Preparation Of Glimepiride Aloe Barbadensis Miller Biology Essay

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PURPOSE: The main purpose of the present study was to prepare Glimepiride matrix tablets with Aloe barbadensis miller leaves mucilage and Povidone and to study its novelty as a matrix forming polymer for controlled release tablet formulations. METHODS: Physicochemical properties of the dried powdered Aloe barbadensis miller mucilage and Povidone blend, drug-excipient compatibility studies, pre formulation studies, post formulation studies, in vitro drug release studies, mathematical modeling of in vitro dissolution data and in vivo hypoglycemic effects in rabbits were performed. RESULTS: Aloe barbadensis miller mucilage was found to have good flow properties, Glimepiride was found compatible with the excipients used. The granules were found to have good flow properties and in vitro drug release pattern. The mathematical modeling proved that the release of drug from the formulations followed zero order release. The reduced hypoglycemic actions were maintained for prolonged time in in vivo hypoglycemic studies. All these values were found to be satisfactory. CONCLUSIONS: The data revealed that the dried Aloe barbadensis miller mucilage and Povidone combination can be used as a matrix forming polymers for making controlled release matrix tablets.

Key words: Glimepiride, Aloe barbadensis miller, Povidone, matrix tablets, in vitro, in vivo controlled release.

INTRODUCTION

The mucilage of Aloe barbadensis miller leaves clinically and experimentally proved anti-diabetic activity (1) and release retardant activity in the present study. Glimepiride is an oral hypoglycemic agent, which is a commonly prescribed drug for the treatment of patients with type II diabetes mellitus. It belongs to sulfonyl urea drug class. Glimepiride is a weak acid with PKa of 6.2. Glimepiride is practically insoluble in water and acidic environment but highly permeable (class 2) according to the Biopharmaceutical classification System (BCS) 92). The oral absorption is uniform, rapid and complete with nearly 100% bioavailability. The normal dose (3) of Glimepiride is 1to2 mg. The pharmacokinetics and dosage schedule supports once daily controlled release formulations for Glimepiride for better control of blood glucose levels to prevent hypoglycemia, enhance clinical efficacy and patient compliance (4). The main objective of present research is to design controlled release tablets of Glimepiride using Aloe barbadensis miller leaves mucilage and Povidone combination and to evaluate both in vitro and in vivo parameters.

MATERIALS AND METHODS

Materials

Glimepiride was a gift sample from Dr. Reddy's Laboratories, Hyderabad, India. Aloe barbadensis miller leaves were collected from plants growing in local areas of Anantapur, India. The plant was authenticated at the Botany Department of Sri Krishnadevaraya University, Anantapur, India. Povidone, Micro crystalline cellulose (Avicel) and Magnesium stearate were procured from SD Fine chemicals (Mumbai, India). All other chemicals used were of AR grade and deionized water was used throughout the experiment.

EXPERIMENTAL METHODS

Extraction of mucilage

The fresh Aloe barbadensis miller leaves were collected and washed with water. Incisions were made on the leaves and left over night. The leaves were crushed and soaked in water for 5-6 h, boiled for 30 min and left to stand for 1h to allow complete release of the mucilage into the water. The mucilage was extracted using a multi layer muslin cloth bag to remove the marc from the solution. Acetone (three times the volume of filtrate) was added to precipitate the mucilage. The so precipitated mucilage was dried in an oven at 400C, collected, ground, passed through a # 80 sieve and stored in desiccator at 300C & 45% relative humidity till use (5).

Purification of the Mucilage

The crude mucilage (1 %) was homogenized (Potter homogenizer) with cold dilute trichloro acetic acid solution (5%). The solution was centrifuged (3500 rpm for 20 min), neutralized with sodium hydroxide by drop wise addition and then dialyzed for 30 h against distilled water. The mucilage was precipitated with ethanol (in the quantities of three times the volumes) and washed successively with ethanol, acetone and diethyl ether (6, 7). The mucilage so obtained was dried under vacuum (less than 1 Torr at 250C for 12h). The so obtained mucilage was passed through a # 80 sieve and stored in desiccator at 300C & 45% relative humidity till use.

Characterization of Mucilage

The collected mucilage was evaluated for physical characteristics (8, 9) viz., Appearance, Odour, Solubility, percentage yield, average particle size, swelling ratio, weight loss on drying, pH, Charring, density and bio burden.

Chemical characteristics for identification test for Carbohydrates test for Tannins, test for chloride, test for sulphate and test for Uronic acid. The limits of unwanted chemicals (9, 10) viz., foreign matter, heavy metal and Arsenic were also performed.

The flow properties ( 11, 12) viz., Angle of repose, Bulk densities, compressibility index and Hausner's ratio were determined. All these evaluations were carried out as per procedures describe in official books.

