Formulation and characterization of zaleplon buccal disks using grafted Moringa oleifera gum

Characterization of ZLP

Drug identification by Fourier transformed infrared (FTIR)

The comparison between the obtained drug sample’s FTIR spectrum with the standard FTIR spectrum of the pure drug and excipients used was performed. The ingredients were analyzed for the interactions and compared with the reported literature (Ahuja et al., 2010).

Differential scanning calorimeter (DSC) study of the drug

The drug’s thermograms were determined by using a DSC (Perkin Elmer). In a standard aluminum pan, a few mg of the finely powdered sample was sealed and heated at 10°C/minute between 40°C and 350°C. The experiment was carried out in a nitrogen environment, with a flow rate of 50 ml/minute (Ahuja et al., 2010).

X-ray diffractometer (XRD) of the drug

Powder XRD studies were used to characterize the drug’s solid state. The diffraction pattern was traced using an XRD (Xpert PRO) at room temperature from 0° to 80° (2?) diffraction angle with a scanning speed of 0.05 minutes⁻¹ (Ahuja et al., 2010).

Scanning electron microscopy (SEM) of the drug

SEM (ZEISS, EVO 18, China) was used to examine the drug’s surface morphology. The powdered sample, under a high vacuum, was mounted on double-sided adhesive carbon tape and sputter-coated with a thin film of gold. At various magnifications, the photomicrographs were taken with a 20 kV accelerating voltage (Ahuja et al., 2010).

Modification of Moringa gum by the grafting technique

Microwave-assisted synthesis of polyacrylamide grafted Moringa gum using CAN as a free radical initiator

1 g of Moringa gum was dissolved in 40 ml distilled water. In 10 ml distilled water, an appropriate amount (5 ml) of acrylamide was dissolved and added to the above solution. They were thoroughly mixed before being transferred to the reaction vessel (250 ml beaker), where a catalytic amount of CAN (0.1 g w/w) was added. The reaction vessel was placed on the turntable of a domestic microwave oven (Samsung, MW71E, India), and microwave irradiation was performed at 800 W power. The microwave irradiation was stopped at one set of boiling (~65°C), and cooled by immersing the reaction vessel in cold water on a regular basis (Salaheldeen et al., 2014).

Figure 1. Chemical structure of M. oleifera gum. [Click here to view]

The microwave irradiation cooling cycle was repeated until a gel-like mass was formed, or until a total of 3 minutes of irradiation time was used (in the absence of gelling). The reaction vessel and its contents were then cooled and left alone to allow the grafting reaction to complete. Later, an excess of acetone (30% v/v) was poured into the gel-like mass in the reaction vessel (Duan et al., 2019). The graft copolymer precipitate was collected, dried at 40°C, pulverized, and sieved with a #100 sieve (Kokilambigai et al., 2017; Shi et al., 2019; Singh et al., 2017). This was followed by purification (Panda, 2014).

The grafting efficiency (percentage GE) of this microwave-assisted synthesized grafted Moringa gum was determined as follows:

$\%\mathrm{GE}=\frac{\mathrm{Wt}\mathrm{.}\mathrm{of}\mathrm{graft}\mathrm{copolymer}\mathrm{–}\mathrm{Wt}\mathrm{.}\mathrm{of}\mathrm{polysaccharide}}{\mathrm{Wt}\mathrm{.}\mathrm{of}\mathrm{monomer}}\times 100$

Characterization of pure Moringa gum and modified Moringa gum

The swelling index (Grewal et al., 2019), total ash, angle of repose (AR) (Elkhodairy et al., 2014), bulk and tap densities, Hausner’s ratio (HR) (Elkhodairy et al., 2014), Carr’s compressibility index (Kumar and Rai, 2012), infrared spectral analysis (Singh and Kumar, 2018), DSC analysis (Rimpy et al., 2017), XRD (Rimpy et al., 2017), and SEM (Singh and Kumar, 2018) parameters of pure and modified Moringa gum were evaluated.

