde, 8.28% w/v pullulan, and 2.14% w/v S. foetida had 88.75±1.18% EE, 80.43±1.2% drug release at 12 h, and 81.73±1.50% mucoadhesion at 12 h, which was considered optimum and was used in an in vivo radiographic study.


Semi-interpenetrating polymer network microspheres loaded with amoxicillin trihydrate were successfully prepared using S. foetida and pullulan. The prolonged retention of microspheres in the stomach with sustained drug release could effectively act against Helicobacter pylori reservoirs in the stomach and improve the therapeutic effect of amoxicillin trihydrate against H. pylori.

Keywords: Sterculia foetida, pullulan, semi-interpenetrating polymeric network, central composite design, in vivo radiographic study


Several disease conditions necessitate the development of controlled and targeted drug delivery systems. Pharmaceutical technology is continuously expanding to provide sustainable, continuous, and therapy-optimal drug levels in the circulation. Drugs whose absorption site is the upper gastrointestinal tract or those that have stability issues can benefit when formulated as mucoadhesive microspheres.1 Mucoadhesive microspheres can adhere in a specific absorptive part of the gastrointestinal region, thereby improving the drug absorption rate and enhancing bioavailability. Additionally, the microsphere size allows them to remain entrapped in the stomach mucosal lining for an extended duration.

Due to their gastroretention, mucoadhesive microspheres may build up a high drug concentration in the stomach mucosa, which may help eradicate peptic ulcers caused by Helicobacter pylori infection.2 Warren and Marshall indicated that H. pylori infection predominantly affects the gastrointestinal tract, causing stomach inflammation, duodenal ulcers, gastric ulcers, and gastric carcinoma. It is reported that H. pylori is responsible for 90-100% of duodenal ulcers and 60-90% of gastric ulcers in patients. A literature survey showed that H. pylori infection increases the likelihood of developing peptic ulcers, and 10-15% of individuals with H. pylori infection have high chances of developing peptic ulcers in their lifetime.3

Amoxicillin trihydrate is a semisynthetic antibiotic widely considered effective in the treatment of peptic ulcers caused by H. pylori. It is a major component of standard therapy and is used in combination with other antibiotics and antacids. Clinically, it is effective, but drug-related problems and increased antibiotic resistance have decreased the success rate of eradicating H. pylori with conventional therapies. Amoxicillin, an acid-soluble drug, dissolves immediately in the stomach and is rapidly absorbed; therefore, it is difficult to retain it in the stomach for long durations unless some rate-controlling means are employed to counter H. pylori. Once H. pylori is colonized in an acidic stomach environment, it penetrates the stomach’s deeper mucus membrane, which is a bacterium niche, owing to its good flagellar motility. Furthermore, H. pylori can attach to lipids and carbohydrates present in the membrane, helping it adhere to the epithelial cells. Several studies and reviews recommended that local drug delivery works more effectively than systemically administered antibiotics to eradicate H. pylori. Therefore, for enhanced therapeutic efficacy, the formulation should release the drug near the stomach lining. Mucoadhesive microspheres can adhere to the gastric mucosa and release the drug constantly for longer periods to the infected cells. Therefore, they can be proven to be very effective against H. pylori.4,5

Natural hydrophilic polymers possess structural flexibility; thus, they can be applied in the preparation of oral controlled-release formulations that provide the required drug release profile.6 Sterculia foetida is a medicinally important polysaccharide obtained in the form of exudates from Sterculia urens, a small-to-medium-sized bushy tree that belongs to the Sterculiaceae family. It fixes itself with water molecules, although not in a completely soluble form. It can swell, increasing its total volume in relation to dry mass, which is 60 times more than the original volume. It possesses a rhamnogalacturonan-type partially acetylated ramified structure, and its molecular weight is 16x103 kDa. Chemically, it is composed of 55-60% unbiased monosaccharide units (galactose and rhamnose), 8% acetyl groups, and 37-0% acid residues (galacturonic and glucuronic acids). The core part of its structure is α-D-galacturonic acid and L-rhamnose units.7 S. foetida has several potential commercial applications, primarily in the food and pharmaceutical industries. It is edible, odorless, and flavorless; thus, it is used as a food additive. As a result of its bioadhesive, non-toxic, non-immunogenic, non-mutagenic, and non-carcinogenic properties,8,9 it is considered promising for the development of drug-loaded microspheres. Moreover, S. foetida exhibits swelling properties and can modify drug release from the polymeric matrix.10 In a previous study, S. foetida was blended with pectin to produce a sustained-release carrier having floating ability and increasing gastric residence.11

