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Phosphorylation of psyllium seed polysaccharide and its characterization
International Journal of Biological Macromolecules 85 (2016) 317–326
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Phosphorylation of psyllium seed polysaccharide and its characterization Monica R.P. Rao ∗ , Deepa U. Warrier, Snehal R. Gaikwad, Prachi M. Shevate Department of Pharmaceutics, AISSMS College of Pharmacy, Near RTO, Kennedy Road, Pune 411001, Maharashtra, India
a r t i c l e
i n f o
Article history: Received 5 November 2015 Received in revised form 11 December 2015 Accepted 13 December 2015 Available online 6 January 2016 Keywords: Psyllium seeds Phosphorylation Swelling index Rheology Mucoadhesion
a b s t r a c t Psyllium is widely used as a medicinally active natural polysaccharide for treating conditions like constipation, diarrhea, and irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis and colon cancer. Studies have been performed to characterize and modify the polysaccharide obtained from psyllium seed husk and to evaluate its use as a pharmaceutical excipient, but no studies have been performed to evaluate the properties of the polysaccharide present in psyllium seeds. The present study focuses on phosphorylation of psyllium seed polysaccharide (PPS) using sodium tri-meta phosphate as the cross-linking agent. The modified phosphorylated psyllium seed polysaccharide was then evaluated for physicochemical properties, rheological properties, spectral analysis, thermal analysis, crosslinking density and acute oral toxicity studies. The modified polysaccharide (PhPPS) has a high swelling index due to which it can be categorized as a hydrogel. The percent increase in swelling of PhPPS as compared to PPS was found to be 90.26%. The PPS & PhPPS mucilages of all strengths were found to have shear thinning properties. These findings are suggestive of the potential use of PhPPS as gelling & suspending agent. PhPPS was found to have a mucoadhesive property which was comparable with carbopol. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Pharmaceutical excipients are substances other than the active pharmaceutical ingredient—that are used in the finished dosage form or may be used as processing aids in the manufacture of active pharmaceutical ingredients [1]. The properties of the final dosage form (i.e., its bioavailability and stability) depend on the excipients chosen, their concentration and interaction with both the active compound and each other. A detailed knowledge of the physical and chemical properties as well as the safety, handling and regulatory status of these materials is essential for formulators throughout the world [2]. Majority of the widely used excipients are obtained from synthetic sources. Although excipients are considered to be pharmacologically inert, it is now well accepted that some have potential for untoward effects. Besides this, problems of incompatibility, stability and high cost are evident in synthetic excipients [3]. There are several polysaccharides of plant origin which have been used as excipients. Polysaccharides consist of a large number of polymeric carbohydrate molecules composed of long chains of monosaccharide units linked together in a long chain by gly-
∗ Corresponding author. E-mail address: monicarp [email protected] (M.R.P. Rao). http://dx.doi.org/10.1016/j.ijbiomac.2015.12.043 0141-8130/© 2015 Elsevier B.V. All rights reserved.
cosidic bond [4]. The pharmaceutical excipients of plant origin, like starch, agar, alginates, carrageenan, guar gum, xanthan gum, gelatin, pectin, acacia, tragacanth, cellulose etc. find applications in the pharmaceutical industry as binding agents, disintegrants, sustaining agents, protective colloids, thickening agents, suspending agents, emulsifiers, gelling agents, bases in suppositories, stabilizers, and coating material [5–10]. Psyllium has been in use as a medicinal agent since ancient times throughout the world and it is official in compendia of many countries. The dried, ripe seeds of Plantago afra (Plantago psyllium), Plantago indica (Plantago arenaria) & Plantago ovata (Plantaginaceae) are used in medicine. Seeds of P. ovata Forsk are commercially referred to as Indian psyllium or Ispaghula [11–13]. Psyllium is used worldwide for the treatment of constipation, diarrhea, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, colon cancer, diabetes and hypercholesterolemia [14,15]. Psyllium seed mucilage contains 22.6% arabinose, 74.65% xylose, traces of other sugars and 35% non-reducing terminal residues [16]. Researchers have studied the physiologically active, gel-forming fraction of the alkali-extractable polysaccharides of P. ovata Forsk seed husk. The polysaccharide from the seed husk has been fractionated and evaluated for gelling ability [17]. Kaith and Kumar carried out grafting of acrylic acid onto psyllium backbone
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Fig. 1. Reaction steps and mechanism of crosslinking.
Fig. 2. Percent swelling indexs.
Fig. 3. RMB absorbed (PhPPS).
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via free radical mechanism and studied its water uptake in oil/water emulsion for use in oil refineries [18]. In the last decade and half, a plethora of novel carriers have been developed which have brought about a phenomenal change in the profile of drug delivery. Among different drug delivery devices, hydrogels, especially based on polysaccharides have attained lot of prominence for achieving controlled release of drugs [19]. Hydrogels are three-dimensional polymeric networks that swell considerably in presence of water [20]. However, hydrogels based on natural polymers have limited applications due to poor mechanical strength of the gel [21]. Thus modification of polysaccharides to develop hydrogels is a powerful tool to control the interaction of the polymer with drugs, to enhance the load capacity and to tailor the release profile of the drug. Grafting and cross linking are common methods to modify and improve the functional properties of polysaccharides [22]. In previous studies, the authors have carried out extraction and characterization of psyllium seed polysaccharide [23]. Swelling studies in different media revealed a manifold increase in volume pointing to its hydrogel properties. Investigation of its rheological properties indicated thixotropic nature with a characteristic spur value at higher polysaccharide concentration. The psyllium seed polysaccharide was also found to have satisfactory binder properties for use in tablets and displayed lower yield values with increasing concentration [24]. In present study an attempt was made by the authors to improve the functionality of the polysaccharide by chemical modification and compare its properties with the parent polysaccharide. In the course of their studies, the authors observed a change in rheological properties of the psyllium seed polysaccharide compared to their previous findings. These changes, though not very significant, could have far reaching consequences in their applicability. It has, of course been reported that natural polymers suffer from disadvantages like uncontrolled rate of hydration, changes in viscosity on storage and microbial contamination [25]. Thus chemical modification of the psyllium seed polysaccharide by phosphorylation was attempted in the present study using sodium tri-meta phosphate as cross-linking agent. The modified phosphorylated psyllium seed polysaccharide was then evaluated for physicochemical properties, rheological properties, spectral analysis, thermal analysis and crosslinking density. Acute oral toxicity studies were also performed for the phosphorylated psyllium seed polysaccharide. 2. Materials and methods 2.1. Materials Psyllium seeds were obtained from Manakarnika Aushadalay, Pune & authenticated at Agarkar Research Institute, Pune. Sodium tri-meta phosphate was a gift sample obtained from Sigma–Aldrich. All the other chemicals, solvents and reagents used were of analytical grade and were procured locally. To perform the acute oral toxicity studies, permission was obtained from Institutional Animal Ethics Committee—Approval No.: CPCSEA/IAEC/PT–21/12–2K13. 2.2. Methods 2.2.1. Extraction of seed gum The psyllium seeds were soaked in deionized water at 80 ◦ C for 2 h and kept overnight. Sodium hydroxide (NaOH) 0.5 M was added while stirring (200 rpm for 15 min) to separate the polysaccharide from seeds and resulting slurry passed through 12#. The psyllium polysaccharide (PPS) was reprecipitated by addition of 2 M HCl to the filtrate. The precipitate was washed with deionized water to remove traces of acid and separated by centrifugation at
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3000 rpm for 15 min. The residue was dried overnight in a tray drier at 50–60 ◦ C. 2.2.2. Chemical crosslinking of PPS Sodium tri-meta phosphate (STMP- 1 g) was dissolved in 50 ml of deionised water. This was added to 5 ml of 0.1 N NaOH. PPS (1 g) in 50 ml of water was then added slowly with stirring. The reaction mixture (100 ml) was stirred for 2 h, poured into each of five petri dishes (20 ml each) and dried at 60 ◦ C for 24 h. The dried complex (modified gum) was powdered, passed through 120 # and used for evaluation. The same procedure was followed by using 0.2 N NaOH as the catalyst [26]. 2.3. Characterization of psyllium seed polysaccharide (PPS) & phosphorylated psyllium seed polysaccharide (Ph PPS) The PPS and PhPPS were characterized for physicochemical properties (solubility studies, swelling index, micromeritic properties, water uptake, and loss on drying), spectral analysis and thermal analysis. Acute oral toxicity studies for PhPPS were also conducted as per OECD Guidelines 423 [27]. 2.3.1. Swelling index Swelling behavior of PPS and PhPPS was studied as a function of pH. It was allowed to swell for 24 h in solutions of different pH such as 0.1 N HCl (pH 1.1), phosphate buffer pH 6.8, deionized water and 0.5 M NaOH (pH 13.7). For this, 1 g of PPS and PhPPS were added separately in 25 ml of the respective media in stoppered volumetric flasks at 37 ◦ C and kept overnight with intermittent shaking. Volume occupied by the PPS was measured using a measuring cylinder and swelling index was calculated from the initial and final volume of PPS and PhPPS respectively [28]. 2.3.2. Solubility profile The solubility of PPS and PhPPS was evaluated at 37 ◦ C in various solvents such as acetone, alcohol, ether, chloroform, dichloromethane, dimethyl amine, tri methyl amine, diethyl ether, ethyl acetate, dimethyl sulfoxide, isopropyl alcohol, 0.5 N sodium hydroxide, N-methyl-2-pyrolidone, N,N-dimethyl formamide, conc. hydrochloric acid and conc. nitric acid. 2.3.3. Physicochemical properties The bulk density, tapped density, angle of repose, Hausner’s ratio and Carr’s index of PPS and PhPPS was evaluated [29]. The moisture content of PPS and PhPPS was determined by using infrared moisture balance (Make: Gurunanak Instruments, Model: 6084). For water uptake studies, PPS and PhPPS was immersed in 25 ml deionized water and were lightly patted with tissue paper to remove excess surface water. The study was carried out at different time intervals (2, 4, 8, 12 & 24 h) and water uptake was computed gravimetrically according to following equation, Wateruptake(%) = (Ts – Ti)/Ti × 10 where Ts is the weight of the swollen polymer and Ti is the initial weight of the polymer. The pH of 1% solution of the PPS and PhPPS was determined using a digital pH meter (EI, DELUX 101). Loss on drying was determined by drying a fixed weight of the samples in an oven for 5 h at 105 ◦ C in glass stoppered bottles. 2.3.4. Crosslinking density The PPS and PhPPS (1 g) were placed separately in dialysis bags (MEMBRA- CELL, MD 77-14 × 100 CLR, SIGMA) and then immersed in 100 ml of methylene blue solution (10−5 mol/l). Concentration of methylene blue in medium outside the dialysis bag was measured
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Fig. 4. Apparent viscosity vs rate of shear for PPS and PhPPS.
by spectrophotometry (Spectrometer UV/vis Lab India) at wavelength of 665 nm until no decrease in methylene blue absorbance was observed. PhPPS prepared using two different concentrations of STMP were investigated i.e., 1 g and 2 g of STMP per gram of PPS. The crosslinking density was estimated by determination of the relative amount of methylene blue (RMBabsorbed ) bound to the particles using equation,
brought toward the mucosa at a constant speed of 0.5 mm/s until a predetermined compressive force of 1 N was applied for 60 s. The probe was then removed at 5 mm/s to a distance of 15 mm and maximum detachment force (kg) was determined for each sample.
