Isolation And Purification Of Natural Mucoadhesive Agents Biology Essay

Plant materials were authenticated and specimens were stored at Botanical Survey of India, Western Regional Centre, Pune, Maharashtra wide voucher number BSI/WRC/TECH/2011/ABGABE1, ABGBAR2, ABGPROJ3, ABGZIM4 and ABGOCA5. Clopidogrel bisulphate (gift sample from Medley Pharmaceutical Ltd., Daman, India). Carboxylic acid derivative of Clopidogrel bisulphate (gift sample from Anant Laboratories, Thane, Mumbai). Atorvastatin (Research Centre). All chemicals used were of analytical reagent grade. Excipient used were of Pharmaceutical grade. HPLC solvents used were of HPLC grade.

5. 1. Isolation and purification of natural mucoadhesive agents.

5.1.1 Isolation and purification of mucoadhesive agent from roots of Abelmoschus esculentus

Fresh roots of Abelmoschus esculents were collected from the fields of Nashik Maharashtra in July month. After removal of the soil, roots were washed with distilled water. Roots were cut into small pieces, were kept in distilled water for four hours at 60oC. It was strained through muslin cloth. Thus obtained filtrate was precipitated with double quantity of acetone. The precipitated mucoadhesive agent was washed thrice with small quantity of acetone. The mucoadhesive agent was dried in oven at 40 oC for six hours and then kept in vacuum desiccator. The dry mucoadhesive agent was reduced to fine powder and stored in an air tight container119.

5.1.2 Isolation and purification of mucoadhesive agent from seeds of Prosopis juliflora

Seeds of Prosopis juliflora were collected from the fields of Nashik, Maharashtra in June month. Seeds were soaked in distilled water for 24 hours, boiled for one hour and were set aside for three hours for release of mucoadhesive agent. The material was filtered through muslin cloth to separate the mark and filtrate. Then the filtrate was precipitated with double quantity of acetone. The precipitated mucoadhesive agent was washed thrice with small quantity of acetone. Mucoadhesive agent was dried in oven at temperature 40oC for six hours and then kept in vacuum desiccator. The dry mucoadhesive agent was reduced to fine powder and stored in an air tight container.

5.1.3 Isolation and Purification of mucoadhesive agent from seeds of Ocimum americanum

Seeds of Ociumum americanum were collected from fields of Nashik, Maharashtra in April month. Seeds were soaked in distilled water for 24 hours. The material was vigorously stirred using heavy duty mechanical stirrer at 2000 rpm and at temp 60°C for two hours. The released mucoadhesive agent was filtered through muslin cloth to obtain clear filtrate. Then double quantity of acetone was added to precipitate the mucoadhesive agent. Precipitated mucoadhesive agent was washed thrice with small quantity of acetone. It was dried in oven at temperature 40 oC for six hours and then kept in vacuum desiccator. The dry mucoadhesive agent was reduced to fine powder and stored in an air tight container.

5.1.4 Isolation and purification of mucoadhesive agent from ripe fruits of Ziziphus mauritiana

Ripe Jujube fruits were collected from fields of Nashik, Maharashtra in December month. Outer covering was removed and seed part was soaked in distilled water for overnight with mechanical stirring at 100 rpm. The mucoadhesive agent was released in distilled water. It was filtered through muslin cloth to separate the mark and filtrate. The filtrate was precipitated with twice quantity of acetone. Precipitate was washed thrice with small quantity of acetone. Mucoadhesive agent was dried in oven at 40ºC for six hours and then kept in vacuum desiccator. The dry mucoadhesive agent was reduced to fine powder and stored in an air tight container.

5.1.5 Isolation and purification of mucoadhesive agent from seeds of Bauhinia racemosa

Seeds were collected from fields of Nashik, Maharashtra in February month. Seeds were boiled for one hour in distilled water to shrink the outer hard covering and soaked in water and stirred in distilled water for overnight at 100 rpm. The material was filtered through muslin cloth. Obtained filtrate was precipitated in double quantity of acetone. It was washed thrice with small quantity of acetone. Mucoadhesive agent was dried in oven at 40ºC for six hours and then kept in vacuum desiccator. Dry mucoadhesive agent was reduced to fine powder and stored in air tight container.

