Materials
Ziziphus mauritiana L. (Indian jujube) and Artocarpus heterophyllus L. (jackfruit ) plants were authenticated by the Botanical Survey of India, Central National Herbarium, AJC Bose Indian Botanic Garden, Howrah, West Bengal, India, and were allotted accession numbers PR-01 and PR-02, respectively. The fruits were obtained from the local market in the month of November 2017. All other chemicals were of analytical grade and were purchased from E. Merck India.
Isolation of polysaccharides
Isolation of ZM gum
The ripe fruit of Indian jujube (250 g) was taken and washed properly. Thereafter, the seeds were removed and the fruit is crushed in a mortar and pestle to prepare the fruit pulp. Five hundred milliliters of distilled water was added to the fruit pulp to prepare the consistency like that of a slurry. Boiling is carried out at a temperature of 90–100 °C with continuous stirring till a viscous solution is obtained [15]. The slurry was subjected to filtration with the help of muslin cloth [16, 17]. Thereafter, a clear solution is obtained which was stand for overnight for settling of the different fibers, cell debris etc. The solution is further centrifuged for 20 min at 5000 rpm. The supernatant obtained is then separated and further treated with twice the volume of ethanol with continuous stirring. This will result in the precipitation of the ZM gum which is then separated and dried at 40–45 °C. The dried film-like material obtained was powdered. Then, it is allowed to pass through sieve no. 120. The final material obtained is stored in a desiccator [4].
Isolation of AH gum
AH gum was obtained from the fruits of jackfruit [15, 17]. About 250 g raw jackfruit was taken and washed properly and rinsed with distilled water. The seeds were removed properly and the fruit was cut into small pieces. The pieces of fruits were mashed with distilled water (fruits to water ratio, 1:3) using pestle and mortar to prepare a slurry. The slurry was subjected to centrifugation for 20 min at 3000 rpm at room temperature. The residue is collected and scraped off. It is then treated with 0.5 M sodium thiosulfate solution in the ratio of 1:1 (residue to solution) for around 24 h, during which it is stirred at regular intervals which will remove protein fractions. The filtrate was then centrifuged for 5 min at 2000 rpm. The residue obtained after centrifugation was subjected to neutralization by the addition of 0.1 M HCl. It is then washed with distilled water for two times. Thereafter, it is again washed with 50% ethanol for two times. Then, the collected material is then subjected to drying at 40–45 °C for overnight. The polysaccharide obtained was then powdered with the use of mortar and pestle. It is then allowed to pass through sieve (0.15-mm mesh size). The isolated AH gum powder was packed properly and stored in desiccators [18].
Determination of yield
The weight of the raw material and isolated gum is taken. The yield was calculated by applying the formula [19, 20]:
$$ \%\mathrm{Yield}\frac{\mathrm{Weight}\ \mathrm{of}\ \mathrm{dried}\ \mathrm{isolated}\ \mathrm{polysaccharide}}{\mathrm{Weight}\ \mathrm{of}\ \mathrm{the}\ \mathrm{whole}\ \mathrm{fresh}\ \mathrm{crude}\ \mathrm{material}}\times \kern0.5em 100 $$
Physicochemical characterization
The different properties of the isolated gum which includes the organoleptic characters, such as odour, colour, and taste, and physicochemical characters, such as solubility, pH of 1% w/v solution at 37 °C, and viscosity of 1% w/v solution at 37 °C, were determined. Since the polysaccharides are obtained in the powder form, so the various powder properties such as tapped density, bulk density, angle of repose, and Carr’s index were also measured. The results of these parameters were compared with HPMC and Carbopol 934. The pH value of the 1% solution of isolated polysaccharide samples were measured using a digital pH meter [21, 22]. Ostwald’s viscometer was used to calculate the viscosity of 1% solution of isolated polysaccharide samples [22].
Phytochemical tests
Various phytochemical tests were performed for the isolated polysaccharides [23] to detect if the samples contain carbohydrates (Molisch’s test), amino acids and proteins, mucilage (ruthenium red test), starch (iodine test), alkaloids (Dragendorff’s test), glycosides (Keller-Killani test), tannins (ferric chloride test), and flavonoids (Shinoda test) [24].
Study of swelling property of mucoadhesive materials
Swelling characteristics of mucilage were tested in distilled water, simulated gastric fluid (0.1 N HCl at pH 1.2), and phosphate buffer (pH 7.4). The swelling index is defined as the volume in milliliter occupied by part (1 g) of the substance. The protocol mentioned in the British Pharmacopoeia is followed for the calculation of swelling index. The test for swelling index follows the procedure in which 1 g of the isolated mucilage was taken in a ground glass stoppered graduated cylinder. Thereafter, 50 ml of distilled water was added. It was then shaken vigorously at an interval of every 10 min, and this process is repeated till 1 h and then left undisturbed for 24 h. After the said time, the volume of the mucilage occupied was measured. The formula used for calculation of swelling index of mucilage powder is as follows:
$$ \mathrm{S}\kern0.5em =\kern0.5em \mathrm{V}2/\mathrm{V}1 $$
where V1 = volume occupied by the mucilage before hydration and V2 = volume occupied by the mucilage after hydration [25].
Shear stress measurement
The shear stress is defined as the measuring of the force that is capable of causing a bioadhesive or mucoadhesive material to slide in accordance to the mucus layer in a direction which is completely parallel to their place of contact for adhesion [26, 27]. The test of shear stress took different concentrations of the mucoadhesive such as 1, 2, and 3% w/v for all the samples of Carbopol 934, HPMC, ZM gum, and AH gum [15]. The solution was prepared in different concentrations and spread on the glass slides. It was covered with another glass slide. A weight of 100 g was placed on the glass slides as it will also help to spread the polymer solution evenly in between the slides. It was kept for the allotted time for 15, 30, and 60 min. After this, one end of the glass slide was fixed hook and the other was collected to a twin passing over a pulley and at the end of pan was attached. Weight was placed in an increasing manner for the defined time till the plates which are attached to the polymer detach itself [28]. The weight at which just the polymer gets detached was noted, and the values were tabulated [13].
Falling sphere method
This method was used to characterize the mucoadhesive strength. In this process, 10% mucus solution is filled in a burette. It is then attached to a stainless steel tube. Mustard grains which can not pass through sieve size #12 were taken and poured in polymer solutions (Carbopol, HPMC, ZM gum, and AH gum) of different concentrations prepared such as 1.0, 2.0, and 3.0% w/v, and then each grain were slowly placed at the top of the mucus layer [28]. The time taken for the mustard grain to fall about 50 divisions in the burette was noted, and the values obtained were tabulated [29, 30].
Fourier transform-infrared (FTIR) spectroscopy analysis
KBr pellets were prepared from the powdered materials in order to perform the Fourier transform–infrared (FTIR) spectroscope (PerkinElmer Spectrum UTR II). The prepared KBr pellet was positioned properly in the sample holder as per protocol, and spectral scanning was carried out with a scan speed of 1 cm/s at a resolution of 4 cm− 1.
1H nuclear magnetic resonance (1H NMR) spectroscopy analysis
1H NMR (600 MHz, 25 °C) spectra of sample in dimethyl sulfoxide (DMSO) were analyzed on a Bruker Avance TM III 500 spectrometer (Bruker Biospin Gmbh, Germany) operating at 500.13 MHz using a 4-mm CP-MAS probe head.