Swelling index

1 g of gum was blended with 96% ethanol (sufficient to damp the gum) and then 25 ml of purified water was added into a graduated cylindrical cylinder. The solution was shaken 1 hour per hand per 10 minutes and allowed to stand for 5 hours. The volume of the enlarged gum was then evaluated (Grewal et al., 2019). The swelling index was represented as 1 g of the prescription after swelling, as volume in ml:

$\frac{{\mathit{V}}_2-{\mathit{V}}_1}{{\mathit{V}}_1}\times 100$

where V₁ = the initial volume of material before hydration and V₂ = the final volume of hydrated material.

Total ash

The ash concentration was obtained after combustion in a furnace at 450°C to measure the remaining residue. The ash was boiled for 5 minutes using 25 ml 2M hydrochloric acid solution, and the insoluble material was filtered, cleaned, and ignited by hot water and the resulting weight was established. We then measured the proportion of insoluble acid ash (Elkhodairy et al., 2014).

Angle of repose

The static rest angle “REAT” was calculated by a set funnel and standing cone system. A funnel was tightened on a flat plane with its tip 2 cm over a graphical paper. The powders were slowly pumped into the funnel before the cone apex reached the edge of the funnel (134) (Elkhodairy et al., 2014). The median diameters of the base of the powder cones were determined and the tangent of the rest angle using the equation was measured as follows:

θ = tan^–1 h/r

where θ = AR, h = height of granules, and r = radius of granules.

Bulk and tap densities

2 g was put in a 10 ml measuring cylinder for each of the powder samples and the volume, V_b, was observed without taping each of the samples. The filled volume, V_t, was read after 100 taps on the table. The bulk and tap densities were determined by the following equations according to the ratio of weight and volume (V_b and V_t, respectively) (Elkhodairy et al., 2014):

Bulk density (BD) = M/V_b

Tapped density (TD) = M/V_t

where M = weight of powder, V_b = volume occupied by powder without tapping, and V_t = volume occupied by powder after tapping.

Hausner’s ratio

HR is an indirect powder flow facility table. The ratio of density to mass density was determined according to the following equation of the specimens (134–135) (Elkhodairy et al., 2014):

Hausner’s ratio = TD/ BD

Carr’s compressibility index

Carr’s compressibility index specified the compression index of the powder blend. It is the straightforward measure for the measurement of the powder BD and TD and the rate it is packed (Kumar and Rai, 2012). The equation was determined as follows:

Carr’s index = TD – BD/ TD ×100

Infrared spectral analysis of pure Moringa gum and modified Moringa gum

The Moringa gum and gum powder samples were triturated using potassium bromide agate and morter with an IR (IR Hydraulic Press CAP-15T, PCI Analytics, Mumbai, India) pressing at an output of 75 kg/cm² per 30 seconds. At 75 kg/cm², the mixture was compressed into dishes. In FTIR (IR affinity I, Shimadzu) in the range of 4,000–400 cm⁻¹ was registered using the FTIR disk spectrum (Singh and Kumar, 2018).

DSC analysis of pure Moringa gum and modified Moringa gum

Moringa gum and grafted Moringa gum thermograms were obtained with a differential calorimeter scanning (Q10, TA systems, USA). A small amount (mg) of fine powder of the sample was sifted into a regular aluminum pan and heated at a rate of 40°C–350°C at 10°C/minute. The inert nitrogen atmosphere (50 ml/minute) analysis was carried out.

XRD of pure Moringa gum and modified Moringa gum

For the solid characterization of Moringa gum and grafted Moringa gum, powder XRD experiments were carried out. The diffractometer configuration was traced from 0°C to 80°C (2 pounds) with a 0.05 minutes to 1.1 scanning speed at room temperature by using XRDs (Miniflex II, Rigaku, Japan) (Rimpy et al., 2017).

