- (2010) Volume 2, Issue 3
First part of work was focused on effect of equipments on stability and dimension characteristics of glycerol monooleate dispersion, while second part was focused on to study effect of formulation parameters on the characteristics of dispersion by applying 42-factorial design at fixed ratio (1:5) of dispersed phase to dispersion medium. In this work lipid was dispersed in previously prepared PVA solution with the help of different equipments. Rest of batches was prepared by using selected equipment. Stability study was conducted using Thermo lab humidity chamber. Finally selected batches were subjected for transmission electron microscopy and X-ray diffraction study to investigate the structure. Propeller stirrer followed by homogenizer (volume mean diameter 1.42 ìm and no phase separation were found), was the best equipment. Concentration of GMO was significant for %fraction of large particles, %lost on paddle, % water lost and volume mean diameter, but not for % recovery while concentration of PVA was significant for % recovery , volume mean diameter, % water lost , %lost on paddle, but not for %fraction of large particles.
Introduction |
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| Fatty acids and monoglycerides possess antiviral and antibacterial activities. It has been demonstrated that enveloped viruses, such as herpes simplex virus type 1 (HSV-1), vesicular stomatitis virus (VSV), and visna virus are inactivated by long-chain unsaturated and medium-chain saturated fatty acids. Microbicidal hydrogel containing monoglyceride as active ingredient was developed against herpes simplex virus type I. In this view, monoglyceride based formulations can be proposed for intra vaginal use as microbicides against sexually transmitted diseases [1]. | ||||||||||
| Beside ability of GMO to disturb viruses (e.g. HSV, HIV) structure it has ability to form different structures. Aqueous dispersions of monoglycerides such as monoolein forms the structures like reverse micelles, reverse hexagonal, bicontinuous cubic and sponge phase. Structure formation depends on the water content and temperature [2]. These structures can be used as carrier to load the active pharmaceutical ingredients and also has ability to control their release. Several examples of drug studied for their sustain release from GMO based liquid crystalline dispersion are cefazolin, cefuroxime, indomethacin, lidocaine hydrochloride etc [3, 4].High pressure homogenization and sonication method was used for preparation of dispersion [5]. During this technique there are chances for degradation of some desire phase (cubic phase) which is more applicable for pharmaceutical point of view. To over come this difficulty author Patrick spicer utilize ethanol as hydrotropic agent for preparation of liquid precursor that form cubic phase spontaneously when come in contact with excess body fluid [6]. | ||||||||||
| In this article we restricted our self for preparation of GMO dispersion by using different equipments like propeller stirrer, magnetic stirrer, homogenization and combination of these equipments. Finally selected equipment was adopted for preparation of further batches. Some preformulatory parameters were evaluated like % water lost, % Lost on Paddle, % Recovery, % fraction of large particles and volume mean diameter. Accelerated stability study was conducted and finally selected batch was subjected for TEM and XRD for structural investigation. | ||||||||||
Material and Methods |
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Materials |
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| Glycerol monooleate (pale yellow liquid) and PVA (80% hydrolyzed, white colored granular powder) were purchased from National chemicals, Vadodara, India. | ||||||||||
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Preparation of PVA Solution |
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| Heat the water at 70 0C, add PVA in required quantity at a time with constant stirring by using the magnetic stirrer at 1200 RPM (Remi Equipments, Mumbai). | ||||||||||
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Equipments study |
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| In first step of process Polar lipid like Glycerol Monooleate (GMO) was heated at 400C after that it was emulsified in prepared PVA solution with the help of different equipments as mentioned in table 1. Same composition was used for all the equipments (GMO: 0.625 g, PVA: 0.375 g, Distilled Water: Quantity sufficient to 50 ml). All the calculation was carried out on weight basis. Prepared batches were evaluated for particle size distribution study and physical stability. | ||||||||||
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Formulation parameters |
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| Once the equipments study was overed, remaining batches were prepared by process described bellow. In the first step of the process, GMO was heated at 400C than added drop wise in PVA solution during constant stirring with the help of propeller stirrer at 700C, 2500 RPM and stirring was continued for 1 hr. After that the dispersion was subjected for homogenization (kalweka, Mumbai) for 3 minutes at 4000 RPM. Filter the dispersion. Particle size distribution study was conducted by using particle size analyzer (Sympatec, Germany); for structure investigation TEM (Philips, Netherlands) and XRD (Philips, Holland) were used. Accelerated stability study was conducted using thermo lab humidity chamber (Mumbai). A 42 factorial design was used to study the effect of concentration of GMO and concentration of PVA on dispersion characteristics at four different levels of GMO (5%, 7.5%, 10%, and 12.5%) and PVA (1%, 2%, 3%, and 4%). Compositions of various batches prepared were given in table 2. | ||||||||||
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Evaluation parameters (For formulation parameter study) |
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| Prepared dispersions were evaluated for % water lost, %lost on paddle, % recovery, % fraction of large particles and volume mean diameter. Water lost: Due to high speed of propeller stirrer (2500 rpm) and temperature (700C) % water lost was calculated with respect to quantity of water added. In calculation water lost on paddle was not considered.% water loss = (weight of water added + weight of GMO + weight of PVA – {weight of dispersion after mixing with propeller stirrer+ weight of disperse phase lost on paddle}) ×100/ weight of water added.%lost on paddle: Lost on paddle was calculated with respect to total weight of dispersion before production. To find out lost on paddle, weight the filter paper and collect the material form paddle and than re weight the filter paper. | ||||||||||