5.1.6 Isolation and purification of mucoadhesive agent from ripe fruits of Cordia myxa

Ripe fruits were collected from fields of Nashik, Maharashtra in June month. Fresh fruits were pressed using tincture press to obtain its viscous juice. Mucoadhesive agent was precipitated with double quantity of acetone. Precipitate was washed thrice with small quantity of acetone. Mucoadhesive agent was dried in oven at 40ºC for six hours and then kept in vacuum desiccator. Dry mucoadhesive agent was reduced to fine powder and stored in air tight container.

5.1.7 Isolation and purification of mucoadhesive agent from seeds of Cassia auriculata

Seeds were collected from fields of Nashik, Maharashtra in April month. Seeds were soaked in distilled water for 24 hours, boiled for one hour and was set aside for three hours for release of mucoadhesive agent, The material was filtered through muslin cloth to separate the mark and filtrate. Then the filtrate was precipitated with double quantity of acetone. The precipitated mucoadhesive agent was washed thrice with small quantity of acetone. Mucoadhesive agent was dried in an oven at temperature 40ºC for six hours and then kept in vacuum desiccators. Dry mucoadhesive agent was reduced to fine powder and stored in air tight container.

5.2. Phytochemical and physicochemical studies on isolated mucoadhesive agents

Phytochemical characterization is subjected to identification of chemical composition and physicochemical characterization subjected to determine suitability of isolated natural mucoadhesive agents as a tablet excipient.

Phytochemical tests like Ruthenium red test, Fehling’s test, Molisch's tests, Ninhydrin test, Test with iodine, Biuret test, Salkowski test, Baljet test, Shinoda test, Wagner’s test, Test with acetic acid solution, ash value and acid insoluble ash value were performed on isolated mucoadhesive agent. Physicochemical studies were performed to determine viscosity, pH, swelling Index, bulk density, tapped density, Carr’s compressibility index, loss on drying and angle of repose.

5.2.1. Phytochemical Tests120, 121, 122

5.2.1.1. Ruthenium red test

This test is subjected to identification of mucilage. 5 mg test substance with ruthenium red solution was shown a pink color confirms it as mucilage.

5.2.1.2. Fehling’s test

This test is subjected to identification of reducing sugars. 1 % w/v dispersion of test substance was prepared in distilled water. 5 ml of felhing’s solution-A and 5 ml of felhing’s solution-B were mixed in one test tube. 2ml mixture was transferred to other test tube and 3 drops of test solution was added. It was kept at 60ºC in water bath for 5-10 min to obtain brick red colored precipitate.

5.2.1.3. Molisch's tests

This test is subjected to identification of carbohydrates. 1 % w/v dispersion of test substance was prepared in distilled water. Small amount of molisch's reagent and 4 ml prepared test substance were combined in a test tube. After mixing, small amount of concentrated sulphuric acid was slowly added along the sides of the slanting test tube to obtain violet ring at junction.

5.2.1.4. Ninhydrin test

This test is subjected to identification of proteins. 2 ml aqueous solution of test substance and 1 ml alcoholic ninhydrin solution was mixed in test tube and heated at 80° C for 5 min to obtain red color.

5.2.1.5. Test with iodine

This test is subjected to identification of non reducing polysachharides (starch). 1 % w/v dispersion of test substance was prepared in distilled water. 3 ml test solution and few drops of dilute Iodine solution were mixed to obtain blue color after cooling and no color after heating.

5.2.1.6. Biuret test

This test is subjected to identification of proteins. 1 % w/v dispersion of test substance was prepared in distilled water. 3 ml test solution, 1 ml 4% sodium hydroxide and 2 drops of copper sulphate solution were mixed to obtain violet to pink.

5.2.1.7. Salkowski test

This test is subjected to identification of steroids. 1 % w/v dispersion of test substance was prepared in distilled water. 2 ml test solution, 2 ml chloroform and 2 ml conc. sulphuric acid were mixed and shaken well to obtain red colored chloroform layer and greenish yellow fluorescence in acid layer.

5.2.1.8. Baljet test

This test is subjected to identification of glycosides. 1 % w/v dispersion of test substance was prepared in distilled water. 2ml test solution and 2 ml sodium picrate was mixed in test tube to obtain yellow to orange color.