SEM of pure Moringa gum and modified Moringa gum

The Moringa gum and grafted Moringa gum surface morphologies were analyzed by SEM (ZEISS, EVO 18, China). The powdered sample was fixed on a double-sided adhesive carbon tape set on a holder and covered in a thin, high-vacuum gold film. At a various magnifications, the photomicrographs had a 20 kV acceleration voltage (Singh and Kumar, 2018).

Formulation of ZLP loaded-buccal disks of Moringa gum and grafted Moringa gum

Buccal disks were made in six batches, each with a different amount of gum or gum copolymer and lactose. To summarize, Moringa gum or grafted Moringa gum (60 mg w/w), ZLP (10 mg w/w), PEG-4000 (25 mg w/w), lactose (45 mg w/w), dicalcium phosphate (45 mg w/w), HPMC K15M (15 mg w/w), microcrystalline cellulose (MCC) (45 mg w/w), talc (1.5 mg w/w), and magnesium stearate (3.5 mg w/w) were homogeneously mixed with the aid of a mortar and pestle, followed by compression in an IR hydraulic press (Hydraulic Press, CAP-15T) under a pressure of 50 kg/cm² for a dwelling time of 30 seconds (Table 1).

Table 1. Composition of different batches of ZLP-loaded buccal disks. [Click here to view]

Evaluation of ZLP loaded-buccal disks

For evaluating ZLP loaded-buccal disks, several physical parameters include weight variation test for uniformity (Jangra et al., 2013; Li and Castillo, 2020; Rani et al., 2012), hardness test for crushing strength, friability test for durability, thickness test, and a percentage drug content for the tablet was performed.

Mucoadhesive strength

The mucoadhesive strength of the formulated disks was determined with a texture analyzer by recording the force needed to separate the disks from freshly excised goat’s gastric mucosal tissue (Rathee et al., 2011). In a nutshell, gastric mucosal tissues from a goat were obtained from a local slaughterhouse and used right away. A cyanoacrylate adhesive tape fixed the test disks to the cylindrical probe (diameter 10 mm). The cylindrical probe (with attached tablet) was lowered down (speed maintained 0.5 mm/second) for making the contact of test disks and mucosal tissue. A force (1 N) was applied to the probe for a specified time (60 seconds). The probe was then moved upward (15 mm distance) with 0.5 mm/seccond speed. The mucoadhesive strength was obtained, i.e., the maximum force was required to separate the disks from the mucosal surface (Shaikh et al., 2011).

Mucoadhesion time

The adhesion of the formulated disks was carried out by determining the mucoadhesion time to the goat’s gastric mucosa (Reddy et al., 2011). The gastric mucosal tissue was first pasted using cyanoacrylate tape onto a slide (glass). On the surface of the test disks, a drop (pH 6.8) was poured, making the disks wet so that they could adhere to the mucosal tissue. A beaker buffer medium of pH 6.8 (200 ml) was filled, and the temperature was maintained to 37°C ± 0.5°C. The slide with the pasted disks on mucosal tissue was kept inside the beaker. The stirrer was allowed to rotate at 50 rpm and the time required for the disks to detach from the goat’s gastric mucosal tissue was recorded as mucoadhesion time (Shaikh et al., 2011).

In-vitro swelling percentage (SP) of tablet formulation in (pH 6.8)

In a petri dish, individual disks were soaked in buffer solutions (pH 6.8) after weighing. After the specified time (2, 4, 6, 8, 12, and 24 hours), the disks (swollen) were removed and dried superficially using blotting paper. The weight of the swollen tablet was then noted, and the SP calculation was done using the following formula:

$\mathrm{SP}=\frac{{\mathit{w}}_{\mathrm{f}}-{\mathit{w}}_{\mathrm{i}}}{{\mathit{w}}_{\mathrm{i}}}\times 100$

where w_f is the final weight of the sample after swelling and w_i is the initial weight of the sample.

Also, the physical observation of the disk’s swelling was recorded by measuring the diameter of the disks using graph paper stuck to the base of a petri dish (Grewal et al., 2019).