Currently, for the pharmaceutical industry, S. foetida is a promising plant polysaccharide. It exhibits acid stability due to acetyl groups in its structure; it also possesses good swelling capacity and good mucoadhesive and biodegradable properties. Various studies have reported the use of S. foetida in the treatment of ulcers, irritable bowel syndrome, persistent colonic diseases, and improving glucose metabolism without adverse effects on mineral balance. Furthermore, many researchers have formulated interpenetrating polymer network (IPN) microspheres using polysaccharides and have reported their use for sustained drug delivery.12

Recently, increasing attention has been given to polysaccharides obtained from microorganisms and yeast. Pullulan is a neutral homopolysaccharide of glucose composed of repeating maltotriose residues connected by α-1.6 glycosidic bonds and some interspersed maltotetraose units produced by Aureobasidium pullulans. Its additional advantages include its biocompatibility, non-toxicity, non-immunogenicity, and non-carcinogenicity. Pullulan is also used as a binder, thickener, lubricant, and gelling agent. Because of its unique linkage pattern, it can capture biomolecules and has exceptional oxygen barrier properties, which increase the stability of these molecules with increased shelf-life. Pullulan is highly soluble in water; thus, it can be used as a drug carrier; however, it involves uncontrolled hydration and drug release-related issues, restricting its applications. The hydrophilicity of pullulan can be reduced by crosslinking it with an anionic polymer such as S. foetida. In addition, pullulan can be easily crosslinked using glutaraldehyde, and adding S. foetida increases its mechanical strength.13

The number of naturally occurring polysaccharides through the formation of IPNs improves mechanical strength and provides the desired release characteristics of the entrapped drug. Several IPNs have been reported thus far and include interactions between Delonix regia gum and sodium alginate,14 calcium pectinate and tamarind seed gum,15 carrageenan-alginate,16 locust bean gum and alginate,17 guar gum and chitosan,18 and carboxymethyl xanthan and alginate.19 However, no attempt has been made to formulate semi-IPN microspheres composed of pullulan with S. foetida. Therefore, in this study, S. foetida-pullulan-based semi-IPN microspheres were formulated for stomach-specific delivery of amoxicillin trihydrate that can provide good mechanical strength, prevent uncontrolled water uptake, and remain in the stomach to provide drug release over long periods for activity against H. pylori. It is proposed that S. foetida has antiulcer properties, which could act synergistically against H. pylori with amoxicillin trihydrate.


Experimental part


Amoxicillin trihydrate was gifted by Ranbaxy Laboratories (Goregaon East, Mumbai, India). Pullulan, having a molecular weight of 200,000 Da, was procured from Kumar Organic Products Limited (Bangalore, India). S. foetida was gifted by Research Lab Fine Chem Industries (Mumbai, India); light liquid paraffin (LLP) was supplied by Hi-Media Laboratories Private Limited (Mumbai, India). Pioneer In-Organics (Delhi, India) supplied Span 80. Glutaraldehyde (25%, v/v) was procured from Merck Limited (Mumbai, India). Dichloromethane was obtained from Loba Chem. Private Limited (Mumbai, India). All chemicals used were of analytical grade.

Preparation of S. foetida–pullulan semi-IPN microspheres

The water-in-oil emulsification-crosslinking method was used to prepare S. foetida-pullulan-based semi-IPN microspheres containing amoxicillin trihydrate (ASP-MPs).20 S. foetida and pullulan were hydrated in distilled water. First, the two polymeric solutions were homogenized together to obtain a uniform solution. The drug was incorporated into this polymeric solution, and the solution was uniformly mixed using a magnetic stirrer until a uniform drug-polymer mixture was obtained. Next, the drug-containing polymeric mixture with 1% w/v Span 80 was added gradually to LLP with constant stirring at 2.000 rpm for 10 min. After the formation of a a milky white emulsion, glutaraldehyde containing 0.5 mL of 1 N HCl was added gradually, and the resulting emulsion was stirred continuously for 2 h to obtain solid microspheres. The microspheres were then filtered and washed with dichloromethane to remove excess LLP and washed several times with water to remove unreacted glutaraldehyde. The resulting microspheres were then allowed to dry under vacuum at 40°C, and finally, they were stored in desiccators until further use.