| % lost on paddle = (weight filter paper after collecting material form paddle - initial weight of filter paper) × 100/ weight of dispersion before production % fraction of large particles: Fraction of large particles after filtration of dispersion was calculated with respect to the weight of the disperse phase before dispersion production. To calculate the % fraction of large particles dispersion was filtered through filter paper. Before filtration note the initial weight of filter paper. The filter paper was then left to desiccate for 24 hrs in order to eliminate as much as possible of water taken up by the particles. Determine the weight of filter paper after desiccation. And finally find the %fraction of large particles. | ||||||||||
| % fraction of large particles = (weight of filter paper after desiccation- initial weight filter paper) × 100/weight of disperse phase % recovery: Recovery was calculated on the basis of weight of dispersion after production and initial weight of dispersion.% recovery: weight of dispersion after production ×100/ initial weight of dispersion | ||||||||||
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Characterization of Dispersions |
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| Dispersions prepared by different equipments were stored in glass vial for 7 days and observed for phase separation phenomena and also subjected for Particle size measurement. GMO-based dispersions were characterized by particle size analyzer, TEM and XRD. | ||||||||||
| Particle size analysis was performed on filtered dispersions using a particle size analyzer (sympatec, Germany) Model: laser diffraction particle size analyzer, Measuring range 0.1 to 35 µm, measuring duration 5 seconds, cycle time 1000 ms. | ||||||||||
| For transmission electron microscopy studies, a drop of the sample was put on an untreated pure copper transmission electron microscopy (TEM) grid (300 mesh, Philips, netherland ,Teenai-20), where most of the liquid was removed with blotting paper leaving a thin film stretched over the grid holes. The TEM was operated at an acceleration voltage of 80 kV. Magnification approximates 30000. | ||||||||||
| X-Ray Diffraction Measurements, Diffraction measurements were carried out using a Philips, Holland Model: X’ Pert (Specification: Source: Cu target X-Ray tube,X-Ray power: 2KW, Detector: Xe-filled Counteract or propotional detector ,Software: JCPD software). | ||||||||||
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Stability study |
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| After production and elimination of large particles by filtration, dispersions were stored in a glass vial at accelerated conditions (Temp 45OC+/-20C; humidity 75 +/-5 %RH) for 45 days in thermo lab stability chamber. In order to asses the physical stability of dispersions, morphological aspect such as phase separation and volume mean diameter were investigated as function of time. | ||||||||||
Results and discussion |
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Equipment study |
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| Selection of equipment was carried out on the basis of dispersion characteristic, here the morphological aspect such as phase separation (measured after 7 days of preparation) and volume mean diameter (measured immediately after preparation) were evaluated. Dispersion having lowest particle size and free form phase separation phenomena was selected. From above result magnetic stirrer was not able to form the stable dispersion. Propeller stirrer, Homogenizer, Propeller stirrer+ homogenizer and magnetic stirrer+ homogenizer gave the stable dispersion. | ||||||||||
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Formulation parameters |
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| All the obtained data were subjected for multivariate test by applying full factorial design using SPSS software. Data were shown in table 5. From table 5, concentration of PVA is significant for %water lost, %lost on paddle, VMD and % recovery but not for % fraction of large particles. Again the B value indicates the magnitude of effect. Here the B value was positive for % water lost and % lost on paddle means increase in concentration of PVA increase in measure responses (% water lost and % lost on paddle). For VMD and % recovery B value was negative it indicates that increase in concentration of PVA decrease in measure responses (VMD and % recovery). Concentration of GMO is significant for %water lost, %lost on paddle, VMD and % fraction of large particles but not for % recovery. Here the B value was positive for % water lost and % lost on paddle and % fraction of large particles it indicates that increase in concentration of GMO increase in measure responses (% water loss and % lost on paddle and % fraction of large particles). For VMD and % recovery B value was negative it indicates that increase in concentration of GMO decrease in measure responses (VMD and % recovery). | ||||||||||
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Stability study |
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| Table 4 No phase separation was found after 45 days. To determine the particle size randomly three batches were selected. It was found that volume mean diameter were increase after 45 days. | ||||||||||
| Batches having the lowest particle size were subjected for TEM.TEM images show the dispersion characterized by spherical particles no clear internal structure was observed. | ||||||||||
| Peak present in X-Ray diffraction graph confirm the existent of liquid crystalline phase though for determination of type of shape (cubosomes or hexosomes) detail characterization with XRD or NMR is required. | ||||||||||
Conclusion |
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| In conclusion, propeller stirrer followed by homogenizer was the best equipment that gave the stable dispersion, having the lowest particle size with the limitation of increase in mean diameter with respect to time. The work performed here enabled an assessment of the standard condition for the preparation of dispersions containing micro particles characterized by spheroid shape and few aggregates. Further the GMO was key ingredient for controlling fraction of large particles and lost on paddle (may be because of sticky nature) while increase in concentration of PVA decrease the recovery because of increase in viscosity of dispersion that makes the process more complicated and also decrease in volume mean diameter but it increase the lost on paddle. | ||||||||||
Acknowledgment |
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| Our sincere thanks to SICART, for providing support during XRD and TEM investigation .We wish to thank Dr. Jolly R Parikh, Dr V.A. Patel and Dr N.P. Chotai for their support in different parts of this work. | ||||||||||
Conflict of Interest |
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| NONE | ||||||||||
Source of Support |
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| NIL | ||||||||||
Tables at a glance |
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Figures at a glance |
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