RMBabsorbed = AMB − APhPPS /AMB – APPS
The Fourier Transform Infrared Spectroscopy (FTIR) spectra of PPS and PhPPS were recorded using JASCO, Japan V 462. The thermograms of PPS and PhPPS were obtained by a Differential Scanning Calorimeter (DSC 821, Make: METTLER) at heating rate of 10 ◦ C/min from 30 to 300 ◦ C in nitrogen atmosphere (30 ml/min). X-ray diffraction patterns were recorded in the reflection mode on a Siemens D-500 X-ray diffractometer. Diffractograms were registered at Bragg angle (2) range of 5–40◦ at a scan rate of 2.5◦ per minute and step size of 0.02◦ .
where, AMB = initial absorbance of methylene blue solution, APhPPS = absorbance of methylene blue solution containing phosphorylated PPS, APPS = absorbance of methylene blue solution containing native PPS. The theoretical crosslinking density was measured using STMP as standard. 2.3.5. Rheological properties The mucilage’s of PPS and PhPPS were prepared in distilled water in strengths of 2, 2.5, 3, 5 and 10% w/v. The mucilages were homogenized using tissue homogenizer to break any lumps and subjected to rheometry studies at different shear rates using Brookfield digital viscometer DV-E (Spindle 61). The rheograms were plotted and the thixotropic nature of the polymers was studied by subjecting the mucilages to increasing and decreasing shear rates [30].
2.4. Spectral and thermal analysis
2.4.1. Scanning electron microscopy Surface topography of PPS and PhPPS was studied using scanning electron microscopy (JSM-6360A-MAKE: JEOL). Each sample was mounted on to an aluminium stub using two-way adhesive tape and then coated with gold-palladium alloy using a sputtering device under a nitrogen atmosphere. The samples were examined with 100× and 2000× magnification. 2.5. Acute oral toxicity studies for PhPPS
2.3.6. Mucoadhesive strength Mucoadhesive property of PPS and PhPPS was evaluated using a texture analyzer (CEB Texture Analyzer, Make-Brookfield Engineering Labs, Inc., Model Texture Pro CT V1.4 Build 17). Fresh sheep mucosa was obtained from the local slaughterhouse. PPS and PhPPS were carefully attached to the cylindrical probe (TA probe) by a bioadhesive tape. The upper platform was moved downward manually near to the mucosa surface and then the polymer sample was
Acute oral toxicity study was conducted according to OECD guidelines 423. The study was performed in healthy nulliparous and non-pregnant adult female albino Swiss mice (20–35 g). Pilot study was carried using six albino Swiss mice in a group with the starting dose of 300 mg/kg body weight. Animals were monitored continuously immediately after dosing up to first 4 h using video tracking system for behavioral, neurological and autonomic param-
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Fig. 5. Shear stress vs rate of shear for PPS and PhPPS.
eters. The animals were under supervision after 24 h and 14 days for any sign of toxicity or mortality. When 300 mg/kg dose was proved non-toxic, in the main study, 2000 mg/kg dose was screened for checking toxicity using the same procedure mentioned above taking 6 animals in a group [31]. 3. Results and discussion 3.1. Crosslinking of psyllium seed polysaccharide Natural polysaccharides lack certain properties and when the natural polymers are crosslinked, the crosslinks tie the macromolecular chains together by primary covalent bonds, transforming each particle into a single, giant molecule [32]. Chemical derivatization of the polysaccharide obtained from psyllium seed husk has been carried out with the objective of improving their physical and physicochemical properties so as to enable their use as multifunctional pharmaceutical excipients. Various cross linkers that have been studied include methacrylamide, N,N-methylene bisacrylamide and polymethacrylamide [33]. Sodium tri-meta phosphate is an efficient cross-linking agent and is a solid of low toxicity with no reports of adverse effects in humans. Phosphorylation using STMP as a crosslinking agent has been used as a method of chemical derivatization for starches [34,35]. Guar gum has been crosslinked using STMP for applications in colon-specific drug delivery [36]. STMP has also been reported as an effective cross-linker of hyaluronan and karaya gum for the purpose of synthesizing gels and sustained release tablets. The crosslinking reaction occurred through the hydroxyl groups of the
polysaccharide and led to ester linkages. Hence, STMP was selected as the cross linker. The advantage of phosphorylation is that di or tri esters can be formed by varying the pH conditions and different grades of crosslinked polymer can be obtained. Changing the amount of phosphorylating agent can regulate the degree of substitution of the products and polymers with adapted degree of substitution can be obtained. Sodium hydroxide provides the necessary alkalinity for the crosslinking reaction with STMP. The mechanism involved in synthesis of modified psyllium seed polysaccharide is reaction between hydroxyl groups of the PPS and metaphosphate groups in STMP leading to phosphate ester (O P O) linkages between two polysaccharide moieties (Fig. 1). The effect of sodium hydroxide concentration on the swelling index of PhPPS was observed (Table 1). The percent swelling of PhPPS was found to increase with increasing concentration of sodium hydroxide from 0.1 to 0.2 N NaOH. This may be due to increase in crosslinking between PPS and STMP at 0.2 N NaOH. Swelling index of PhPPS in deionized water prepared using 0.1 N NaOH as catalyst was found to be 1720 ± 0.12% and that of PhPPS prepared using 0.2 N NaOH as catalyst was found to be 1876 ± 0.18%. 3.2. Characterization of psyllium seed polysaccharide (PPS) and phosphorylated psyllium seed polysaccharide (PhPPS) 3.2.1. Swelling profile of PPS and PhPPS Swelling is defined as the uptake of liquid by a polymer with an increase in volume. By controlling the swelling of the polymer, the dosage form can release the drug in a controlled manner to a desired
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Table 1 Percent swelling index. Medium
Swelling index of PhPPS prepared by using 0.1 N NaOH as catalyst (%)
Deionised water Phosphate buffer pH 6.8 0.1 N HCl 0.5 N NaOH
1720 412 846 292
± ± ± ±
3.12 2.23 3.13 2.25
Swelling index of PhPPS prepared by using 0.2 N NaOH as catalyst (%) 1876 441 948 341
± ± ± ±
2.18 2.19 3.24 2.23
n = 3.
site. The swelling profile of PPS and PhPPS in different media is shown in Fig. 2. For PPS, the maximum swelling was found in phosphate buffer pH 6.8 with 1242% swelling, whereas no swelling was observed in 0.5 N NaOH. For PhPPS, the maximum swelling was found in deionized water with 1876% swelling and swelling was also observed in 0.5 N NaOH with 341% swelling. In all the solutions except phosphate buffer pH 6.8, the swelling of PhPPS was found to be higher than that of corresponding PPS. The percent increase in swelling of PhPPS as compared to PPS was found to be 90.26%. The decreased swelling in 0.5 N NaOH may be attributed to ionization of the phosphate groups grafted on the polymer leading to electrostatic repulsions between the chains of PPS. This could further lead to reduced water entrapment and hence lower swelling. At acidic pH, it may be presumed that H+ ions will negate the effect of phosphate anions and diminish the repulsive interactions between the chains of PPS, hence the structural integrity of polymer is maintained and more water may be entrapped. Therefore, swelling of polysaccharide is more in 0.1N HCl. Bejenariu et al. observed similar effect for xanthan gum [37]. At pH 13, the swelling of STMP crosslinked xanthan was found to be maximum. The extensive swelling that PPS and PhPPS exhibit in water and 0.1N HCl is indicative of their hydrogel nature which can be exploited to design sustained release dosage forms. Swelling could be a result of entanglement of the polysaccharide chains and development of intraand inter-molecular hydrogen bonds between the polysaccharide and water causing greater amount of water to be entrapped within macromolecular chains. Due to joining of two PPS molecules by phosphate ester groups, the synthetic bridges so formed, reinforce the natural hydrogen bonds, delaying the speed of granule swelling and reducing the rupture of the swollen polysaccharide molecules. It can also be presumed that crosslinking interferes with free access of water to PPS hydroxyl groups. However, for PhPPS, the gradual increase in swelling observed is caused by chain straightening which enhances water penetration into the polymer’s network. 3.2.2. Solubility profile Solubility can be defined as the spontaneous interaction of two or more substances to form a homogenous molecular dispersion. The PPS was found to be soluble in strong ammonia solution and dimethyl sulfoxide after 15 min sonication at room temperature whereas, PhPPS was found to be soluble only in conc. hydrochloric acid and conc. nitric acid. PhPPS was found to form a gel in chloroform and 0.5 N NaOH. 3.2.3. Physicochemical properties Evaluation of physicochemical properties of PPS and PhPPS revealed a remarkable difference between the two polysaccharides (Table 2). However the micromeritic properties of PPS and PhPPS were found to be almost similar. The angle of repose of PPS was found to be 29.74◦ and that of PhPPS was found to be 24.97◦ . The study of Carr’s index and Hausner’s ratio further supported the results of angle of repose. These findings reveal that the flow properties of PhPPS are good as compared to PPS and are suggestive of its use as a directly compressible excipient. The moisture content of PhPPS as determined by loss on drying method was lower as compared to PPS. This further supports the better flow properties
of PhPPS. The pH of PPS in deionised water was found to be 4.46 and that of PhPPS was found to be 7.90. The lower pH of PPS is due to its acidic nature. It may be proposed that due to the crosslinking reaction of PPS with STMP, sodium and phosphate groups are introduced in the PPS, due to which the pH of PhPPS increased. 3.2.4. Crosslinking density The crosslinking of polymer chains is of primary importance in controlling many polymer properties. The crosslinking density signifies the quantity of crosslinking agent that has been incorporated in the polymer. Methylene blue (MB) is a cationic molecule with a high affinity for negatively charged solids and phosphate groups are anionic in nature [38]. An absorption kinetic study was carried out to determine the equilibrium time after which no further reduction in MB concentration could be detected. The number of phosphate groups was found to increase with the level of crosslinking agent and reached a limiting value of absorbed methylene blue at 7 h at both levels of the cross linker (Fig. 3). A 14% increase in crosslinking density was evident with PhPPS at higher level of STMP after 7 h. 3.2.5. Rheological properties Rheology is an important physical property affecting the physical stability and ease of use of any liquid and semisolid preparation. The PPS and PhPPS mucilages of all strengths were found to have shear thinning properties as evident from the graphs (Fig. 4) of apparent viscosity vs. rate of shear. The results indicated pseudoplastic behavior i.e., decrease in viscosity with increase in shear rate which is characteristic of polymeric systems. The decrease in viscosity resulted from the shearing action on long-chain molecules of the polymer. As the shearing stress is increased, normally disarranged molecules begin to align their long axes in the direction of flow. This orientation reduces internal resistance of the material and allows a greater rate of shear at each successive shearing stress. In addition, some of the solvent associated with the molecules may be released, resulting in an effective lowering of both the concentration and the size of the dispersed molecules. This too decreases the apparent viscosity. These findings are suggestive of the potential use of PhPPS as a gelling agent and suspending agent [39]. The rheogram (Fig. 5) for shear rate vs. shear stress for both PPS and PhPPS mucilage were found to produce a characteristic bulge in the upcurve. The bulge indicated swelling of PPS and structural integrity at low shear values which suddenly lose their rigidity as shear is increased. This loss in rigidity is not gradual and leads to a bulge. The rheogram for shear rate vs. shear stress for 5 and 10% PPS and PhPPS mucilages showed a characteristic spur value indicating a sudden breakdown in the structure at a low shear rate. At higher strengths, the mucilage has a very rigid structure which can be attributed to the formation of hydrogen bonds between the polysaccharide and water. The spur value was found to be greater than 100/s for both PPS and PhPPS with PhPPS exhibiting a spur value of approximately 120/s and PPS showing spur value at nearly 135/s. However, at lower strengths, spur value was absent which was indicative of the greater structural flexibility. Thixotropy is a desirable property in liquid pharmaceutical systems that ideally should have a high consistency in the container as well as pour or spread easily. The PPS and PhPPS mucilages of
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Table 2 Micromeritic properties of PPS and PhPPS.