Experimental design for optimizing the ASP-MP formulation

ASP-MPs were prepared and optimized using three factors and a three-level randomized central composite design to determine the effect of formulation variables on the prepared microspheres. The three independent variables, i.e., the volume of crosslinker glutaraldehyde (A), amount of pullulan (B), and amount of S. foetida (C), were varied at three levels (Table 1). The statistical trial design was generated and evaluated using Design-Expert version software (Stat-Ease Inc., USA). Percent drug entrapment efficiency (DEE), cumulative percent drug release for 12 h, and percent mucoadhesion were selected as dependent variables. Design matrices showing the effect of the formulation variables on the studied response variables are shown in Table 2, and response surface plots are shown in Figure 1a, b, c. Optimization was performed by modeling the effect of independent variables on the responses using a quadratic mathematical model, as shown in equation 1, and constraints on responses were maximum EE, maximum drug content, and maximum mucoadhesion.21,22

Yo = bo + b1A + b2B + b3C + b12AB + b13AC + b23BC + b11A2 + b22B2 + b33C2                         equation   (1)

where Y is the response; b0 is the intercept; and A, B, and C are the independent formulation variables.

Characterization of ASP-MPs

Fourier-transform infrared spectroscopy (FTIR)

FTIR spectra of S. foetida gum, pullulan, amoxicillin trihydrate, and a physical mixture of S. foetida and pullulan with amoxicillin trihydrate and ASP-MPs were obtained between 4.000 and 400 cm-1 using KBr pellets (FTIR-84005, Shimadzu Asia Pacific Pvt. Ltd. Singapore) (Figure 2).

Differential scanning calorimetry (DSC)

The thermal and crystalline properties of S. foetida gum, pullulan, and a physical mixture of S. foetida and pullulan with amoxicillin trihydrate and ASP-MPs were examined using DSC (Mettler Toledo AG, Analytical, Switzerland) at a heating rate of 10°C/min from 0°C to 400°C with continuous nitrogen purge at a rate of 50 mL/min (Figure 3).

X-ray diffraction (XRD)

XRD (Bruker model D8 advance theta/2theta) was conducted to study the crystalline properties of amoxicillin trihydrate upon entrapment in the microspheres. The radiation source was CuKα, at k¼1.5406 Å. The samples were spread over a low background sample holder (amorphous silica) and set in a goniometer. Scanning was performed at a scan speed of 5°/min with increments at 0.02 and 2Ɵ from 0° to 80°. The current and voltages were 40 mV and 35 mA, respectively (Figure 4).

Determination of particle size and surface morphology

The required quantity of microspheres was distributed in an insoluble solvent, and the suspension was diluted as needed. One drop of the suspension was placed on a glass slide and examined under a microscope; a digital picture was also taken (DMWBf model, Motic, China). The sample was placed on a piece of adhesive carbon tape set on a brass stub for scanning electron microscopy (SEM). The sample was then gold-coated using a sputtering unit (model: JFC1600, USA) for 10 s at 10 mA. Finally, the gold-coated sample was placed in an SEM chamber (Jeol, JSM 6390LA), and secondary electron images were recorded (Figure 5).

Determination of percent drug loading and percent DEE

Fifty milligrams of microspheres were finely powdered and soaked in 50 mL of simulated gastrointestinal fluid [(SGF), pH 1.2] for 24 h. The resulting suspension was filtered, suitably diluted, and analyzed at 229 nm. Percent drug loading and DEE were then determined according to the following relationship:23

% Drug loading=Amount of drug in microspheresAmount of microspheres ×100                  equation (2)

DEE (%)=Actual drug content in microspheresTheoretical drug content in microspheres ×100                  equation (3)

Evaluatin of mucoadhesive properties in vitro

Goat intestinal mucosa was used to study the mucoadhesive properties of semi-IPN microspheres. First, a dispersion of semi-IPN microspheres was sprayed on goat mucosa mounted on a glass slide. The glass slide was previously incubated in desiccators (90% relative humidity) for 20 min and then placed in the cell. Finally, the cell was attached to the outer assembly at an angle of 45°. Next, SGF (pH 1.2) was circulated through the cell over the microspheres at a rate of 1 mL/min using a peristaltic pump. The washings were collected after fixed time intervals, and centrifuged and separated microspheres were dried at 40°C.