5.2.1.9. Shinoda test

This test is subjected to identification of flavonoids. 1 % w/v dispersion of test substance was prepared in distilled water. 3 ml test substance, 5 ml 95% ethanol, 2 drops of Conc. HCl and 0.5 g magnesium turnings were mixed to obtain pink color.

5.2.1.10. Wagner’s test

This test is subjected to identification of alkaloids. 1 % w/v dispersion of test substance was prepared in distilled water and filtered. 3 ml test solution filtrate was mixed with 2 drops of Wagner’s reagent to obtain reddish brown precipitate.

5.2.1.11. Test with acetic acid solution

This test is subjected to identification of alkaloids. 1 % w/v dispersion of test substance was prepared in distilled water. 2 ml test solution and acetic acid solution were mixed to obtain red color.

5.2.2. Physicochemical studies123

5.2.2.1. Viscosity

1% w/v solution of test substance was prepared in distilled water and 0.1 N HCl separately. Solutions were kept at 37 ± 2 °C for 6 hours. Viscosity of test solutions determined using Brookfield viscometer using spindle no. 3 at 50 rpm (Model No. DV-E).

5.2.2.2. pH determination124

1 % w/v dispersion of test substance in distilled water was prepared by shaking for 30 min and the pH determined using a pH meter (Elico).

5.2.2.3. Swelling Index88

Swelling index is the volume in ml occupied by 1 g of a drug, together with any adhering mucilage, following it has swollen in distilled water for 4 hours.

1 g test substance (Fine powder) was placed in 25 ml ground glass stoppered measuring cylinder with graduations in 0.5 ml divisions. The test substance was moistened with 1.0 ml of 96% ethanol and 25 ml distilled water was added. Cylinder was closed using stopper. Cylinder was shaken every 10 min time interval for 1 hour and then was allowed to stand for 3 hours. Volume occupied by the test sample was determined in ml after decanting the supernatant clear liquid. Swelling index was calculated from average of three tests. Same experiment was performed using 25 ml 0.1 N hydrochloric acid instead of distilled water. Swelling index was calculated using following equation.

Vt = Volume occupied by test substance after hydration, at time 3 hrs.

V0 = Volume occupied by test substance before hydration, at time zero.

5.2.2.4. Bulk density123

Bulk Density was determined using Borosil 50 ml measuring cylinder. The volume of 5 gm mucilage powder was noted. Value of Bulk density was calculated by,

5.2.2.5. Tapped density123

Tapped Density was determined using Borosil 50 ml measuring cylinder. The volume of known weight 5gm was noted. The cylinder was given 250 taps on using tapped density apparatus. Value of Tapped density was calculated by

5.2.2.6. Carr’s compressibility index123

Carr’s compressibility index of AER mucilage powder was calculated from respective tapped density and bulk density by,

5.2.2.7. Loss on drying88

In flat bottom dish, 50 mm in diameter and 30 mm in height, 1 g fine powdered test substance dried in oven at 100-105 C for 3 hours. The test substance was allowed to cool in desiccator over diphosphorus pentoxide for 24 hours and weighed. Percentage loss on drying was calculated by following equation.

Weight of empty dish = A g

Weight of test substance taken = Y g

Weight of dish containing test substance after drying and placing in desiccator = B g

Weight of test substance after drying = (B – A) g

5.2.2.8. Angle of repose123

Angle of repose was measured using fixed funnel method and was calculated by following method.

A funnel was clamped with its tip 2 cm below which the graph paper placed on a flat horizontal surface. The powder was slowly poured through the funnel until top of the cone (h) reached the tip of funnel. The mean diameter and thus radius (r) of the base of the powder cone was determined and tangent of the angle of repose was calculated using following equation.

Where, α = Angle of repose

H= Height of cone

R= Radius of base of conical pile.

5.2.1.12. Total Ash value125

Flat, thin porcelain dish was weighed and ignited. 2 g of powdered test substance was weighed into empty porcelain dish. Porcelain dish was heated with the help of burner until all the carbon burnt off. Dish was cooled in desiccator. Total residue (% w/w) was weighed and percentage of total ash with reference to air dried sample of test substance was determined.

