Extraction and purification of Khaya senegalensis gum
The method of Ofori-Kwakye et al. [14] was used in the extraction of the khaya gum. Briefly, one kilogram of dried crude Khaya senegalensis exudate was reduced and macerated in chloroform-water double strength and allowed to stand for 120 h with intermittent stirring. The gum mucilage was then strained with a muslin cloth to remove any insoluble impurities. Absolute ethanol was used to precipitate the mucilage. The mucilage was then collected and washed twice with diethyl ether and dried in hot air oven (Uniscope SM9053, England) at 40 oC for 24 h. The extracted gum was dried at 40 oC for 24 h. The dried gum was then pulverized using pestle and mortar and the powdered gum was passed through a sieve with mesh size of 210 μm. Thereafter, it was stored in an air-tight container until needed.
Characterization of gum sample
Moisture content
The moisture content of the gum sample was determined using a moisture analyser (Sartorius AG, Germany). Two grams of the gum powder was placed into the moisture balance and evenly spread on the tray. The readings were recorded at 15 min.
pH
A 5.0% w/v dispersion of the gum powder was made with distilled water and the pH measured using a pH meter (Hanna instruments, USA).
Ash value
A 2 g weight of gum powder sample was poured into a nickel crucible which was initially heated at 105 °C in a furnace (Carbolite, Germany) to a constant weight. The temperature was then increased to 600 oC and heated for 6 h. The sample temperature was allowed to cool to 100 oC and removed from the furnace, cooled in a desiccator over silica gel and reweighed using an analytical balance (Adventurer-Pro, Ohaus, USA). The weight of the residues (carbon-free ash) was determined and expressed as percentage of the initial sample.
For determination of the acid-insoluble ash content of the gum sample, the ash obtained from the above experiment was boiled with 25 mL of 2M HCl for 5 min. The insoluble residue was separated by centrifugation at 2000 rpm for 10 min using a centrifuge (Thermo Scientific, Germany). The sediment obtained was re-suspended in hot water and evaporated to dryness in a tarred crucible. The weight of the residue was expressed as a percentage of the initial weight of the sample.
Phytochemical analysis
Phytochemical analyses were carried out to test for the presence of secondary plant metabolites in the gum powder. Secondary metabolites are various chemical compounds produced by plants from the materials derived from primary metabolites. Molisch’s test, Fehling’s test, frothing test, lead acetate test, ferric chloride test, Shinoda test, Keller-Kiliani test, Borntrager’s test, Salkowski’s test, Liebermann-Burchard test, and Dragendorff’s test were carried out using methods reported in previous studies to determine the presence of carbohydrates, reducing sugar, saponin, phenolic compounds, tannin, flavonoid, cardiac glycoside, anthraquinone, steroid, terpenoid, and alkaloid, respectively [15, 16].
Elemental analysis
Chromium (Cr), cadmium (Cd), copper (Cu), lead (Pb), nickel (Ni), and cobalt (Co) contents in the gum sample were determined using an Atomic Absorption Spectrometer (iCE 3000, Thermo Fisher). Five grams of oven dried samples were weighed into a crucible and placed in an incinerator at 600 °C and left to ash for 2 h and then cooled to 105 °C and the gum powder weighed. This process was repeated until a constant weight was obtained. The ashed samples (0.5 g) were weighed and transferred into the digestion tube. The ashed sample was mixed in the digestion tube with 5 mL each of distilled water, 1 M perchloric acid and concentrated HNO3. The contents were digested on a heating mantle and the temperature was set at 150 °C for 120 min followed by the addition of 1 mL of 2 M H2SO4. Distilled water was added to the tube to make up to the mark, mixed, and filtered. The elements were determined by using an atomic absorption spectrophotometer (AAS) at an appropriate wavelength, temperature and lamp-current for the different elements.
Micromeritics and rheological properties
The bulk density, tapped density, Carr’s index, and Hausner’s ratio of the gum powder were determined according to the methods described by Azubuike et al. [17].
The viscosity of the gum powder was determined using a digital viscometer (DV-E, China). The viscosity values of 2%, 5%, and 10 % w/v aqueous dispersion of the gum were determined at 50 rpm and 25 oC. The swelling index of khaya gum was determined using the method employed by Ologunagba et al. [18].
Fourier-transform infrared spectroscopy (FTIR) analysis
A 5 mg quantity of khaya gum sample, metformin, and metformin-loaded microspheres were individually blended with 50 mg of solid KBr and compressed into discs. The scanned range was from 500 to 4000 cm−1 in FTIR spectroscopy (Cary 630, Agilent Technologies, USA) under dry air at room temperature.
Differential scanning calorimetry (DSC)
The thermal behaviour of the gum powder was determined using differential scanning calorimeter (Mettler Toledo, USA) as described by Sindhu and Khatkar [19]. The gum powder (6 mg) was weighed into an aluminum pan. The pan was hermetically sealed and equilibrated at room temperature for 1 h, then heated at the rate of 10 °C/min from 30 °C to 250 °C with an empty-sealed pan as a reference. The DSC studies were also carried out for the pure metformin powder and the different formulations of the microspheres.