The weight of the washed microspheres was measured, and mucoadhesion (%) was calculated as follows:23

Percentage mucoadhesion=Qa-Ql × 100/Qa             equation   (4)

where Qa=weight of microspheres applied initially and Ql=weight of microspheres leached out.

In vitro drug release study

A type I USP dissolution apparatus (Electrolab, Mehsana, Gujrat, India) was used to measure the drug release in SGF (pH 1.2, 900 mL) at 37°C±0.5°C with a rotational speed of 100 rpm. Microspheres containing 500 mg of drug were placed in SGF. Every hour, 1 mL of the sample was suitably diluted and analyzed at 229 nm (Shimadzu/UV-1700, Japan). The aliquots withdrawn were replaced with drug-free buffer. Dissolution testing was performed for 12 h (Figure 6). Kinetic models such as the zero-order,24 first-order,25 Higuchi,26 and Peppas27 were used to explore the kinetics and mechanics of drug release.

In vivo radiographic study

To determine the location and extent of gastrointestinal transit, ASP-MPs were administered orally to rats and monitored using a radiological method. The Institutional Animal Ethics Committee approved the study (protocol no: 535/02/a/CPCSEA/S/Jan.2002). Six healthy albino Wistar rats (either sex, 400-500 g) were kept in individual cages in a sanitized room maintained at 27°C±0.5°C at a 12 h light/dark cycle. Food was abstained for 12 h prior to the study, whereas water was allowed ad libitum. Radiopaque ASP-MPs were prepared by adding 50 mg of barium sulfate to the polymeric solution (the composition was the same as that of the optimized formulation) following the procedure used in the formulation of ASP-MPs. ASP-MPs were administered orally through the gastric tube along with 2 mL of water. The animals were subjected to fasting throughout the study (up to 12 h) with 1  mL of water allowed every hour. ASP-MPs were monitored in the gastric region of the rats at a predetermined time interval of 10 h using X-ray imaging (Siregraph-B, Siemens, Germany) (Figure 7).28

Statistical analysis

Statistical optimization of the formulations was conducted using Design-Expert® version (Stat-Ease Inc.). Each measurement was performed in triplicate (n=3) and represented as mean ± standard deviation. Student’s t-test was performed to compare the two groups.29,30

Stability testing

Stability testing of the optimized formulation was performed according to the ICH guidelines for 6 months.31 The formulation was packed in laminated aluminum foil and placed in a stability chamber at 40°C±2°C and 75±5% relative humidity. The samples were examined at the end of 1, 3, and 6 months to check for significant differences in their mucoadhesion property, EE, and drug content. Differences were regarded as significant at p<0.05.


Formulation of microspheres

In this study, ASP-MPs containing amoxicillin trihydrate were prepared using glutaraldehyde as a crosslinking agent. Pullulan is a highly water-soluble polymer; therefore, when it is used alone, it exhibits uncontrolled hydration, leading to instant release of high amounts of the drug. Therefore, to control hydration, S. foetida was coupled on a pullulan backbone with the help of glutaraldehyde. Water-soluble glutaraldehyde was selected as the crosslinking agent because of its ability to facilitate the formation of linkages from hydroxyl groups. The -CHO groups of glutaraldehyde (covalent crosslinker) react with the -OH groups of S. foetida, forming an acetal linkage that creates a three-dimensional IPN structure (Figure 2). The obtained microspheres were water-insoluble and had a three-dimensional network with amoxicillin entrapped in them. In an investigation conducted due to toxicity concerns of glutaraldehyde as a crosslinker, flavonoid-like rutin was used to crosslink gelatin microspheres, and a comparison was performed with glutaraldehyde. However, the crosslinking efficiency of rutin was low, and the glutaraldehyde-crosslinked microspheres were as safe as rutin-crosslinked microspheres.32 The microspheres were washed several times with water to remove unreacted glutaraldehyde.