In-vitro drug dissolution studies

Dissolution studies of various mixtures and marketed formulations (control) were carried out in 900 ml of phosphate buffer (pH 6.8) at 37°C ± 5°C and a stirrer rotation speed of 50 rpm using a United States Pharmacopoeia dissolution apparatus equipped with a paddle stirrer (Type II). At 1, 2, 4, 6, 8, 10, and 12 hours, a 5ml aliquot of dissolution medium was removed and replaced with the same amount of buffer using a pipette. The samples were appropriately prepared and analyzed spectrophotometrically at a wavelength of 254 nm (Jangra et al., 2013; Rani et al., 2012).

Mathematical models for drug release

The cumulative amount of ZLP released from formulated disks at various time intervals was fitted to zero-order kinetics, first-order kinetics, the Higuchi model, the Hixon–Crowel model, and the Korsmeyer–Peppas model to characterize the mechanism of drug release.

Data analysis

The results of the one-way analysis of variance (ANOVA) (Sigma Stat 3.5 software) were analyzed using the student’s t-test. The difference was considered statistically significant if it was less than the probability level of 0.05.

Stability studies for the optimized batch

For the determination of the effect of temperature, humidity, moisture, and light on the drug’s quality, stability studies were carried out. These findings are used to determine the storage conditions, the test period, and shelf life (Li and Castillo, 2020). The optimized formulation (F3) was stored in a stability chamber at 40°C ±2°C and 75% ± 5% relative humidity for a period of 1 month. The samples were analyzed for various parameters after 1 month by spectrophotometer at 254 nm.

Drug identification by FTIR

The IR spectrum of the drug clearly shows the peaks of the various functional groups present (Ahuja et al., 2010). The IR spectrum exhibits characteristic bands of the stretching vibration of C-H stretch vibrations at 3,087.7 cm⁻¹ and 2,933.7 cm⁻¹. It also exhibits characteristic bands of stretch vibrations of –C=N (alcohol) at 1,614.4 cm⁻¹ and 1,223.5 cm⁻¹ for C-N. It shows stretch bands of –C=C- (aromatic) at 1,576.3 cm⁻¹. ZLP exhibits strong absorption peaks at 2,232.7 cm⁻¹ and 1,651 cm⁻¹ suggesting the existence of cyanide and amide carbonyl groups, respectively. The IR spectrum of a pure drug (ZLP) obtained is shown in Figure 2 (Singh and Kumar, 2018).

Differential scanning calorimeter

In the case of pure drug, a sharp endotherm was observed at 184.94°C (Fig. 3) (Rimpy et al., 2017). The observed peak was compared to the drug’s standard peak, which indicates the purity of the drug.

SEM studies of the pure drug (ZLP)

The drug particles show a spherical shape, smooth surface, and crystalline geometry as observed both at 2,000 and 8,000× (Fig. 4) (Rimpy et al., 2017).

XRD of the drug

From the diffractogram of the drug, sharp peaks can be observed at 2θ = 10, 14, 16, 18, 21, 22, 25, 26, and 29, which show its crystalline nature in the pure form shown in (Fig. 5) (Ahuja et al., 2010). This shows that the drug, which was in crystalline form, has reduced to an amorphous form.

Modification of Moringa gum by grafting technique: synthesis of grafted Moringa gum

The properties of Moringa gum were modified via microwave-assisted graft copolymerization of acrylamide onto Moringa gum (Elkhodairy et al., 2014). Moringa gum is composed of galactose (25.9%), rhamnose (5.6%), and traces of uronic acid with pendent hydroxyl groups. When polysaccharides are microwave irradiated, the pendant hydroxyl groups exhibit localized rotation, resulting in dielectric heating and increasing the reaction rates at these groups (Elkhodairy et al., 2014). Additionally, microwave irradiation decreases the activation Gibbs energy of the reactions, resulting in faster reaction rates. Additionally, CAN, which was used as a free radical initiator in the reaction, generates free radicals that abstract hydrogen atoms from polysaccharides molecules and generate macro radicals (Elkhodairy et al., 2014). These free radicals then reacted with the acrylamide monomer to form acrylamide free radicals, initiating a chain reaction that results in the formation of Moringa gum-g-poly (acrylamide) and poly(acrylamide) homopolymers.