Weight of empty porcelain dish = X g

Weight of test substance taken = Y g

Weight of the porcelain dish + Residue (after incineration) = Z g

Weight of total ash = (Z-X) g

Therefore total ash value of test substance was calculated by,

5.2.1.13. Acid insoluble ash value125

Residue obtained in total ash value determination was washed using 25 ml dilute HCL (2M) into 100 ml beaker. The mixture was boiled for 5 minutes. It was filtered through ash less filter paper and residue was washed twice with hot water. Clean porcelain dish was ignited, cooled and weighed. Residue containing ash less filter paper placed in the porcelain dish and burn on burner until all carbon was removed. Dish was cooled in desiccator. Residue containing dish was weighed and the acid insoluble ash (% w/w) was determined with reference to air dried sample of test substance.

Weight of empty porcelain dish = A g

Weight of test substance taken = Y g

Weight of porcelain dish containing acid insoluble residue = B g

Acid insoluble ash = (B-A) g

5.3. Mucoadhesion studies on isolated mucoadhesive agents

Mucoadhesion studies were performed on isolated natural mucoadhesive agents and compared with Carbopol 934P.

5.3.1 In vitro mucoadhesion methods

5.3.1.1 Wihelmy’s method

Wihelmy’s method was studied to determine mucoadhesion strength of isolated mucoadhesive agents in terms of detachment weight required to break the mucus-polymer bond against adhesion. Small glass plates of dimension 2×5cm were coated by spreading 1%, 2 % and 3% w/v solution of isolated mucoadhesive agent and Carbopol 934P. These solutions were uniformly coated to small glass plates respectively. Coated plates were dried at 40 ºC. The mucus gel was collected from goat stomach and kept at 20 ºC. Thread was attached at one end of coated glass plate. Coated glass plate was dipped into mucus gel while other side of thread was passed through pulley and end of thread was attached to small plastic bag. Plates were dipped into mucus gel for 10, 20 and 30 minutes. Weight was added into small polyethylene bag with small increments of water till the surfaces get pull out and separated vertically from mucus gel. Glass plate weight was minimized from the final detachment weight. Weight in gram required to pull out the glass plate from the mucus gel represents the force required to break the mucus-polymer bond against adhesion. Six times study was performed 8, 21, 126.

5.3.1.2 Falling Sphere method

Falling sphere method was studied to determine mucoadhesive strength in terms of time in second required for mucoadhesive agent coated grain to fall 50 divisions in the burette filled with 10 % mucus solution. 1%, 2%, 3% w/v solutions of isolated mucoadhesive agents and Carbopol 934P respectively were prepared. Mustard grains of diameter 1.0-1.1 mm were taken and then coated with above mucoadhesive agent solutions. Grains were dried at 30 ºC. Each grain was slowly placed at the top layer of 10 % mucus solution in the burette. Time in seconds taken by the mucoadhesive agent coated grain to fall 50 divisions in the burette was noted. Six times study was performed127, 128.

5.3.1.3 Shear Stress Method

Shear stress method was studied to determine the mucoadhesion strength of mucoadhesive polymer. In this method shear required to pull the glass plate from a base plate in parallel direction was taken as mucoadhesive strength. Two smooth, transparent, polished glass plates of dimensions 2x5 cm were selected. One plate was fixed with cynoacrylate adhesive on glass block which was previously fixed on horizontal leveled table top. To this fixed glass plate goat mucosa was stick using cynoacrylate adhesive. One end of other glass slide was tied with thread. Thread was passed down through pulley. At the end of thread a small polyethylene bag was attached. Length of the tread from pulley to plastic bag was 8cm. 1%, 2%, 3% w/v solutions of isolated natural mucoadhesive agent and Carbopol 934P were prepared in distilled water. A fixed volume (0.5ml) solution of above mucoadhesive agents were kept on the centre of fixed glass plate with pipette and then thread attached glass plate was placed on the first plate and pressed by applying 100 gm of weight to form uniform film in between both plate. Air bubble between plates was observed, its presence may affect the results. After keeping for fixed time intervals of 10, 20 and 30 min, gradually, purified water was added into polyethylene bag until thread attached glass plate slide over the fixed base plate. Correction was made for weight of glass plate. The weight of water required for detachment was noted. Six times study was performed129.