Bulk density Tapped density Angle of repose Hausner’s ratio Carr’s index Moisture content pH Water uptake (end of 24 h) Loss on drying
Psyllium seed polysaccharide
Phosphorylated psyllium seed polysaccharide
0.7142 g/cc ± 0.23 0.833 g/cc ± 0.25 29.74◦ ± 1.72 1.1667 ± 0.38 14.301% ± 1.37 1% ± 0.27 4.46 ± 0.56 884.32% ± 18.19 0.16% ± 0.06
0.6418 g/cc ± 0.28 0.6891 g/cc ± 0.19 24.97◦ ± 1.68 1.0738 ± 0.25 6.86402% ± 1.28 0.5% ± 0.11 7.9 ± 0.52 1463.2% ± 16.23 0.14% ± 0.04
n = 3.
Fig. 6. Hysteresis loop for PPS and PhPPS.
all strengths were subjected to increasing and decreasing shear rates and the rheograms were plotted. Presence of a hysteresis loop was indicative of thixotropic nature of the mucilages (Fig. 6). The rheogram shows that for all the mucilages, the down curve is displaced to the left of the upcurve. This indicated a breakdown of structure (and hence shear thinning) that does not reform immediately when stress is reduced. The degree of thixotropy is represented by the area of hysteresis loop. The area of hysteresis loop was calculated using planimeter method. The area of hysteresis loop for 3% PPS mucilage was found to be 11,236 mm2 whereas for the 5% PPS mucilage it was 9604 mm2 . The area of hysteresis loop for 3% PhPPS mucilage was found to be 15,376 mm2 whereas for the 5% PhPPS mucilage it was 12,100 mm2 . The PPS and PhPPS mucilages of lower concentration had greater degree of thixotropy than the PPS and PhPPS mucilages of higher concentration. This
can be due to the fact that mucilages of higher concentration have high structural rigidity which leads to lesser breakdown in structure during shearing. The presence of hysteresis loop is a desirable feature while formulating a gel because the gel can be easily applied to a surface (through structure breakdown in spreading) and can, at rest rebuild its structure and viscosity so that it does not drip and run [40]. 3.2.6. Mucoadhesive strength Mucoadhesion is considered to occur in four major stages: wetting, interpenetration, adsorption and formation of secondary chemical bonds between mucus membrane and polymers. The mucoadhesive strength is affected by molecular weight of polymer, contact time with membrane and degree of swelling of the polymer. The mucoadhesive strength of PPS and PhPPS was stud-
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Table 3 Mucoadhesive force of polymers.
Table 4 Acute oral toxicity observations at a dose of 2000 mg/kg body weight of animal.
Polymer
Mucoadhesive force (g)
Sr. no. Parameter
Observation (During 4 h) Observation (After 24 h)
PPS PhPPS Carbopol
5.2 ± 1.4 6.7 ± 1.5 7.2 ± 1.8
1) 2) 3) 4) 5) 6) 7) 8) 9) 10)
Normal Normal Normal Normal Observed Observed Normal Not observed Not observed Not observed
ied using sheep intestinal mucosa. The adhesive force was obtained from software TexturePro CT V1.4 Build 17 Brookfield Engineering Labs, Inc. The results for mucoadhesive force are shown in Table 3. It was found that PhPPS had higher mucoadhesive strength than PPS and comparable mucoadhesion with carbopol which is widely used for this purpose. Mucoadhesion is a complex process with many theories being reported in literature [41]. The mucoadhesive substance should be capable of favoring both chemical and mechanical interactions with mucous such as molecules with hydrogen bond building groups (–OH, –COOH), an anionic surface charge, high molecular weight, flexible chains and surface-active properties, which help in spreading throughout the mucus layer, can present mucoadhesive properties [42]. The mucoadhesive properties of PhPPS may be attributed to the anionic nature of phosphate groups which are capable of forming ionic interactions with biomolecules which are present in mucous. Besides this the free hydroxyl groups of the polysaccharides may also form hydrogen bonds with the mucosal surface. Thus, PhPPS could be investigated further as a mucoadhesive agent to formulate gastro retentive mucoadhesive gels and microspheres. 3.2.7. Interpretation of crosslinked polymer 3.2.7.1. Fourier Transform Infrared Spectroscopy. Infra red spectrum of the PPS and PhPPS was recorded on Jasco FTIR- 401, Japan at transmittance mode. FTIR studies revealed the various characteristic peaks of OH, C O, O , and CH2 . As the original material does not contain any phosphate group in its structure, the typical stretching vibrations of P O & P O (at about 1200–1100/cm) were observed in the spectra of crosslinked sample. It was also noticeable that bands assigned to hydroxyl stretching vibration (3400/cm) were decreased in intensity after the crosslinking reaction. 3.2.7.2. Differential scanning calorimetry. DSC is a thermal analysis instrument that determines the temperature and heat flow associated with material transitions as a function of time and temperature. Stability of the native conformation is based on the thermodynamic relationships between enthalpy (H) and entropy (S). The more negative H, the greater is the stability [43]. The DSC thermograms (Fig. 7) of unmodified PPS showed endothermic peaks at 57.59 ◦ C, 77.95 ◦ C, 127.49 ◦ C and 151.74 ◦ C. The first endothermic peak in the thermogram of PPS may be due to decomposition of PPS. Shallow and broad endothermic peaks are obtained in the thermogram of PhPPS. Disappearance of the peak, larger enthalpy value and a shift in the last endothermic peak is observed in the thermogram of PhPPS. The decomposition temperature for PhPPS was higher as compared to PPS. This indicated that PhPPS had better thermal stability which was further supported by the higher enthalpy values for PhPPS. The enthalpy values for PPS were found to be −5.58 mW, −4.71 mW, −3.51 mW and −1.64 mW and for PhPPS were found to be −3.61 mW, −3.67 mW, −2.23 mW. Thus, both FT-IR and DSC indicated that PPS structure was modified by crosslinking. 3.2.7.3. X-Ray diffraction studies. Crystalline polymers are associated with partial alignment of their molecular chains. X-ray diffraction is one of the analytical methods to determine the crystalline nature of polymers. Regular arrangement of atoms and molecules produce sharp diffraction peaks whereas amorphous
Skin fur Eyes Mucous membrane Behavioral patterns Sleep Lethargy Salivation Diarrhea Coma Tremors
Normal Normal Normal Normal Normal Normal Normal Not observed Not observed Not observed
regions result in broad halos [44]. The PXRD study (Fig. 8) identified the crystalline nature of the PPS and PhPPS. PPS showed distinctive peaks at 2 angles of 30, 45, 55, 75 and 85◦ . PhPPS showed distinctive peaks at 2 angles of 111, 221, 232 and 265◦ . The diffractogram of PhPPS displayed peaks of greater intensity and at different 2 values than the parent polysaccharide. 3.2.7.4. Scanning electron microscopy. Scanning electron microscopy determines the amorphous or crystalline nature of the polymer chains and their influence on each other. The SEM micrographs (Fig. 9) indicate the crystalline structure of PPS and PhPPS. The micrographs of PPS suggested needle shaped crystal structure while the SEM micrographs of PhPPS indicated plate like crystalline layers. 3.2.8. Acute oral toxicity studies In the absence of human data, research with experimental animals is the most reliable means of detecting important toxic properties of chemical substances and for estimating risks to human and environmental health. The scientific rationale behind acute toxicity studies include examination of adverse effects that may occur on first exposure to a single dose of a substance and to identify whether toxicity occurs after continuous exposure to a substance [45]. The acute oral toxicity studies for PhPPS were conducted as per OECD guidelines 423. In principle, the method is not intended to allow the calculation of a precise LD50, but allows for the determination of defined exposure ranges where lethality is expected since death of a proportion of the animals is still the major endpoint of this test. To perform the acute oral toxicity studies, permission was obtained from Institutional Animal Ethics Committee—Approval No.: CPCSEA/IAEC/PT – 21/12–2K13. In acute oral toxicity studies, the mice showed sedation and lethargy during first four hours of observation (Table 4). A dose of 2000 mg/kg body weight of mice did not produce any mortality even after 14 days. This indicates that PhPPS is safe for human use. 4. Conclusion Psyllium polysaccharide has potential to be used as an excipient to develop novel formulation for the delivery of therapeutic agents. In this study, the polysaccharide was extracted from psyllium seeds (P. ovata) and chemical modification i.e., phosphorylation of the seed polysaccharide was carried out using Sodium tri-meta phosphate as the cross-linking agent. The modified polysaccharide (PhPPS) was found to have a high swelling index due to which it can be categorized as a hydrogel. The percent increase in swelling of PhPPS as compared to PPS was found to be 90.26%. The PPS and PhPPS mucilages of all strengths were found to have shear thinning properties. These findings are suggestive of the potential use of PhPPS as gelling and suspending agent which needs to be further investigated. PhPPS was found to have a mucoadhesive property
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Fig. 7. DSC spectra of PPS and PhPPS.