Influence of formulation composition on percent DEE (Y1)

The percent DEE increased from 60.37±1.85% to 88.24±2.46% as the concentration of polymers increased. From the response surface curve, it was determined that the encapsulation efficiency of the drug increased with an increase in the polymer concentration. The polynomial equation correlating percent DEE with independent variables is as follows.

EF(Y1)= +82.11-6.30*A+5.88*B+3.81*C-0.3525*AB+0.7350*AC-1.08*BC-2.36*A2 -2.46*B2-1.48*C2                               equation (5)

where A is the volume of glutaraldehyde and B and C are the amounts of pullulan and S. foetida, respectively. The randomized central composite design showed a good fit. A high f value of 28.94 and a p value of <0.0500 indicated that the model was significant.

The influence of independent formulation variables on EE is depicted using response surface plots is shown in Figure 1a. The EE increased, possibly due to the high viscosity of the polymer blend, consequently reducing drug seepage during microsphere preparation. Further, the high polymer concentration may have caused increased entrapment of the drug inside the S. foetida-pullulan network. However, the encapsulation efficiency decreased with an increase in the glutaraldehyde concentration; this decrease might be due to an increase in the crosslinking density of the polymer matrix. This caused the formation of a rigid structure with a consequent reduction in free volume within the polymer matrix for drug entrapment.33,34

Influence of formulation composition on in vitro mucoadhesion

The percent mucoadhesion in SGF of all batches was between 60.78±1.62% and 87.35±1.83%. As the polymer concentration increased, mucoadhesion increased. The polynomial equation correlating percent mucoadhesion with the independent variables is shown below:

Percent mucoadhesion= +76.072.81*A+6.59*B+3.86*C                  equation (6)

The model f value of 35.43 and p value of <0.05 indicated that the model was significant. The predicted R2 of 0.7856 was in agreement with the adjusted R2 of 0.8446 as the difference between them was <0.2. The influence of independent formulation variables on mucoadhesion (%) is depicted using response surface plots in Figure 1b. An increase in mucoadhesion with polymer concentration may have occurred due to the availability of more OH groups for hydrogen bond formation with the mucus membrane.29 Mucoadhesion decreases with an increase in glutaraldehyde levels. The decrease in mucoadhesion may be because of the formation of a strongly crosslinked polymeric matrix, causing decreased swelling and weaker interactions with the mucosal surface. The mucoadhesive properties of S. foetida are attributable to its capacity to form a mucoadhesive bond with mucin and interpenetration of the polymer chain in the interfacial area.2,33,34

Influence of formulation composition on percent drug release

All the formulations showed sustained release of amoxicillin over 12 h in SGF (pH 1.2). The cumulative drug release from semi-IPN microspheres after 12 h was between 60.32±2.09% and 89.41±1.99% (Table 2, Figure 6). The drug release rate decreased with an increase in polymer concentration. The correlation between drug release at 12 h and different independent variables is explained by the following polynomial equation:

Drug release= +82.15-6.18*A-4.17*B-4.53*C+1.30*AB+0.1912*AC-0.2513*BC-2.148A2-4.54*B2-3.23*C2                 equation (7)

The impact of formulation variables on percent drug release is depicted using surface response plots in Figure 1c. Increased S. foetida content in the blend membrane reduced the rate of drug release. The hydrophobicity of the microsphere matrix increased with S. foetida content due to an increase in ester linkages and methyl groups. Additionally, the reduction in the rate of drug release could be caused by a decrease in the molecular volume of S. foetida in the polymer matrix due to its decreased swelling and hydration capacity. Similarly, the drug release pattern was highly dependent on glutaraldehyde levels.33 When the glutaraldehyde volume was increased from 2 mL to 4 mL, percent drug release was decreased from 89.41±1.99% to 60.32±2.09% in 12 h. This behavior may be a result of fewer free spaces in the glutaraldehyde-treated IPN matrices for unhindered drug diffusion through the polymer network.34 The drug release followed Hixson-Crowell kinetics as it showed maximum R2 (0.9964). The drug release mechanism followed super case-II (n=1.33 of Korsmeyer-Peppas), i.e., diffusion and chain relaxation. There could be a dominant role of chain relaxation at later time points due to waning of crosslinks over time.