5.3.1.4 Spring Balance Method

Spring balance method was studied to determine mucoadhesion strength of natural mucoadhesive agents and Carbopol 934P. Tablets (thickness 1mm and diameter 8.75 mm) of natural mucoadhesive agent and Carbopol 934P were prepared by direct compression method. Previously calibrated spring balance was used. Fresh goat stomach mucosa was collected from slaughter house and was stored in 0.1 N HCl for moistening at 37 ºC before use. The sliding stage was fitted vertically on the stand. Spring balance was vertically attached to bottom of the sliding stage. To the bottom of spring balance, plastic closure was attached using thin metal wire. The freshly excised mucosa was cleaned and cut to closure size. The mucosa was attached to plane surface of plastic closure by using cynoacrylate adhesive. Tablet was stick to the previously fixed glass plate on horizontal table using cynoacrylate adhesive. Mucosa attached closure surface along with spring balance was kept vertically over tablet, slightly pressed and allowed to form a bond between mucosa and mucoadhesive agent for ten minute. With the help of sliding stage, the spring balance was slowly raised until the mucosa detached from the tablet surface. Reading was observed on the balance scale and noted. Six times study was performed. The weight required to detach the disc was measured in grams and taken as mucoadhesion strength130, 131. The force of adhesion (kgm/s2) was calculated by, Force of Adhesion = Weight required for detachment (kg) x acceleration (m/s2).

5.3.1.5 Modified physical balance method

Modified physical balance method was studied to determine mucoadhesive strength in terms of detachment force. Tablets (thickness 1mm and diameter 8.75 mm) of natural mucoadhesive agent and Carbopol 934P were prepared by direct compression method. Fresh goat stomach mucosa was collected from slaughter house and stored in 0.1 HCl at 37 ºC before use. Pans of both arms of physical balance were removed. Mucosa was placed on teflon block and tightly fixed using cynoacrylate adhesive. Mucosa fixed block was placed in a beaker. To the beaker 0.1 N HCl was added up to surface of mucosa for moistening. Prepared tablet was stick to plastic bottle closure surface using cynoacrylate adhesive and was attached to one arm of modified physical balance using thread, below which mucosa attached block was affixed. On the opposite side of balance arm, small polyethylene bag was attached and both arms were balanced. Block with attached mucosa in a beaker was raised up to tablet surface. Tablet was slightly pressed on mucosa to allow mucoadhesion for ten minute. Then small increments of water added in polyethylene bag to detach the surfaces50, 106, 132. The weight in gram required to detach the surfaces noted. Six times study was performed. The force of adhesion (kgm/s2) was calculated by, Force of Adhesion = Weight required for detachment (kg) x acceleration (m/s2).

5.3.1.6 Retention time method

Method was studied to determine the time required for complete detachment of tablet from the mucosa was recorded. An adhesive tablet of isolated mucoadhesive agent was prepared. It was slightly pressed over goat gastric mucosa after previously being held on a glass plate and was deep in a beaker containing 500 ml of 0.1 N HCl at 37±0.5 ºC. One stirrer was fitted at a distance of 5 cm from the tablet and rotated at 25 rpm. Time required for complete detachment of tablet from mucosal surface was noted. Six times study was performed93, 106, 133.

5.3.2 In vivo mucoadhesion method

5.3.2.1 In vivo mucoadhesion study (X- Ray)

Study protocol was approved from institutional animal ethics committee (IAEC No. 03PH15JUN11). Retention time of isolated natural mucoadhesive agent in rabbit stomach was determined using prepared barium sulphate tablets. Mucoadhesive agents isolated from Prosopis juliflora seed and Abelmoschus esculentus root were selected and compared with Carbopol 934P for the x- ray study. 100 mg tablet was prepared using 50 mg barium sulphate (instead of drug), 50 mg isolated natural mucoadhesive agent or Carbopol 934P by direct compression technique. Nine healthy male rabbits weighing 2.0 – 2.5 kg were selected in three groups. In each group three rabbits were selected. Tablets were administered orally along with 50 ml water with the help of pharmacologist. X-ray photographs were taken at 0, 2, 4, 6, 8 and 10 hours time interval129, 40.