Fig. 8. PXRD spectra of PPS and PhPPS.
Fig. 9. SEM of PPS and PhPPS.
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which was comparable with carbopol. Acute oral toxicity studies also indicate the safety of the modified product. Thus we can conclude that the modified psyllium seed polysaccharide (PhPPS) can be used as a multifunctional excipient. Conflict of interest There is no conflict of interest, financial or otherwise associated with this project. Acknowledgement The authors would like to thank Dr. Ashwini Madgulkar Principal, AISSMS College of Pharmacy, Pune, Maharashtra, India for providing necessary facilities to carry out the research work. References [1] Ministry of Health and Family Welfare, Indian Pharmacopoeia, Government of India, Published by the Indian Pharmacopoeia Commission. 3 (2010) 2153. [2] R.C. Rowe, et al., Handbook of Pharmaceutical Excipients, 6th ed., Pharmaceutical Press, USA, 2009, pp. 1. [3] J. Swarbick, J.C. Boylan, Encyclopedia of Pharmaceutical Technology, Marcel Dekker, Inc., New York, 2002, pp. 1151. [4] A. Florence, D. Atwood, Physicochemical Principles of Pharmacy, 4th ed., Pharmaceutical Press, London, 2002, pp. 274. [5] S.R.J. Robbins, Gum arabic. In A review of recent trends in selected markets for water soluble gums, ODNRI Bulletin. 108 (1998) 18–33. [6] M. Nakano, Y. Nakamura, K. Juni, et al., Sustained release of sulfamethizole from agar beads after oral administration to humans, Chem. Pharm. Bull. 28 (1980) 2905–2908. [7] D.F. Durso, Handbook of Water Soluble Gums and Resins, McGraw Hill, Kingsport Press, New York, 1980, pp. 12. [8] T.R. Bhardwaj, M. Kanwar, M.R. Lal, et al., Natural gums and modified natural gums as sustained release carriers, Drug Dev. Ind. Pharm. 26 (2000) 1025–1038. [9] A. Desai, S. Shidhaye, S. Malke, et al., Use of natural release retardant in drug delivery system, Indian Drugs 42 (2005) 565–575. [10] B. Sujitha, et al., Natural polysaccharides, Int. J. Pharm. Technol. 4 (2013) 2347–2362. [11] G.E. Trease, W.C. Evans, A Text book of Pharmacognosy, 16th ed. W.B. Saunders, Indian ed., Noida (2008) 168–215. [12] United States Pharmacopoeia, The Official Compendia of Standards, Asian Edition, 2006, 1863–1864. [13] British Pharmacopoeia, Published on the recommendation of the Medicines Commission pursuant to the Medicines Act 1968, 3rd ed., 15–34. [14] A. Madgulkar, M.R.P. Rao, D. Warrier, in: Jean-Michelle Merillon, Kishan G. Ramavat (Eds.), Characterization of Psyllium (Plantago ovata) Polysaccharide and its Uses in ‘Polysaccharides, Bioactivity and Biotechnology’, Springer International Publishing, Switzerland, 2014, http://dx.doi.org/10.1007/978-3319-03751-6 49-1. [15] Truestar Health: http://www.truestarhealth.com/Notes/ 2150006html#Traditional-Use (accessed on 10.11.15). [16] H.M. Fischer, Y. Nanxiong, R.G.J. Ralph, L. Anderson, J.A. Marletta, The gel forming polysaccharide of psyllium husk (Plantago ovata Forsk), Carbohydr. Res. 339 (2004) 2009–2017. [17] J.F. Kennedy, J.S. Sandhu, Structural data for the carbohydrate of isapghula husk Plantago ovata Forsk, Carbohydr. Res. 75 (1979) 265–274. [18] B.S. Kaith, B.K. Kumar, Preparation of psyllium mucilage and acrylic based hydrogels and their application in selective absorption of water from different oil/water emulsion, Iran. Polym. J. 16 (8) (2007) 529–538. [19] B. Singh, V. Sharma, Design of psyllium-PVA-acrylic acid based novel hydrogels for use in antibiotic drug delivery, Int. J. Pharm. 389 (2010) 94–106. [20] S.J. Kim, K.J. Lee, S.I. Kim, Electrostimulus responsive behavior of poly(acrylic acid)/polyacrylonitrile semi-interpenetrating polymer network hydrogels, J. Appl. Polym. Sci. 92 (2004) 1473–1477.
[21] Q. Tang, X. Sun, Q. Li, J. Wu, J. Lin, Fabrication of a high strength hydrogel with an interpenetrating network structure, Colloids Surf. A: Physicochem. Eng. Aspects 346 (2009) 91–98. [22] V.D. Athawale, S.C. Rathi, Graft polymerization: starch as a model substrate, J. Macromol. Sci.: Polym. Rev. 39 (1999) 445–480. [23] M.R.P. Rao, M.P. Khambete, H.N. Lunavat, Study of rheological properties of psyllium polysaccharide and its evaluation as suspending agent, Int. J. Pharm. Technol. Res. 3 (2) (2011) 1191–1197. [24] M.R.P. Rao, P. Sadaphule, M. Khambete, H. Lunawat, K. Thanki, N. Gabhe, Characterization of psyllium (Plantago ovata) polysaccharide and its use as a binder in tablets, Ind. J. Pharm. Edu. Res. 47 (2) (2013). [25] S. Pal, R. Das, in: Susheel Kalia, M.W. Sabaa (Eds.), Polysaccharide Based Graft Copolymers for Biomedical Applications in Polysaccharide Based Graft Copolymers, Springer-Verlag, Berlin, Heidelberg, 2015, http://dx.doi.org/10. 1007/978-3-642-36566-9 9,2013. [26] V. Dulong, S. Lack, et al., Hyaluronan-based hydrogels particles prepared by crosslinking with trisodium trimetaphosphate, synthesis and characterization, Carbohydr. Polym. 57 (2004) 1–6. [27] Organization of Economic Co-operation & Development (OECD), The OECD guidelines for testing of chemical: 420 Acute Oral Toxicity. France (2001). [28] K.R. Khandelwal, Practical Pharmacognosy, Techniques and experiments. 19th ed. Nirali Prakashan, Pune (MH) (2008) 159. [29] D. Krishnarajan, et al., Effect of cellulose and non-cellulose polymers on ciprofloxacin extended release tablets, J. Chem. Pharm. Res. 4 (7) (2012) 3617–3623. [30] S. Patrick, Martin’s Physical Pharmacy and Pharmaceutical Sciences, 5th ed., Lippincott Williams and Wilkins, Philadelphia, 2000, pp. 587–588. [31] V. Chinampudur, S.R. Boddapati, H.S. Srikanth, J.R. Edwin, A.J. Joshua, et al., Evaluation of turmeric polysaccharide extract: assessment of mutagenicity and acute oral toxicity, BioMed Res. Int. (2013), http://dx.doi.org/10.1155/ 2013/158348, Article ID 158348. [32] A.R. Gennaro, Remington: The Science and Practice of Pharmacy, vol. 1, 20th ed., Lippincott Williams and Wilkins, Printed in India, 2000, pp. 409–412. [33] S.P. Basavaraj, S. Vinayak, A.R. Kulkarni, Development and evaluation of psyllium seed husk polysaccharide based wound dressing films, Orient. Pharm. Exp. Med. 11 (2011). [34] T. Kasemsuwan, T. Bailey, J. Jane, Preparation of clear noodles with mixtures of tapioca and high-amylose starches, Carbohydr. Polym. 32 (3–4) (1998) 301–312. [35] K. Muhammad, F. Hussin, Y.C. Ghazali, J.F. Kennedy, Effect of pH on phosphorylation of sago starch, Carbohydr. Polym. 42 (1) (2000) 85–90. [36] G. Kabir, B. Yagen, A. Penhasi, A. Rubinstein, Phosphated crosslinking guar for colon—specific drug delivery I, preparation and physical characterization, J. Control. Release 63 (1–2) (2000) 121–127. [37] A. Bejenariu, et al., Trisodium trimetaphosphate crosslinked xanthan networks: synthesis, swelling, loading and releasing behavior, Polym. Bull. (2009) 1–14. [38] L. Beng-zheng, W. Li-jun, L. Dong, Z. Zhing-jie, et al., Physical properties and loading capacity of starch-based microparticles cross-linked with trisodium trimetaphosphate, J. Food Eng. 92 (2009) 255–260. [39] A. Martin, J. Swarbrick, A. Cammarata, Physical Pharmacy-Physical Chemical Principles Pharmaceutical Sciences, 503, Varghese publishing house, Bombay, 2001, pp. 513–514. [40] A. Barnes Howard, Thixotropy a review, J. Non-Newton. Fluid Mech. 70 (1997) 1–33. [41] B.M. Boddupalli, Z.N.K. Mohammed, R.A. Nath, B. David, Mucoadhesive drug delivery system: an overview, J. Adv. Pharm. Technol. Res. 1 (4) (2010) 381–387. [42] H. Hagerstrom, K. Edsman, M. Stromme, Low-frequency dielectric spectroscopy as a tool for studying the compatibility between pharmaceutical gels and mucus tissue, J. Pharm. Sci. 92 (2003) 1869–1881. [43] G. Hohne, W. Hemminger, H.J. Flammersheim, Differential Scanning Calorimetry: An Introduction for Practitioners, Springer, Berlin, Germany, 1996. [44] S. Kavesh, J.M. Schultz, Meaning and measurement of crystallinity in polymers: a review, Polym. Eng. Sci. 9 (5) (1969) 331–338. [45] E. Walum, Acute Oral Toxicity, Environmental Health Perspectives 106, Suppl. 2 April (1998) 497–503.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Phosphorylation of psyllium seed polysaccharide and its characterization Monica R.P. Rao ∗ , Deepa U. Warrier, Snehal R. Gaikwad, Prachi M. Shevate Department of Pharmaceutics, AISSMS College of Pharmacy, Near RTO, Kennedy Road, Pune 411001, Maharashtra, India
a r t i c l e
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Article history: Received 5 November 2015 Received in revised form 11 December 2015 Accepted 13 December 2015 Available online 6 January 2016 Keywords: Psyllium seeds Phosphorylation Swelling index Rheology Mucoadhesion
a b s t r a c t Psyllium is widely used as a medicinally active natural polysaccharide for treating conditions like constipation, diarrhea, and irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis and colon cancer. Studies have been performed to characterize and modify the polysaccharide obtained from psyllium seed husk and to evaluate its use as a pharmaceutical excipient, but no studies have been performed to evaluate the properties of the polysaccharide present in psyllium seeds. The present study focuses on phosphorylation of psyllium seed polysaccharide (PPS) using sodium tri-meta phosphate as the cross-linking agent. The modified phosphorylated psyllium seed polysaccharide was then evaluated for physicochemical properties, rheological properties, spectral analysis, thermal analysis, crosslinking density and acute oral toxicity studies. The modified polysaccharide (PhPPS) has a high swelling index due to which it can be categorized as a hydrogel. The percent increase in swelling of PhPPS as compared to PPS was found to be 90.26%. The PPS & PhPPS mucilages of all strengths were found to have shear thinning properties. These findings are suggestive of the potential use of PhPPS as gelling & suspending agent. PhPPS was found to have a mucoadhesive property which was comparable with carbopol. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Pharmaceutical excipients are substances other than the active pharmaceutical ingredient—that are used in the finished dosage form or may be used as processing aids in the manufacture of active pharmaceutical ingredients [1]. The properties of the final dosage form (i.e., its bioavailability and stability) depend on the excipients chosen, their concentration and interaction with both the active compound and each other. A detailed knowledge of the physical and chemical properties as well as the safety, handling and regulatory status of these materials is essential for formulators throughout the world [2]. Majority of the widely used excipients are obtained from synthetic sources. Although excipients are considered to be pharmacologically inert, it is now well accepted that some have potential for untoward effects. Besides this, problems of incompatibility, stability and high cost are evident in synthetic excipients [3]. There are several polysaccharides of plant origin which have been used as excipients. Polysaccharides consist of a large number of polymeric carbohydrate molecules composed of long chains of monosaccharide units linked together in a long chain by gly-
∗ Corresponding author. E-mail address: monicarp [email protected] (M.R.P. Rao). http://dx.doi.org/10.1016/j.ijbiomac.2015.12.043 0141-8130/© 2015 Elsevier B.V. All rights reserved.