Optimization by desirability function

Optimization was perfomed based on the desirability function. Microspheres were prepared under the model-predicted optimal condition with glutaraldehyde 4% v/v, pullulan 8.28% w/v, and S. foetida 2.14% w/v. Optimization was performed by placing constraints on the dependent variables. The response variables of the formulated microspheres under optimized conditions were nearly equal (low percentage bias) to the predicted values, ensuring reliability of the model (Table 3).

Influence of formulation composition on particle size

The microspheres had sizes of 57.99±1.53-121.90±1.38 µm. The variation in particle size due to formulation variables indicates that microsphere size was dependent on the glutaraldehyde amount and polymer concentration. When the amount of glutaraldehyde increased, the particle size decreased. This may be the result of the increased crosslink density, which caused shrinking of the internal phase to a greater extent, creating a low void space. Alternatively, when the polymer ratio was increased at a given quantity of glutaraldehyde, the particle size also increased. This could be because of increased drug entrapment in the voids present in the polymeric network. Additionally, when the drug concentration was increased, it needed more space in the polymeric networks and voids, consequently increasing the size.

Characterization of ASP-MPs


Figure 2 depicts the FTIR spectrum of amoxicillin trihydrate, showing peaks at 1616.24 cm-1 for aromatic C=C stretching and 1685.67 cm-1 and 1577.66 cm-1 for C=O stretching and N-H deformation of amide, respectively. The peak at 1774.39 cm-1 was due to b-lactam C=O stretching, and the peaks at 2968.24 and 3039.60 cm-1 corresponded to C-H stretching for CH3 and aromatic C-H stretching, respectively. All the prominent peaks of amoxicillin trihydrate were retained in the spectrum of a physical mixture of S. foetida and pullulan, indicating no interaction of the drug with the polymer. The FTIR spectrum of ASP-MP formulation revealed a broad absorption band from 3527.56-1 to 3105 cm-1 due to the OH group of S. foetida and CHO group of glutaraldehyde, which proves the formation of glutaraldehyde-crosslinked semi-IPN microspheres. Furthermore, bands from 3475-1 to 3620 cm-1 due to O-H stretching in the FTIR spectrum of S. foetida had shifted to the lower wavenumber from 3423-1 to 3519 cm-1 in the FTIR spectrum of APS-MPs. This shift can be attributed to hydrogen bond formation between S. foetida and amoxicillin in the microspheres. A similar finding was reported by Üstündağ Okur et al.35


Thermograms of amoxicillin, pullulan, S. foetida gum, physical mixture of S. foetida-pullulan with amoxicillin trihydrate, and ASP-MPs are shown in Figure 3. The pure drug exhibited a characteristic broad peak starting from 60°C to 140°C due to trihydrate dehydration and a broad endothermic peak starting from 185°C to 210°C with a peak at 199°C due to melting. DSC of pullulan revealed an endothermic peak from 0°C to 110°C, whereas  S. foetida showed an endothermic peak from 0°C to 110°C due to dehydration. The endothermic peak of amoxicillin trihydrate due to the loss of water molecule is revealed in the DSC of the physical mixture, showing a lack of incompatibility. In contrast, the endothermic melting peak was diminished due to the masking of the drug in the polymers. The absence of a characteristic endothermic peak of amoxicillin in the ASP-MP thermogram might have accurred because of the entrapment of the drug in the microspheres, making it undetectable.


Sharp peaks with high intensity from 2ɵ 10°-30° in the amoxicillin trihydrate XRD pattern indicated crystalline properties. The characteristic peaks present in the drug from 2ɵ 15°-30° had broadened in microspheres, indicating a loss of crystallinity upon entrapment in microspheres (Figure 4).


SEM was performed to visualize the morphology of the ASP-MPs. The formed microspheres were slightly irregular and aggregated, with a rough surface (Figure 5). The rough surface might be due to the shrinkage of crosslinked Sterculia gum during drying of the IPN microspheres.

In vivo radiographic studies

X-ray images revealed the presence of microspheres in the stomach at 6 h after administration. The ASP-MPs were retained for an extended period of time due to mucoadhesion to the stomach mucosal lining (Figure 7).