Graft copolymer synthesis is a combination of microwave-assisted and conventional methods, in that it is based on a free radical mechanism that uses microwave radiation in conjunction with a chemical-free radical initiator (CAN) to create extra radical sites on the Moringa gum backbone. By varying the concentrations of CAN and acrylamide (monomer), different grades of the graft copolymer were formed. In each case, the reaction mixture was microwave irradiated until it solidified into a viscous gel-like mass. The optimized grade was determined by its higher grafting percentage. When the microwave power is kept at 800 W, the grafting is optimized with a 5 g acrylamide and a 0.1 g CAN in the reaction mixture (~50 ml). The optimal batch with a concentration of CAN is an electron-deficient molecule. As a result, it borrows electrons from Moringa gum’s alcoholic oxygen to form a new bond. The optimal calculated parameters were 1.0 g Moringa gum, 0.1 g CAN, and 5.0 g acrylamide, which resulted in a graft copolymer with a GE of 65%. This bond is more polar (than the OH bond), and it breaks easily in the presence of microwave irradiation, forming a free radical site on the backbone of Moringa gum.

Figure 2. FTIR spectrum of the drug (ZLP), pure gum, and grafted gum. [Click here to view]

Figure 3. DSC showing of drug (ZLP), pure gum, and grafted gum. [Click here to view]

Figure 4. SEM images of pure drug showing shape and surface topology. (a) 2,000× and (b) 8,000×. [Click here to view]

Swelling index

An optimized method was used to calculate the swelling index (Koirala et al., 2021). The grafted gum shows a marginally higher SP in phosphate buffer (pH 6.8) compared with pure Moringa gum (Kumar and Rai, 2012; Singh et al., 2017).

Powder flow properties

The BD, TD, AR, HR, and Carr’s index of pure gum as well as modified Moringa gum are shown in Table 2. Dried gum powder shows excellent flow properties and compressibility, making them suitable for direct compression tableting (Singh et al., 2017).

Ash content

The total ash content of pure Moringa gum and modified Moringa gum is presented in tabular form (Table 2).

In-vitro SP of buccal disks in (pH 6.8)

The weight gain and swelling were proportional. The SPs of ZLP buccal disks (pH 6.8) are shown in Figure 9.

In-vitro dissolution study

The in-vitro dissolution study of ZLP mucoadhesive buccal disk (MF1–MF6) with control (marketed formulation) is shown in Figure 10. The optimized batch (MF3) showed a drug release of 95.22% in 12 hours.

Drug release kinetics and mechanism

Data from ZLP buccal disks drug release kinetics fitted to zero order, first order, Korsmeyer–Peppas, Higuchi, and Hixon–Crowel release models are shown (Fig. 11). The “n” values obtained ranged from 0.54 to 0.80. The best-fitted model, as depicted in, was found to be the Korsmeyers–Peppas model. The mechanism involved in the Korsmeyers–Peppas model was found to be a combination of diffusion and erosion-based drug release.

ANOVA- analysis of variance

The analysis of variance indicated that swelling behavior, mucoadhesion strength, mucoadhesion time, and %CDR (12 hours) of ZLP-loaded buccal disks were found to be significant (p < 0.05) and adequate, without a significant lack of fit.

Figure 10. In-vitro drug release data of drug-loaded formulation (MF1–MF6) a(mean ± SD; n = 3). [Click here to view]

Figure 11. Drug release model of ZLP buccal disks. [Click here to view]

Table 3. Stability data of the optimized batch of ZLP buccal disks. [Click here to view]

Stability study for optimized batch (MF3)

The accelerated stability studies’ results showed no appreciable modification observed in hardness, drug content, and cumulative percentage drug release in the stored tablets under specified conditions (Table 3) (Li and Castillo, 2020).