cosidic bond [4]. The pharmaceutical excipients of plant origin, like starch, agar, alginates, carrageenan, guar gum, xanthan gum, gelatin, pectin, acacia, tragacanth, cellulose etc. find applications in the pharmaceutical industry as binding agents, disintegrants, sustaining agents, protective colloids, thickening agents, suspending agents, emulsifiers, gelling agents, bases in suppositories, stabilizers, and coating material [5–10]. Psyllium has been in use as a medicinal agent since ancient times throughout the world and it is official in compendia of many countries. The dried, ripe seeds of Plantago afra (Plantago psyllium), Plantago indica (Plantago arenaria) & Plantago ovata (Plantaginaceae) are used in medicine. Seeds of P. ovata Forsk are commercially referred to as Indian psyllium or Ispaghula [11–13]. Psyllium is used worldwide for the treatment of constipation, diarrhea, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, colon cancer, diabetes and hypercholesterolemia [14,15]. Psyllium seed mucilage contains 22.6% arabinose, 74.65% xylose, traces of other sugars and 35% non-reducing terminal residues [16]. Researchers have studied the physiologically active, gel-forming fraction of the alkali-extractable polysaccharides of P. ovata Forsk seed husk. The polysaccharide from the seed husk has been fractionated and evaluated for gelling ability [17]. Kaith and Kumar carried out grafting of acrylic acid onto psyllium backbone
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Fig. 1. Reaction steps and mechanism of crosslinking.
Fig. 2. Percent swelling indexs.
Fig. 3. RMB absorbed (PhPPS).
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via free radical mechanism and studied its water uptake in oil/water emulsion for use in oil refineries [18]. In the last decade and half, a plethora of novel carriers have been developed which have brought about a phenomenal change in the profile of drug delivery. Among different drug delivery devices, hydrogels, especially based on polysaccharides have attained lot of prominence for achieving controlled release of drugs [19]. Hydrogels are three-dimensional polymeric networks that swell considerably in presence of water [20]. However, hydrogels based on natural polymers have limited applications due to poor mechanical strength of the gel [21]. Thus modification of polysaccharides to develop hydrogels is a powerful tool to control the interaction of the polymer with drugs, to enhance the load capacity and to tailor the release profile of the drug. Grafting and cross linking are common methods to modify and improve the functional properties of polysaccharides [22]. In previous studies, the authors have carried out extraction and characterization of psyllium seed polysaccharide [23]. Swelling studies in different media revealed a manifold increase in volume pointing to its hydrogel properties. Investigation of its rheological properties indicated thixotropic nature with a characteristic spur value at higher polysaccharide concentration. The psyllium seed polysaccharide was also found to have satisfactory binder properties for use in tablets and displayed lower yield values with increasing concentration [24]. In present study an attempt was made by the authors to improve the functionality of the polysaccharide by chemical modification and compare its properties with the parent polysaccharide. In the course of their studies, the authors observed a change in rheological properties of the psyllium seed polysaccharide compared to their previous findings. These changes, though not very significant, could have far reaching consequences in their applicability. It has, of course been reported that natural polymers suffer from disadvantages like uncontrolled rate of hydration, changes in viscosity on storage and microbial contamination [25]. Thus chemical modification of the psyllium seed polysaccharide by phosphorylation was attempted in the present study using sodium tri-meta phosphate as cross-linking agent. The modified phosphorylated psyllium seed polysaccharide was then evaluated for physicochemical properties, rheological properties, spectral analysis, thermal analysis and crosslinking density. Acute oral toxicity studies were also performed for the phosphorylated psyllium seed polysaccharide. 2. Materials and methods 2.1. Materials Psyllium seeds were obtained from Manakarnika Aushadalay, Pune & authenticated at Agarkar Research Institute, Pune. Sodium tri-meta phosphate was a gift sample obtained from Sigma–Aldrich. All the other chemicals, solvents and reagents used were of analytical grade and were procured locally. To perform the acute oral toxicity studies, permission was obtained from Institutional Animal Ethics Committee—Approval No.: CPCSEA/IAEC/PT–21/12–2K13. 2.2. Methods 2.2.1. Extraction of seed gum The psyllium seeds were soaked in deionized water at 80 ◦ C for 2 h and kept overnight. Sodium hydroxide (NaOH) 0.5 M was added while stirring (200 rpm for 15 min) to separate the polysaccharide from seeds and resulting slurry passed through 12#. The psyllium polysaccharide (PPS) was reprecipitated by addition of 2 M HCl to the filtrate. The precipitate was washed with deionized water to remove traces of acid and separated by centrifugation at
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3000 rpm for 15 min. The residue was dried overnight in a tray drier at 50–60 ◦ C. 2.2.2. Chemical crosslinking of PPS Sodium tri-meta phosphate (STMP- 1 g) was dissolved in 50 ml of deionised water. This was added to 5 ml of 0.1 N NaOH. PPS (1 g) in 50 ml of water was then added slowly with stirring. The reaction mixture (100 ml) was stirred for 2 h, poured into each of five petri dishes (20 ml each) and dried at 60 ◦ C for 24 h. The dried complex (modified gum) was powdered, passed through 120 # and used for evaluation. The same procedure was followed by using 0.2 N NaOH as the catalyst [26]. 2.3. Characterization of psyllium seed polysaccharide (PPS) & phosphorylated psyllium seed polysaccharide (Ph PPS) The PPS and PhPPS were characterized for physicochemical properties (solubility studies, swelling index, micromeritic properties, water uptake, and loss on drying), spectral analysis and thermal analysis. Acute oral toxicity studies for PhPPS were also conducted as per OECD Guidelines 423 [27]. 2.3.1. Swelling index Swelling behavior of PPS and PhPPS was studied as a function of pH. It was allowed to swell for 24 h in solutions of different pH such as 0.1 N HCl (pH 1.1), phosphate buffer pH 6.8, deionized water and 0.5 M NaOH (pH 13.7). For this, 1 g of PPS and PhPPS were added separately in 25 ml of the respective media in stoppered volumetric flasks at 37 ◦ C and kept overnight with intermittent shaking. Volume occupied by the PPS was measured using a measuring cylinder and swelling index was calculated from the initial and final volume of PPS and PhPPS respectively [28]. 2.3.2. Solubility profile The solubility of PPS and PhPPS was evaluated at 37 ◦ C in various solvents such as acetone, alcohol, ether, chloroform, dichloromethane, dimethyl amine, tri methyl amine, diethyl ether, ethyl acetate, dimethyl sulfoxide, isopropyl alcohol, 0.5 N sodium hydroxide, N-methyl-2-pyrolidone, N,N-dimethyl formamide, conc. hydrochloric acid and conc. nitric acid. 2.3.3. Physicochemical properties The bulk density, tapped density, angle of repose, Hausner’s ratio and Carr’s index of PPS and PhPPS was evaluated [29]. The moisture content of PPS and PhPPS was determined by using infrared moisture balance (Make: Gurunanak Instruments, Model: 6084). For water uptake studies, PPS and PhPPS was immersed in 25 ml deionized water and were lightly patted with tissue paper to remove excess surface water. The study was carried out at different time intervals (2, 4, 8, 12 & 24 h) and water uptake was computed gravimetrically according to following equation, Wateruptake(%) = (Ts – Ti)/Ti × 10 where Ts is the weight of the swollen polymer and Ti is the initial weight of the polymer. The pH of 1% solution of the PPS and PhPPS was determined using a digital pH meter (EI, DELUX 101). Loss on drying was determined by drying a fixed weight of the samples in an oven for 5 h at 105 ◦ C in glass stoppered bottles. 2.3.4. Crosslinking density The PPS and PhPPS (1 g) were placed separately in dialysis bags (MEMBRA- CELL, MD 77-14 × 100 CLR, SIGMA) and then immersed in 100 ml of methylene blue solution (10−5 mol/l). Concentration of methylene blue in medium outside the dialysis bag was measured
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Fig. 4. Apparent viscosity vs rate of shear for PPS and PhPPS.
by spectrophotometry (Spectrometer UV/vis Lab India) at wavelength of 665 nm until no decrease in methylene blue absorbance was observed. PhPPS prepared using two different concentrations of STMP were investigated i.e., 1 g and 2 g of STMP per gram of PPS. The crosslinking density was estimated by determination of the relative amount of methylene blue (RMBabsorbed ) bound to the particles using equation,
brought toward the mucosa at a constant speed of 0.5 mm/s until a predetermined compressive force of 1 N was applied for 60 s. The probe was then removed at 5 mm/s to a distance of 15 mm and maximum detachment force (kg) was determined for each sample.