Semi-IPN microspheres loaded with amoxicillin trihydrate were successfully prepared using S. foetida and pullulan by the emulsion crosslinking method using glutaraldehyde as the crosslinker. Microspheres prepared under optimized conditions (2.14% w/v S. foetida, 8.28% w/v pullulan, and 4% v/v glutaraldehyde) showed predicted values of EE, drug release, and mucoadhesion, with low percentage bias. Additionally, X-ray images revealed the presence of microspheres for 10 h. Thus, the prolonged retention of microspheres in the stomach with sustained drug release could effectively act against the H. pylori reservoir in the stomach and improve the therapeutic effectiveness of amoxicillin trihydrate against H. pylori.

Conflict of interest: No conflict of interest was declared by the authors. The authors are solely responsible for the content and writing of this paper.

  1. Benita S. Microencapsulation: methods and industrial applications. New York: Marcel Dekker Inc.;1996.
  2. Patil S, Talele GS. Gastroretentive mucoadhesive tablet of lafutidine for controlled release and enhanced bioavailability. Drug Deliv. 2015;22:312-319.
  3. Arora S, Bisen G,  Budhiraja RD. Mucoadhesive and muco-penetrating delivery for eradication of Helicobacter pyroli. Asian J Pharm. 2012;6:18-30.
  4. Patel J, Patel MM. Stomach specific anti-helicobacter pylori therapy: preparation and evaluation of amoxicillin-loaded chitosan mucoadhesive microspheres. Curr Drug Deliv. 2007;4:41-50.
  5. Liu Z, Lua W, Qian L, Zhang X, Zeng P, Pan J. In vitro and in vivo studies on mucoadhesive microspheres of amoxicillin. J Control Release. 2005:102:135-144.
  6. Rajinikanth PS, karunagaran LN, Balasubramaniam J, Mishra B. Formulation and evaluation of Clarithromycin microspheres for eradication of Helicobacter pylori. Chem Pharm Bull. 2008;56:1658-1664.
  7. Gabriel A, Janeth MV, Ana claudia LC, Alberto J. Karaya gum: general topics and applications, macromolecules. MMAIJ. 2013;9:111-116.
  8. Phillips GO, Williams PA, Handbook of hydrocolloids. In: Phillips GO, Williams Pai, eds. (2nd ed). Cambridge, England: Woodhead Publishing Limited; 2009.
  9. Anderson DMW, Wang WP. The tree exudate gums permitted in foodstuffs as emulsifiers, stabilisers and thickeners. Chem Ind For Prod. 1994;14:73-83.
  10. Singh B, Sharma N. Design of sterculia gum based double potential antidiarrheal drug delivery system. Colloids Surf B Biointerfaces. 2011;82:325-332.
  11. Bera H, Boddupalli S, Nayak A. Mucoadhesive-floating zinc-pectinate-sterculia gum interpenetrating polymer network beads encapsulating ziprasidone HCl, Carbohydr Polym. 2015;131:108-118.
  12. Prajapati VD, Jani GK, Khanda SM. Pullulan: an exopolysaccharide and its various applications. Carbohydr Polym. 2013;5:95:540-549.
  13. Yua L, Deana K, Li L. Polymer blends and composites from renewable resources. Prog Polym Sci. 2006;31:576-602.
  14. Dias RJ, Ghorpade VS, Havaldar VD, Mali KK, Hindurao N. Development and optimization of interpenetrating network beads of Delonix regia gum and sodium alginate using response surface methodology. J Appl Pharm Sci. 2015;5:56-64.
  15. Nakay AK, Hasnain MS, Pal D. Gelled microspheres/Beads of sterculia gum and tamarind gum for sustained drug release. Polymer Gels. 2018:361-415.
  16. Mohamadnia Z, Zohuriaan Mehr MJ, Kabiri K. pH-Sensitive IPN hydrogel beads of carrageenan-Alginate for controlled drug delivery. J Bioact Compat Polym. 2007;22:342-356.