Scanning electron microscopy (SEM)
The granule morphology of the gum powder was observed in a scanning electron microscope (Pro X, The Netherlands). The gum powder was mixed with ethanol to obtain a 1% suspension. The suspension (one drop) was then smeared on aluminium stub with double-sided adhesive tape and the sample gum powder was coated with gold powder to avoid charging under the electron beam when the acetone has volatilized. An accelerating potential (15 kV) was used during micrography [20].
Preparation of metformin hydrochloride microspheres
Polymer blends {khaya gum:sodium alginate (2:3, 9:11, and 1:1) respectively} consisting of the different ratios of khaya gum and sodium alginate were used. The choice of the ratio blends of the polymers were based on preformulation studies carried out. Microspheres embedded with metformin were prepared by the ionic gelation technique as described by Odeku et al. [21]. Briefly, appropriate quantities of metformin (2% w/v) were added and stirred continuously until a dispersion was obtained (2:1 ratio of polymer and drug respectively). The resulting dispersion was extruded into zinc chloride solution (5% w/v) using a 5 mL hypodermic syringe with a 23 G needle at a dropping rate of 2 mL/min and stirring speed of 300 rpm. The formed microspheres were allowed 30 min curing time and then left standing for 1 h to allow further cross-linking. The microspheres were collected by decantation, washed with distilled water, and then dried for 18 h in hot air oven at 40 oC. The microspheres were then stored in airtight containers until needed.
Characterization of microspheres
Size distribution of microspheres
The sizes of microspheres were determined using an optical microscope (Leica, Germany) fitted with an ocular micrometer. The mean diameter reported was obtained from a total of more than 100 microspheres.
Morphology of microspheres
The morphology of the microspheres was analysed by scanning electron microscopy. The microspheres were dusted onto double sided tape on an aluminium stub and coated with gold using a cold sputter coater to a thickness of 400 Å and then imaged using a 15 kV electron beam. Photographs were taken within a range of 190–250 magnifications [22].
Swelling index
A 100 mg quantity of microspheres was soaked in 20 mL of phosphate buffer, pH 6.8 for 3 h. After the 3 h, the microspheres were then removed and excess buffer was wiped using a dry filter paper and their final weights were determined. Swelling index was calculated by using equation (1).
$$ s\mathrm{welling}\ \mathrm{in}\mathrm{dex}\ \left(\%\right)=\frac{\mathrm{change}\ \mathrm{in}\ \mathrm{weight}\ \left(\mathrm{mg}\right)}{\mathrm{original}\ \mathrm{weight}\ \left(\mathrm{mg}\right)}\times 100\kern0.5em $$
(1)
Entrapment efficiency
Appropriate quantity of drug-loaded microspheres was crushed in a glass mortar with a pestle, weighed to obtain 100 mg (equivalent to 50 mg of metformin) and then suspended in 50 mL of phosphate buffer, pH 6.8. After 24 h, the solution was filtered and the filtrate was appropriately diluted with phosphate buffer, pH 6.8 and analysed using UV spectrophotometer (Biomate 3, USA) at 233 nm. The drug entrapment efficiency (E) was then calculated using equation (2).
$$ E\ \left(\%\right)=\frac{\mathrm{practical}\ \mathrm{drug}\ \mathrm{content}}{\mathrm{theoretical}\ \mathrm{drug}\ \mathrm{content}}\times 100\kern1.00em $$
(2)
In vitro dissolution studies and kinetic modelling of release profiles
The in vitro dissolution studies were carried out using the basket method USP type II rotated at 50 rpm in 900 mL of phosphate buffer, pH 6.8, maintained at 37 ± 0.5 oC. The microspheres equivalent to 500 mg of metformin was placed in the dissolution medium. Samples (5 mL) were withdrawn at 0, 30 min and then at 1 h intervals for 9 h and replaced each time with equal amounts of fresh medium. The samples withdrawn were filtered through a 0.45-micron membrane filter (Nunc, New Delhi, India). The amount of metformin released was determined after suitable dilution at wavelength of 233 nm, using a UV spectrophotometer (Biomate 3, USA). Dissolution test was also carried out using the same conditions for a sustained release marketed brand of metformin tablets (Panfor® SR 500 mg, Mega Lifesciences Pty. Ltd., Thailand).
Kinetic modelling of the release data for the three microspheres formulations was carried out by fitting to different kinetic equations. The results of drug release from the microsphere formulations was fitted into drug release kinetic models—zero order, first order, Higuchi, and Korsmeyer-Peppas. The model of best fit was identified by comparing the values of correlation coefficients.
Statistical analysis
All the tests were carried out in triplicates. All the values were expressed as mean ± standard deviation (SD). Differences with p < 0.05 were considered statistically significant. GraphPad Prism-7 software (GraphPad Software Inc., USA) was used for statistical analysis.