RMBabsorbed = AMB − APhPPS /AMB – APPS
The Fourier Transform Infrared Spectroscopy (FTIR) spectra of PPS and PhPPS were recorded using JASCO, Japan V 462. The thermograms of PPS and PhPPS were obtained by a Differential Scanning Calorimeter (DSC 821, Make: METTLER) at heating rate of 10 ◦ C/min from 30 to 300 ◦ C in nitrogen atmosphere (30 ml/min). X-ray diffraction patterns were recorded in the reflection mode on a Siemens D-500 X-ray diffractometer. Diffractograms were registered at Bragg angle (2) range of 5–40◦ at a scan rate of 2.5◦ per minute and step size of 0.02◦ .
where, AMB = initial absorbance of methylene blue solution, APhPPS = absorbance of methylene blue solution containing phosphorylated PPS, APPS = absorbance of methylene blue solution containing native PPS. The theoretical crosslinking density was measured using STMP as standard. 2.3.5. Rheological properties The mucilage’s of PPS and PhPPS were prepared in distilled water in strengths of 2, 2.5, 3, 5 and 10% w/v. The mucilages were homogenized using tissue homogenizer to break any lumps and subjected to rheometry studies at different shear rates using Brookfield digital viscometer DV-E (Spindle 61). The rheograms were plotted and the thixotropic nature of the polymers was studied by subjecting the mucilages to increasing and decreasing shear rates [30].
2.4. Spectral and thermal analysis
2.4.1. Scanning electron microscopy Surface topography of PPS and PhPPS was studied using scanning electron microscopy (JSM-6360A-MAKE: JEOL). Each sample was mounted on to an aluminium stub using two-way adhesive tape and then coated with gold-palladium alloy using a sputtering device under a nitrogen atmosphere. The samples were examined with 100× and 2000× magnification. 2.5. Acute oral toxicity studies for PhPPS
2.3.6. Mucoadhesive strength Mucoadhesive property of PPS and PhPPS was evaluated using a texture analyzer (CEB Texture Analyzer, Make-Brookfield Engineering Labs, Inc., Model Texture Pro CT V1.4 Build 17). Fresh sheep mucosa was obtained from the local slaughterhouse. PPS and PhPPS were carefully attached to the cylindrical probe (TA probe) by a bioadhesive tape. The upper platform was moved downward manually near to the mucosa surface and then the polymer sample was
Acute oral toxicity study was conducted according to OECD guidelines 423. The study was performed in healthy nulliparous and non-pregnant adult female albino Swiss mice (20–35 g). Pilot study was carried using six albino Swiss mice in a group with the starting dose of 300 mg/kg body weight. Animals were monitored continuously immediately after dosing up to first 4 h using video tracking system for behavioral, neurological and autonomic param-
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Fig. 5. Shear stress vs rate of shear for PPS and PhPPS.
eters. The animals were under supervision after 24 h and 14 days for any sign of toxicity or mortality. When 300 mg/kg dose was proved non-toxic, in the main study, 2000 mg/kg dose was screened for checking toxicity using the same procedure mentioned above taking 6 animals in a group [31]. 3. Results and discussion 3.1. Crosslinking of psyllium seed polysaccharide Natural polysaccharides lack certain properties and when the natural polymers are crosslinked, the crosslinks tie the macromolecular chains together by primary covalent bonds, transforming each particle into a single, giant molecule [32]. Chemical derivatization of the polysaccharide obtained from psyllium seed husk has been carried out with the objective of improving their physical and physicochemical properties so as to enable their use as multifunctional pharmaceutical excipients. Various cross linkers that have been studied include methacrylamide, N,N-methylene bisacrylamide and polymethacrylamide [33]. Sodium tri-meta phosphate is an efficient cross-linking agent and is a solid of low toxicity with no reports of adverse effects in humans. Phosphorylation using STMP as a crosslinking agent has been used as a method of chemical derivatization for starches [34,35]. Guar gum has been crosslinked using STMP for applications in colon-specific drug delivery [36]. STMP has also been reported as an effective cross-linker of hyaluronan and karaya gum for the purpose of synthesizing gels and sustained release tablets. The crosslinking reaction occurred through the hydroxyl groups of the
polysaccharide and led to ester linkages. Hence, STMP was selected as the cross linker. The advantage of phosphorylation is that di or tri esters can be formed by varying the pH conditions and different grades of crosslinked polymer can be obtained. Changing the amount of phosphorylating agent can regulate the degree of substitution of the products and polymers with adapted degree of substitution can be obtained. Sodium hydroxide provides the necessary alkalinity for the crosslinking reaction with STMP. The mechanism involved in synthesis of modified psyllium seed polysaccharide is reaction between hydroxyl groups of the PPS and metaphosphate groups in STMP leading to phosphate ester (O P O) linkages between two polysaccharide moieties (Fig. 1). The effect of sodium hydroxide concentration on the swelling index of PhPPS was observed (Table 1). The percent swelling of PhPPS was found to increase with increasing concentration of sodium hydroxide from 0.1 to 0.2 N NaOH. This may be due to increase in crosslinking between PPS and STMP at 0.2 N NaOH. Swelling index of PhPPS in deionized water prepared using 0.1 N NaOH as catalyst was found to be 1720 ± 0.12% and that of PhPPS prepared using 0.2 N NaOH as catalyst was found to be 1876 ± 0.18%. 3.2. Characterization of psyllium seed polysaccharide (PPS) and phosphorylated psyllium seed polysaccharide (PhPPS) 3.2.1. Swelling profile of PPS and PhPPS Swelling is defined as the uptake of liquid by a polymer with an increase in volume. By controlling the swelling of the polymer, the dosage form can release the drug in a controlled manner to a desired
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Table 1 Percent swelling index. Medium
Swelling index of PhPPS prepared by using 0.1 N NaOH as catalyst (%)
Deionised water Phosphate buffer pH 6.8 0.1 N HCl 0.5 N NaOH
1720 412 846 292
± ± ± ±
3.12 2.23 3.13 2.25
Swelling index of PhPPS prepared by using 0.2 N NaOH as catalyst (%) 1876 441 948 341
± ± ± ±
2.18 2.19 3.24 2.23
n = 3.
site. The swelling profile of PPS and PhPPS in different media is shown in Fig. 2. For PPS, the maximum swelling was found in phosphate buffer pH 6.8 with 1242% swelling, whereas no swelling was observed in 0.5 N NaOH. For PhPPS, the maximum swelling was found in deionized water with 1876% swelling and swelling was also observed in 0.5 N NaOH with 341% swelling. In all the solutions except phosphate buffer pH 6.8, the swelling of PhPPS was found to be higher than that of corresponding PPS. The percent increase in swelling of PhPPS as compared to PPS was found to be 90.26%. The decreased swelling in 0.5 N NaOH may be attributed to ionization of the phosphate groups grafted on the polymer leading to electrostatic repulsions between the chains of PPS. This could further lead to reduced water entrapment and hence lower swelling. At acidic pH, it may be presumed that H+ ions will negate the effect of phosphate anions and diminish the repulsive interactions between the chains of PPS, hence the structural integrity of polymer is maintained and more water may be entrapped. Therefore, swelling of polysaccharide is more in 0.1N HCl. Bejenariu et al. observed similar effect for xanthan gum [37]. At pH 13, the swelling of STMP crosslinked xanthan was found to be maximum. The extensive swelling that PPS and PhPPS exhibit in water and 0.1N HCl is indicative of their hydrogel nature which can be exploited to design sustained release dosage forms. Swelling could be a result of entanglement of the polysaccharide chains and development of intraand inter-molecular hydrogen bonds between the polysaccharide and water causing greater amount of water to be entrapped within macromolecular chains. Due to joining of two PPS molecules by phosphate ester groups, the synthetic bridges so formed, reinforce the natural hydrogen bonds, delaying the speed of granule swelling and reducing the rupture of the swollen polysaccharide molecules. It can also be presumed that crosslinking interferes with free access of water to PPS hydroxyl groups. However, for PhPPS, the gradual increase in swelling observed is caused by chain straightening which enhances water penetration into the polymer’s network. 3.2.2. Solubility profile Solubility can be defined as the spontaneous interaction of two or more substances to form a homogenous molecular dispersion. The PPS was found to be soluble in strong ammonia solution and dimethyl sulfoxide after 15 min sonication at room temperature whereas, PhPPS was found to be soluble only in conc. hydrochloric acid and conc. nitric acid. PhPPS was found to form a gel in chloroform and 0.5 N NaOH. 3.2.3. Physicochemical properties Evaluation of physicochemical properties of PPS and PhPPS revealed a remarkable difference between the two polysaccharides (Table 2). However the micromeritic properties of PPS and PhPPS were found to be almost similar. The angle of repose of PPS was found to be 29.74◦ and that of PhPPS was found to be 24.97◦ . The study of Carr’s index and Hausner’s ratio further supported the results of angle of repose. These findings reveal that the flow properties of PhPPS are good as compared to PPS and are suggestive of its use as a directly compressible excipient. The moisture content of PhPPS as determined by loss on drying method was lower as compared to PPS. This further supports the better flow properties
of PhPPS. The pH of PPS in deionised water was found to be 4.46 and that of PhPPS was found to be 7.90. The lower pH of PPS is due to its acidic nature. It may be proposed that due to the crosslinking reaction of PPS with STMP, sodium and phosphate groups are introduced in the PPS, due to which the pH of PhPPS increased. 3.2.4. Crosslinking density The crosslinking of polymer chains is of primary importance in controlling many polymer properties. The crosslinking density signifies the quantity of crosslinking agent that has been incorporated in the polymer. Methylene blue (MB) is a cationic molecule with a high affinity for negatively charged solids and phosphate groups are anionic in nature [38]. An absorption kinetic study was carried out to determine the equilibrium time after which no further reduction in MB concentration could be detected. The number of phosphate groups was found to increase with the level of crosslinking agent and reached a limiting value of absorbed methylene blue at 7 h at both levels of the cross linker (Fig. 3). A 14% increase in crosslinking density was evident with PhPPS at higher level of STMP after 7 h. 3.2.5. Rheological properties Rheology is an important physical property affecting the physical stability and ease of use of any liquid and semisolid preparation. The PPS and PhPPS mucilages of all strengths were found to have shear thinning properties as evident from the graphs (Fig. 4) of apparent viscosity vs. rate of shear. The results indicated pseudoplastic behavior i.e., decrease in viscosity with increase in shear rate which is characteristic of polymeric systems. The decrease in viscosity resulted from the shearing action on long-chain molecules of the polymer. As the shearing stress is increased, normally disarranged molecules begin to align their long axes in the direction of flow. This orientation reduces internal resistance of the material and allows a greater rate of shear at each successive shearing stress. In addition, some of the solvent associated with the molecules may be released, resulting in an effective lowering of both the concentration and the size of the dispersed molecules. This too decreases the apparent viscosity. These findings are suggestive of the potential use of PhPPS as a gelling agent and suspending agent [39]. The rheogram (Fig. 5) for shear rate vs. shear stress for both PPS and PhPPS mucilage were found to produce a characteristic bulge in the upcurve. The bulge indicated swelling of PPS and structural integrity at low shear values which suddenly lose their rigidity as shear is increased. This loss in rigidity is not gradual and leads to a bulge. The rheogram for shear rate vs. shear stress for 5 and 10% PPS and PhPPS mucilages showed a characteristic spur value indicating a sudden breakdown in the structure at a low shear rate. At higher strengths, the mucilage has a very rigid structure which can be attributed to the formation of hydrogen bonds between the polysaccharide and water. The spur value was found to be greater than 100/s for both PPS and PhPPS with PhPPS exhibiting a spur value of approximately 120/s and PPS showing spur value at nearly 135/s. However, at lower strengths, spur value was absent which was indicative of the greater structural flexibility. Thixotropy is a desirable property in liquid pharmaceutical systems that ideally should have a high consistency in the container as well as pour or spread easily. The PPS and PhPPS mucilages of
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Table 2 Micromeritic properties of PPS and PhPPS.