5.4. Fourier Transform Infrared Spectroscopy Studies (FTIR)

Mucoadhesive agents were dried at 40 ºC for 24 hours and then kept in vacuum desiccator for 3 days. 20 mg of the samples were grounded with KBr and pellets were formed under a hydraulic pressure of 10 tones/cm2. FTIR spectra of mucoadhesive agents were taken at room temperature using FTIR (Bruker) and obtained spectrum was studied for identification of functional134, 135, 136.

5.5. Differential Scanning Calorimetric Studies (DSC)

Mucoadhesive agents were dried at 40 ºC for 24 hours and then kept in vacuum desiccator for 3 days before DSC study. Mucoadhesive agent was weighed into aluminum crucible. Differential scanning calorimeter (Mettler Toledo, DSC 823e) was used to analyze the sample. Sample was heated at scanning rate of 10 °C/ min over a temperature range 20-300 °C under nitrogen flow of 40 ml/min. DSC curve was studied for powder properties137.

5.6. Preformulation studies:

5.6.1 Characterization of Clopidogrel bisulphate

Clopidogrel Bisulphate (United States Pharmacopoeia 29 NF24) was tested as per official monograph.

5.6.2 Standard Calibration Curve of Clopidogrel Bisulphate in 0.1 N HCl

1 gm Clopidogrel bisulphate was dried in oven at 40 °C for four hours, cooled and kept in desiccator for overnight. 1000 μg/ml stock solution of clopidogrel bisulphate was prepared in 0.1 N HCl. From the stock solutions 0, 5, 10, 20, 30, 40, 50, 60 μg/ ml concentrations were prepared. Spectrum of 30 μg/ ml was obtained on double beam Uv - visible spectrophotometer (Labindia 3000+). Maximum absorption was obtained at 218 nm. It was taken as absorption maxima. Absorbance of all the concentrations was measured at 218 nm. Procedure was repeated six times.

5.6.3 Physicochemical characterization of Carbopol 934P

Carbopol 934P (British Pharmacopoeia, 2004) was characterized for viscosity, bulk density, tapped density, moisture content, pH and solubility studies.

5.6.4 Drug excipient compatibility study

Compatibility was assured through Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC). Clopidogrel Bisulphate and isolated natural mucoadhesive agent were physically mixed in 1:1 proportions, triturated and were kept in desiccator for seven days.

FTIR spectrum was obtained by preparing pellets of above mixture in potassium bromide. DSC curve was obtained for the above mixtures.

5.7. Formulation development and evaluation of Clopidogrel bisulphate tablets

Clopidogrel bisulphate was selected as model drug in present research work. Tablet formulations of 225 mg weight were developed using Clopidogrel bisulphate (CLPB) 98 mg equivalent to 75 mg of Clopidogrel, Natural mucoadhesive agents isolated from Abelmoschus esculentus root (AER), Bauhina racemosa seed (BRS), Prosopis juliflora seed (PJS), Zizisphus mauritiana fruit (ZMF) and Ocimum americanum seed (OAS) and anhydrous lactose as diluent.

Three formulations were developed using each mucoadhesive agent. Carbopol 934P formulation was developed for comparison. From these formulations granules were prepared using wet granulation technique. Alcohol was used as granulating liquid and incorporated mucoadhesive agents act as binder. Wet mass was granulated by passing it manually through 16# sieve. Granules were dried at 40 ºC in oven for six hours and again sieved through 12# sieve. Prepared granules were stored in air tight container. Talc (6 mg) was added as flow promoter and lubricating agent before tablet compression. Tablets were prepared by compressing granules in tablet press (RIMEK MINIPRESS-II, Karnavati) for constant thickness138.

Table 1 showing formulations of Clopidogrel bisulphate mucoadhesive tablet

5.7.1. Evaluation of Granules

Granules were evaluated for granule size, bulk density, tapped density, Carr’s compressibility index and angle of repose139, 140.