  17. Prajapati VD, Jani GK, Moradiya NG, Randeria NP, Maheriya PM, Nagar BJ. Locust bean gum in the development of sustained release mucoadhesive macromolecules of aceclofenac. Carbohydr Polym. 2014;113:138-148.
  18. Mallikarjuna Reddy K, Ramesh Babu V, Sairam M, Subha MCS, Mallikarjuna NN, Kulkarni PV, Aminabhavi TM. Development of chitosan-guar gum semiinterpenetrating polymer network microspheres for controlled release of cefadroxil. Des Monomers Polym. 2006;9:491-501.
  19. Ray S, Banerjee S, Maitis S, Laha B, Barik S, Sa B, Bhattacharyya UK. Novel interpenetrating network microspheres of xanthan gum-poly (vinyl alcohol) for the delivery of diclofenac sodium to the intestine-in vitro and in vivo evaluation in Drug Deliv. 2010;17:508-519.
  20. Kulkarni RV, Mutalik S, Mangond BS, Nayak UY. Novel interpenetrated polymer network microbeads of natural polysaccharides for modified release of water soluble drug: in-vitro and in-vivo evaluation. J Pharm Pharmacol. 2012;64:530-540.
  21. Nayak AK, Pal D, Santra K. Development of calcium pectinate-tamarind seed polysaccharide mucoadhesive beads containing metformin HCl. Carbohydr Polym. 2014;101:220-230.
  22. Hooda A, Nanda A, Jain M, Kumar V, Rathee P. Optimization and evaluation of gastroretentive ranitidine HCl microspheres by using design expert software. Int J Biol Macromol. 2012;51:691-700.
  23. Venkateswaramurthy N, Sambathkumar R, Perumal P. Design development and evaluation of amoxicillin trihydrate mucoadhesive microspheres for Helicobactor pylori eradication therapy. Int J Appl Pharm. 2010:2:23-25.
  24. Costa P, Sousa LJM, Modelling and comparison of dissolution profiles. Eur J Pharm Sci. 2001;13:123-133.
  25. Costa FO, Sousa JJ, Pais AA, Fornosinho SJ. Comparison of dissolution profile of ibuprofen pellets. J Control Release. 2003:89:199-212.
  26. Higuchi T. Mechanism of sustained action medication theoretical analysis of rate of release of solid drugs dispersed in solid matrices. J Pharm Sci. 1963;52:1145-1149.
  27. Peppas NA. Analysis of fickian and non-fickian drug release from polymers. Pharm Acta Helv. 1985;60:110-111.
  28. Jain A, Pandey V, Ganeshpurkar A, Dubey N, Bansal D. Formulation and characterization of floating microballoons of Nizatidine for effective treatment of gastric ulcers in murine model. Drug Deliv. 2015;22:306-311.
  29. Montgomery DC. Design and Analysis of Experiments. (50th ed). New York: Wiley; 2001.
  30. Montgomery DC. Introduction to Statistical Quality Control. (40th ed). New Delhi: Wiley India (P) Ltd; 2008.
  31. Srivastava AK, Wadhwa S, Ridhurkar D, Mishra B. Oral sustained delivery of atenolol from floating matrix tablets-formulation and in vitro evaluation. Drug Dev Ind Pharm. 2005;31:367-374.
  32. Fabiana G, Thalita MC, Camila AO, Daniela DP, Michele GI, Joana M, Catarina R, Vladi OC, Telma MK, Maria VR, André R. Gelatin-based microspheres crosslinked with glutaraldehyde and rutin oriented to cosmetics. Brazil J Pharm Sci. 2016;52:603-612.
  33. Raizaday A, Yadav HK, Kumar SH, Kasina S, Navya M, Tashi C. Development of pH sensitive microparticles of Karaya gum: by response surface methodology. Carbohydr Polym. 2015;134:353-363.
  34. Guruguntla N, Palla KB, Yeggada M, Areti P, Chintha M. Interpenetrating polymer network hydrogel membranes of Karaya gum and sodium alginate for controlled release of Flutamide Drug, J Appl Pharm Sc. 2016;6:011-019.
  35. Üstündağ Okur N, Hökenek N, Okur ME, Ayla Ş, Yoltaş A, Siafaka PI, Cevher E. An alternative approach to wound healing field; new composite films from natural polymers for mupirocin dermal delivery. Saudi Pharm J. 2019;27:738-752.