Bulk density Tapped density Angle of repose Hausner’s ratio Carr’s index Moisture content pH Water uptake (end of 24 h) Loss on drying
Psyllium seed polysaccharide
Phosphorylated psyllium seed polysaccharide
0.7142 g/cc ± 0.23 0.833 g/cc ± 0.25 29.74◦ ± 1.72 1.1667 ± 0.38 14.301% ± 1.37 1% ± 0.27 4.46 ± 0.56 884.32% ± 18.19 0.16% ± 0.06
0.6418 g/cc ± 0.28 0.6891 g/cc ± 0.19 24.97◦ ± 1.68 1.0738 ± 0.25 6.86402% ± 1.28 0.5% ± 0.11 7.9 ± 0.52 1463.2% ± 16.23 0.14% ± 0.04
n = 3.
Fig. 6. Hysteresis loop for PPS and PhPPS.
all strengths were subjected to increasing and decreasing shear rates and the rheograms were plotted. Presence of a hysteresis loop was indicative of thixotropic nature of the mucilages (Fig. 6). The rheogram shows that for all the mucilages, the down curve is displaced to the left of the upcurve. This indicated a breakdown of structure (and hence shear thinning) that does not reform immediately when stress is reduced. The degree of thixotropy is represented by the area of hysteresis loop. The area of hysteresis loop was calculated using planimeter method. The area of hysteresis loop for 3% PPS mucilage was found to be 11,236 mm2 whereas for the 5% PPS mucilage it was 9604 mm2 . The area of hysteresis loop for 3% PhPPS mucilage was found to be 15,376 mm2 whereas for the 5% PhPPS mucilage it was 12,100 mm2 . The PPS and PhPPS mucilages of lower concentration had greater degree of thixotropy than the PPS and PhPPS mucilages of higher concentration. This
can be due to the fact that mucilages of higher concentration have high structural rigidity which leads to lesser breakdown in structure during shearing. The presence of hysteresis loop is a desirable feature while formulating a gel because the gel can be easily applied to a surface (through structure breakdown in spreading) and can, at rest rebuild its structure and viscosity so that it does not drip and run [40]. 3.2.6. Mucoadhesive strength Mucoadhesion is considered to occur in four major stages: wetting, interpenetration, adsorption and formation of secondary chemical bonds between mucus membrane and polymers. The mucoadhesive strength is affected by molecular weight of polymer, contact time with membrane and degree of swelling of the polymer. The mucoadhesive strength of PPS and PhPPS was stud-
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Table 3 Mucoadhesive force of polymers.
Table 4 Acute oral toxicity observations at a dose of 2000 mg/kg body weight of animal.
Polymer
Mucoadhesive force (g)
Sr. no. Parameter
Observation (During 4 h) Observation (After 24 h)
PPS PhPPS Carbopol
5.2 ± 1.4 6.7 ± 1.5 7.2 ± 1.8
1) 2) 3) 4) 5) 6) 7) 8) 9) 10)
Normal Normal Normal Normal Observed Observed Normal Not observed Not observed Not observed
ied using sheep intestinal mucosa. The adhesive force was obtained from software TexturePro CT V1.4 Build 17 Brookfield Engineering Labs, Inc. The results for mucoadhesive force are shown in Table 3. It was found that PhPPS had higher mucoadhesive strength than PPS and comparable mucoadhesion with carbopol which is widely used for this purpose. Mucoadhesion is a complex process with many theories being reported in literature [41]. The mucoadhesive substance should be capable of favoring both chemical and mechanical interactions with mucous such as molecules with hydrogen bond building groups (–OH, –COOH), an anionic surface charge, high molecular weight, flexible chains and surface-active properties, which help in spreading throughout the mucus layer, can present mucoadhesive properties [42]. The mucoadhesive properties of PhPPS may be attributed to the anionic nature of phosphate groups which are capable of forming ionic interactions with biomolecules which are present in mucous. Besides this the free hydroxyl groups of the polysaccharides may also form hydrogen bonds with the mucosal surface. Thus, PhPPS could be investigated further as a mucoadhesive agent to formulate gastro retentive mucoadhesive gels and microspheres. 3.2.7. Interpretation of crosslinked polymer 3.2.7.1. Fourier Transform Infrared Spectroscopy. Infra red spectrum of the PPS and PhPPS was recorded on Jasco FTIR- 401, Japan at transmittance mode. FTIR studies revealed the various characteristic peaks of OH, C O, O , and CH2 . As the original material does not contain any phosphate group in its structure, the typical stretching vibrations of P O & P O (at about 1200–1100/cm) were observed in the spectra of crosslinked sample. It was also noticeable that bands assigned to hydroxyl stretching vibration (3400/cm) were decreased in intensity after the crosslinking reaction. 3.2.7.2. Differential scanning calorimetry. DSC is a thermal analysis instrument that determines the temperature and heat flow associated with material transitions as a function of time and temperature. Stability of the native conformation is based on the thermodynamic relationships between enthalpy (H) and entropy (S). The more negative H, the greater is the stability [43]. The DSC thermograms (Fig. 7) of unmodified PPS showed endothermic peaks at 57.59 ◦ C, 77.95 ◦ C, 127.49 ◦ C and 151.74 ◦ C. The first endothermic peak in the thermogram of PPS may be due to decomposition of PPS. Shallow and broad endothermic peaks are obtained in the thermogram of PhPPS. Disappearance of the peak, larger enthalpy value and a shift in the last endothermic peak is observed in the thermogram of PhPPS. The decomposition temperature for PhPPS was higher as compared to PPS. This indicated that PhPPS had better thermal stability which was further supported by the higher enthalpy values for PhPPS. The enthalpy values for PPS were found to be −5.58 mW, −4.71 mW, −3.51 mW and −1.64 mW and for PhPPS were found to be −3.61 mW, −3.67 mW, −2.23 mW. Thus, both FT-IR and DSC indicated that PPS structure was modified by crosslinking. 3.2.7.3. X-Ray diffraction studies. Crystalline polymers are associated with partial alignment of their molecular chains. X-ray diffraction is one of the analytical methods to determine the crystalline nature of polymers. Regular arrangement of atoms and molecules produce sharp diffraction peaks whereas amorphous
Skin fur Eyes Mucous membrane Behavioral patterns Sleep Lethargy Salivation Diarrhea Coma Tremors
Normal Normal Normal Normal Normal Normal Normal Not observed Not observed Not observed
regions result in broad halos [44]. The PXRD study (Fig. 8) identified the crystalline nature of the PPS and PhPPS. PPS showed distinctive peaks at 2 angles of 30, 45, 55, 75 and 85◦ . PhPPS showed distinctive peaks at 2 angles of 111, 221, 232 and 265◦ . The diffractogram of PhPPS displayed peaks of greater intensity and at different 2 values than the parent polysaccharide. 3.2.7.4. Scanning electron microscopy. Scanning electron microscopy determines the amorphous or crystalline nature of the polymer chains and their influence on each other. The SEM micrographs (Fig. 9) indicate the crystalline structure of PPS and PhPPS. The micrographs of PPS suggested needle shaped crystal structure while the SEM micrographs of PhPPS indicated plate like crystalline layers. 3.2.8. Acute oral toxicity studies In the absence of human data, research with experimental animals is the most reliable means of detecting important toxic properties of chemical substances and for estimating risks to human and environmental health. The scientific rationale behind acute toxicity studies include examination of adverse effects that may occur on first exposure to a single dose of a substance and to identify whether toxicity occurs after continuous exposure to a substance [45]. The acute oral toxicity studies for PhPPS were conducted as per OECD guidelines 423. In principle, the method is not intended to allow the calculation of a precise LD50, but allows for the determination of defined exposure ranges where lethality is expected since death of a proportion of the animals is still the major endpoint of this test. To perform the acute oral toxicity studies, permission was obtained from Institutional Animal Ethics Committee—Approval No.: CPCSEA/IAEC/PT – 21/12–2K13. In acute oral toxicity studies, the mice showed sedation and lethargy during first four hours of observation (Table 4). A dose of 2000 mg/kg body weight of mice did not produce any mortality even after 14 days. This indicates that PhPPS is safe for human use. 4. Conclusion Psyllium polysaccharide has potential to be used as an excipient to develop novel formulation for the delivery of therapeutic agents. In this study, the polysaccharide was extracted from psyllium seeds (P. ovata) and chemical modification i.e., phosphorylation of the seed polysaccharide was carried out using Sodium tri-meta phosphate as the cross-linking agent. The modified polysaccharide (PhPPS) was found to have a high swelling index due to which it can be categorized as a hydrogel. The percent increase in swelling of PhPPS as compared to PPS was found to be 90.26%. The PPS and PhPPS mucilages of all strengths were found to have shear thinning properties. These findings are suggestive of the potential use of PhPPS as gelling and suspending agent which needs to be further investigated. PhPPS was found to have a mucoadhesive property
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Fig. 7. DSC spectra of PPS and PhPPS.