5.7.1.1. Granule size distribution

10 gm granules were accurately weighed and placed upon the sieves having close fitting with mesh size 12, 16, 22, 25, 30, 44, 60 and 85. The nest of sieves was shaken in rotary horizontal and vertically tapping, using mechanical sieve shaker for 10 minutes. The oversize fraction (retained) on each mesh was weighed141. Three runs were carried out. Average granule diameter was determined by,

Where, F= Mean % retained

X= Arithmatic mean size of opening

5.7.1.2. Bulk density

Granules were passed through # 22 and then 25 gram granules were accurately weighed. These granules were slowly poured into 100 ml Borosil measuring cylinder. The mark of ml was noted and bulk density was determined by,

5.7.1.3. Tapped density

After measuring the bulk density, tapped density was measured by tapping cylinder method. After 250 taps from constant height, the mark of ml was noted and tapped density was determined by,

5.7.1.4. Carr’s compressibility index

% Carr’s compressibility index (CCI) was obtained from the bulk density and tapped density by,

Where, P= Tapped density

A= Bulk density

5.7.1.5. Angle of repose

Static angle of repose was measured using funnel method. The angle of repose for 20 gram granules was obtained by,

Where α = Angle of repose

H= Height of cone

R= Radius of base of conical pile.

5.7.2. Evaluation of Tablets

All prepared tablets and marketed tablet were evaluated for tablet properties as per Indian Pharmacopoeia 1996 and 2007 procedures and compared with Carbopol 934P tablet. Tablets were evaluated for tablet thickness, tablet weight variation test, tablet hardness test, tablet friability test, tablet content uniformity test and tablet swelling index142, 143.

5.7.2.1. Tablet thickness

20 tablets were randomly selected from each formulation. Thickness in mm was measured by digital caliper (Aerospace). Average and standard deviation of F1-F16 tablet formulations were calculated.

5.7.2.2. Tablet weight variation test

Tablet formulations F1-F16 were evaluated for average weight. 20 tablets of each batch were selected to determine average weight of compressed tablets in triplicate. % Average weight and standard deviation was calculated144

5.7.2.3. Tablet hardness test

The tablet hardness of 10 prepared tablets was determined immediately and intermittently after compression, using Pfizer hardness tester

5.7.2.4. Tablet friability test

Tablet Friability of prepared mucoadhesive tablets was determined by IP 2007 method. Twenty weighed tablets were used and rotated in a Friability tester at 25 rpm for 4 minutes. The % Friability values were obtained by,

5.7.2.5. Tablet Assay

Tablet formulations F1- F16 were tested to determine % purity of drug. 20 Tablets were powdered from each formulation batch. Powder weight equivalent to 98 mg of Clopidogrel bisulphate was accurately weighed and added in 100 ml 0.1N HCL with stirring at 200 rpm for 1 hour. The prepared solution was filtered, diluted with 0.1N HCl and absorbance was measured using UV spectrophotometer at 218 nm. Drug concentration present in the sample was determined using previously constructed calibration curve.

5.7.2.6. Tablet swelling index (SI)

Mucoadhesive tablets were weighed individually (W0). Tablets were positioned separately in beaker containing 200 ml of 0.1 N HCl. After each interval (1, 2, 3, 4, 5, 6, 7 and 8 hrs) tablet was removed from beaker, excess surface water was removed using filter paper and weighed again (Wt). The swelling index was calculated by following equation145, 146.

Where, S. I.= Tablet Swelling Index

W0 = Weight of tablet before placing in the beaker

Wt = Weight of tablet at time t

5.8. In vitro mucoadhesion studies on prepared tablets

Tablets prepared from formulations F1-F16 were evaluated for mucoadhesion strength and retention time using modified physical balance method 136, 137 and retention time method 93, 133 respectively. In these methods, tablets prepared from pure mucoadhesive agents were replaced by tablets prepared from formulations F1-F16. Six times study was performed.

5.9. In vitro drug release study

In vitro drug release of matrix tablets were studied using dissolution test apparatus (DISSO 2000, LabIndia), USP paddle type- II equilibrated at temperature 37±0.5 °C and 50 rpm speed. The dissolution study was carried out in 1000 ml 0.1 N HCl. 5 ml of sample was withdrawn after one hour interval and was replaced by an equal volume of fresh dissolution medium kept at 37±0.5 °C. Samples were filtered through Whatman filter paper No. 41 and absorbance of samples were measured using double beam Uv- visible spectrophotometer at 218 nm and percent cumulative drug release was calculated using standard calibration curve.