Fig. 8. PXRD spectra of PPS and PhPPS.
Fig. 9. SEM of PPS and PhPPS.
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which was comparable with carbopol. Acute oral toxicity studies also indicate the safety of the modified product. Thus we can conclude that the modified psyllium seed polysaccharide (PhPPS) can be used as a multifunctional excipient. Conflict of interest There is no conflict of interest, financial or otherwise associated with this project. Acknowledgement The authors would like to thank Dr. Ashwini Madgulkar Principal, AISSMS College of Pharmacy, Pune, Maharashtra, India for providing necessary facilities to carry out the research work. References [1] Ministry of Health and Family Welfare, Indian Pharmacopoeia, Government of India, Published by the Indian Pharmacopoeia Commission. 3 (2010) 2153. [2] R.C. Rowe, et al., Handbook of Pharmaceutical Excipients, 6th ed., Pharmaceutical Press, USA, 2009, pp. 1. [3] J. Swarbick, J.C. Boylan, Encyclopedia of Pharmaceutical Technology, Marcel Dekker, Inc., New York, 2002, pp. 1151. [4] A. Florence, D. Atwood, Physicochemical Principles of Pharmacy, 4th ed., Pharmaceutical Press, London, 2002, pp. 274. [5] S.R.J. Robbins, Gum arabic. In A review of recent trends in selected markets for water soluble gums, ODNRI Bulletin. 108 (1998) 18–33. [6] M. Nakano, Y. Nakamura, K. Juni, et al., Sustained release of sulfamethizole from agar beads after oral administration to humans, Chem. Pharm. Bull. 28 (1980) 2905–2908. [7] D.F. Durso, Handbook of Water Soluble Gums and Resins, McGraw Hill, Kingsport Press, New York, 1980, pp. 12. [8] T.R. Bhardwaj, M. Kanwar, M.R. Lal, et al., Natural gums and modified natural gums as sustained release carriers, Drug Dev. Ind. Pharm. 26 (2000) 1025–1038. [9] A. Desai, S. Shidhaye, S. Malke, et al., Use of natural release retardant in drug delivery system, Indian Drugs 42 (2005) 565–575. [10] B. Sujitha, et al., Natural polysaccharides, Int. J. Pharm. Technol. 4 (2013) 2347–2362. [11] G.E. Trease, W.C. Evans, A Text book of Pharmacognosy, 16th ed. W.B. Saunders, Indian ed., Noida (2008) 168–215. [12] United States Pharmacopoeia, The Official Compendia of Standards, Asian Edition, 2006, 1863–1864. [13] British Pharmacopoeia, Published on the recommendation of the Medicines Commission pursuant to the Medicines Act 1968, 3rd ed., 15–34. [14] A. Madgulkar, M.R.P. Rao, D. Warrier, in: Jean-Michelle Merillon, Kishan G. Ramavat (Eds.), Characterization of Psyllium (Plantago ovata) Polysaccharide and its Uses in ‘Polysaccharides, Bioactivity and Biotechnology’, Springer International Publishing, Switzerland, 2014, http://dx.doi.org/10.1007/978-3319-03751-6 49-1. [15] Truestar Health: http://www.truestarhealth.com/Notes/ 2150006html#Traditional-Use (accessed on 10.11.15). [16] H.M. Fischer, Y. Nanxiong, R.G.J. Ralph, L. Anderson, J.A. Marletta, The gel forming polysaccharide of psyllium husk (Plantago ovata Forsk), Carbohydr. Res. 339 (2004) 2009–2017. [17] J.F. Kennedy, J.S. Sandhu, Structural data for the carbohydrate of isapghula husk Plantago ovata Forsk, Carbohydr. Res. 75 (1979) 265–274. [18] B.S. Kaith, B.K. Kumar, Preparation of psyllium mucilage and acrylic based hydrogels and their application in selective absorption of water from different oil/water emulsion, Iran. Polym. J. 16 (8) (2007) 529–538. [19] B. Singh, V. Sharma, Design of psyllium-PVA-acrylic acid based novel hydrogels for use in antibiotic drug delivery, Int. J. Pharm. 389 (2010) 94–106. [20] S.J. Kim, K.J. Lee, S.I. Kim, Electrostimulus responsive behavior of poly(acrylic acid)/polyacrylonitrile semi-interpenetrating polymer network hydrogels, J. Appl. Polym. Sci. 92 (2004) 1473–1477.
[21] Q. Tang, X. Sun, Q. Li, J. Wu, J. Lin, Fabrication of a high strength hydrogel with an interpenetrating network structure, Colloids Surf. A: Physicochem. Eng. Aspects 346 (2009) 91–98. [22] V.D. Athawale, S.C. Rathi, Graft polymerization: starch as a model substrate, J. Macromol. Sci.: Polym. Rev. 39 (1999) 445–480. [23] M.R.P. Rao, M.P. Khambete, H.N. Lunavat, Study of rheological properties of psyllium polysaccharide and its evaluation as suspending agent, Int. J. Pharm. Technol. Res. 3 (2) (2011) 1191–1197. [24] M.R.P. Rao, P. Sadaphule, M. Khambete, H. Lunawat, K. Thanki, N. Gabhe, Characterization of psyllium (Plantago ovata) polysaccharide and its use as a binder in tablets, Ind. J. Pharm. Edu. Res. 47 (2) (2013). [25] S. Pal, R. Das, in: Susheel Kalia, M.W. Sabaa (Eds.), Polysaccharide Based Graft Copolymers for Biomedical Applications in Polysaccharide Based Graft Copolymers, Springer-Verlag, Berlin, Heidelberg, 2015, http://dx.doi.org/10. 1007/978-3-642-36566-9 9,2013. [26] V. Dulong, S. Lack, et al., Hyaluronan-based hydrogels particles prepared by crosslinking with trisodium trimetaphosphate, synthesis and characterization, Carbohydr. Polym. 57 (2004) 1–6. [27] Organization of Economic Co-operation & Development (OECD), The OECD guidelines for testing of chemical: 420 Acute Oral Toxicity. France (2001). [28] K.R. Khandelwal, Practical Pharmacognosy, Techniques and experiments. 19th ed. Nirali Prakashan, Pune (MH) (2008) 159. [29] D. Krishnarajan, et al., Effect of cellulose and non-cellulose polymers on ciprofloxacin extended release tablets, J. Chem. Pharm. Res. 4 (7) (2012) 3617–3623. [30] S. Patrick, Martin’s Physical Pharmacy and Pharmaceutical Sciences, 5th ed., Lippincott Williams and Wilkins, Philadelphia, 2000, pp. 587–588. [31] V. Chinampudur, S.R. Boddapati, H.S. Srikanth, J.R. Edwin, A.J. Joshua, et al., Evaluation of turmeric polysaccharide extract: assessment of mutagenicity and acute oral toxicity, BioMed Res. Int. (2013), http://dx.doi.org/10.1155/ 2013/158348, Article ID 158348. [32] A.R. Gennaro, Remington: The Science and Practice of Pharmacy, vol. 1, 20th ed., Lippincott Williams and Wilkins, Printed in India, 2000, pp. 409–412. [33] S.P. Basavaraj, S. Vinayak, A.R. Kulkarni, Development and evaluation of psyllium seed husk polysaccharide based wound dressing films, Orient. Pharm. Exp. Med. 11 (2011). [34] T. Kasemsuwan, T. Bailey, J. Jane, Preparation of clear noodles with mixtures of tapioca and high-amylose starches, Carbohydr. Polym. 32 (3–4) (1998) 301–312. [35] K. Muhammad, F. Hussin, Y.C. Ghazali, J.F. Kennedy, Effect of pH on phosphorylation of sago starch, Carbohydr. Polym. 42 (1) (2000) 85–90. [36] G. Kabir, B. Yagen, A. Penhasi, A. Rubinstein, Phosphated crosslinking guar for colon—specific drug delivery I, preparation and physical characterization, J. Control. Release 63 (1–2) (2000) 121–127. [37] A. Bejenariu, et al., Trisodium trimetaphosphate crosslinked xanthan networks: synthesis, swelling, loading and releasing behavior, Polym. Bull. (2009) 1–14. [38] L. Beng-zheng, W. Li-jun, L. Dong, Z. Zhing-jie, et al., Physical properties and loading capacity of starch-based microparticles cross-linked with trisodium trimetaphosphate, J. Food Eng. 92 (2009) 255–260. [39] A. Martin, J. Swarbrick, A. Cammarata, Physical Pharmacy-Physical Chemical Principles Pharmaceutical Sciences, 503, Varghese publishing house, Bombay, 2001, pp. 513–514. [40] A. Barnes Howard, Thixotropy a review, J. Non-Newton. Fluid Mech. 70 (1997) 1–33. [41] B.M. Boddupalli, Z.N.K. Mohammed, R.A. Nath, B. David, Mucoadhesive drug delivery system: an overview, J. Adv. Pharm. Technol. Res. 1 (4) (2010) 381–387. [42] H. Hagerstrom, K. Edsman, M. Stromme, Low-frequency dielectric spectroscopy as a tool for studying the compatibility between pharmaceutical gels and mucus tissue, J. Pharm. Sci. 92 (2003) 1869–1881. [43] G. Hohne, W. Hemminger, H.J. Flammersheim, Differential Scanning Calorimetry: An Introduction for Practitioners, Springer, Berlin, Germany, 1996. [44] S. Kavesh, J.M. Schultz, Meaning and measurement of crystallinity in polymers: a review, Polym. Eng. Sci. 9 (5) (1969) 331–338. [45] E. Walum, Acute Oral Toxicity, Environmental Health Perspectives 106, Suppl. 2 April (1998) 497–503.