The study was performed on prepared mucoadhesive tablets of formulation F3, F6, F9, F12, F15, F16 and marketed tablets. Study was performed in triplicate. Percent cumulative drug released versus time in hours was plotted. Drug release data was studied through zero order kinetics, first order kinetics, Higuchi matrix, Hixson-crowell cube root law and Korsmeyer-Peppas exponential equation 147.

5.10. In vivo Bioavailability study

In vivo bioavailability study was performed on tablet formulation F9 containing mucoadhesive agent isolated from Prosopis juliflora seed and marketed product of Clopidogrel (CLOPITAB, Mfg by Lupin Ltd.). Bioavailability study protocol was approved from the institutional animal ethics committee (IAEC No. 03PH15JUN11). Six male rabbits of weight 2.0 to 2.5 Kg were randomly selected. Rabbits were kept without food for six hours before tablet administration. Rabbits were allowed drinking of water during the study138. One group (three rabbits) was administered F9 formulation and other group (three rabbits) was administered marketed product. After administration of tablet, 0.5 ml blood samples was collected using cannula from marginal vein at 0.25, 0.5, 0.75, 1.0, 2.0, 4.0, 6.0, 8.0, 12.0, 16.0, 24.0 and 48.0 hours intervals. Plasma was separated by centrifugation at 3000 rpm for 10 min at 25+5 °C. Samples were immediately analysed. Pending samples were stored at freezing temperature. Samples were analysed by previously reported high performance liquid chromatography method148.

Preparation of working solutions

3,20,000 ng/ml stock solution of carboxylic acid metabolite of clopidogrel was prepared in water: methanol: acetonitrile (40:40:20) v/v mixed solvent system. From prepared stock solution, working solutions were prepared in 1.25 to 1,60,000 ng/ml range using above mixed solvent system. Working solution of atorvastatin (internal standard) having concentration 3,20,000 ng/ml was prepared in above mixed solvent system. All solutions were kept at 2 to 8 °C.

Preparation of calibration standards

Calibration standard concentrations of carboxylic acid metabolite of clopidogrel 125, 250, 2000, 4000, 16000 and 32000 ng/ml were prepared using 160 µl of plasma (drug free) 20 µl working solution of internal standard and 20 µl working solution of appropriate concentration. These solutions were centrifuged using microcentrifuge.

Sample preparation procedure

To the calibration standards, 2.5 ml of extraction solvent (ethyl acetate: dichloro methane (80:20) v/v) was added and centrifuged for about 10 min at 3000 rpm. After centrifugation, 2 ml of supernatant was transferred to the evaporation tube. The supernatant was evaporated to dryness in the thermostatically controlled water bath maintained at 55°C. The dry residue was dissolved in 100 µl of water: methanol: acetonitrile (40:40:20 v/v) mixture and transferred to 1 ml sample tube for injection into HPLC.

High performance liquid chromatographic (HPLC) analysis

The level of carboxylic acid metabolite of clopidogrel present in blood plasma of rabbits was estimated by following method using HPLC. A simple gradient method was used, in which percentage of solvent A (0.05% trifluroacetic acid in water) and solvent B (Acetonitrile) was varied. The total run time was 20 min and flow rate was 1 ml/min. For first six minutes 90A: 10B, for 7 to 10 min 50A: 50B, for 11 to 12 min 30A: 70B, for 13 to 16 min. 10A: 90B and for 17 to 20 min 90A: 10B solvent were varied. Separation was achieved on Kromasil ODS, analytical column maintained at 30°C and detection at 220 nm. 50 µl of sample was kept at autosampler at 15°C was injected into HPLC for each chromatographic run. Area under the curve of calibration standards was compared to obtain concentration of drug in plasma of rabbit. Pharmacokinetic parameters Cmax, Tmax and AUC(0-t) were determined from plasma concentration Vs time curve and compared with marketed product.

5.11. Stability Studies

Accelerated stability studies were performed on prepared tablets according to International Conference of Harmonization guidelines at 40+1 °C and 75+ 1% RH. Tablets were stored into an air tight high density polyethylene container and were placed in environment chamber. Tablets were evaluated at 0 day, 90 days and 180 days for mucoadhesive strength, assay and drug release study149