Drug-excipient compatibility studies

DSC analysis was performed using DSC 60, Shimadzu, Japan. A 1:1 ratio of drug and excipient was weighed into aluminum crucible. And sample was analyzed by heating at a scanning rate of 200C over a temperature range 200-3000 under nitrogen environment.

FTIR spectra were recorded (Hitachi-270-30 IR spectrophotometer, Japan) on samples prepared in potassium bromide disks which were compressed by hydrostatic press at 7 tons pressure. The samples were scanned at frequency of 500 to 4000 cm-1.

Preparation of matrix tablets

Controlled release Glimepiride matrix tablets with Aloe barbadensis miller leave mucilage and Povidone were prepared by using different drug: mucilage ratios. Aloe barbadensis miller leaves mucilage and Povidone were used as matrix formers, while microcrystalline cellulose as a diluent and Magnesium stearate as a lubricant. All ingredients used were passed through a # 100 sieve, weighed and blended. Wet granulation technique was adopted in preparing the granules and compressed by using 10 mm flat faced punches (13). The formulae of different matrix tablets were shown in Table 4.

Post compression parameters

Swelling behavior of matrix tablets

F-1, F-2, F-3, F-4 and F-5 formulations were subjected to swelling behavior studies. One tablet from each formulation was kept in a Petri dish containing phosphate pH 7.4. At the end of 2 h, the tablet was withdrawn, kept on tissue paper and weighed, repeated for every 2 h till the end of 12 h (14). The % weight gain by the tablet was calculated by the equation 1.

S.I = {(Mt-M0) / M0} X 100 (1)

Where, S.I = Swelling Index, Mt = Weight of tablet at time 't' and Mo = Weight of tablet at time 0.

Thickness and diameter

The thickness and diameter of the tablets was measured by Vernier Calipers. It is expressed in mm.

Weight Variation

20 tablets were selected at random and average weights were determined. Then individual tablet weights were compared with mean tablet weight (15).

Hardness

The hardness of the tablet was determined using a Pfizer hardness tester (15). It is expressed in kg / cm2.

Friability

Roche Friabilator was used for the determination of the friability of the tablets. It is expressed in percentage (%). 20 tablets were initially weighed (Winitial) and transferred into the friabilator. The friabilator was operated at 25 rpm per min for 4 min (100 revolutions). The tablets were weighed again (Wfinal). The % friability was then calculated by equation 2 (15).

F = Winitial - Wfinal / Winitial X 100 (2)

Content uniformity

Twenty tablets were taken and amount of drug present in each tablet was determined. The tablets were crushed in a mortar and the powder equivalent to 2mg of drug was transferred to 100mL standard flask. The powder was dissolved in 5mL of Methanol and made up to volume with 0.1N HCl. The sample was mixed thoroughly and filtered through a 0.45μ membrane filter. The filtered solution was diluted suitably and analyzed for drug content by UV spectrophotometer at a λ max of 230 nm using 0.1 N Hydrochloric acid as blank.

Estimation of Glimepiride

An ultraviolet spectrophotometric method based on measurement of absorbance at 230 nm in Phosphate buffer of pH 7.4. The concentration range of 1-20 µg/mL was obeyed Beer-Lambert's law. The accuracy and Precision were found to be 96% and 1.14% respectively (number of trials=5). No interference was observed during this study with excipients used.

In vitro drug release studies

Release of Glimepiride from the matrix tablets was studied in 900 mL phosphate buffer (pH 7.4) using United States Pharmacopoeia (USP) 8-station Dissolution Rate Test Apparatus (Model Electro lab, TDT- 06T, Mumbai, India) with 50 rpm paddle speed and 37 ± 0.50C. A sample of Glimepiride matrix tablets (2 mg of Glimepiride) was used in each test. Samples of dissolution fluid were withdrawn through a filtered (0.45 μm) at different time intervals and were assayed at 230 nm for Glimepiride content (16) using a UV/visible double-beam spectrophotometer (Elico SL 210, Mumbai, India). The in vitro drug release studies were conducted in triplicate (n = 3).

Drug release kinetics

To analyze the mechanism of drug release from the prepared formulations, the data obtained from in vitro release studies were subjected to, Zero order, first order, Higuchi, Korsmeyer peppa's and Hixson Crowell's models (17-20).

Scanning Electron Microscopy

Scanning Electron Microscopy (SEM) (JSM 5610 LV SEM, JEOL, Datum Ltd., Japan) of tablet (F5) was performed before and different intervals of dissolution. The morphological characters of these scans were compared to hypothesize the mechanism of drug release.

In Vivo study

Normal healthy rabbits weighing 1.5 to 2.0 kg each were used for the in vivo studies. The Institutional Animal Ethics Committee's approval was obtained before the commencement of the study. The study was conducted as per standard institutional guidelines. Two groups of rabbits with 5 in each were fasted 12h before the study (21). Before the administration of drug, a blood sample was taken from the marginal ear vein each rabbit (control). The blood glucose levels of the collected samples were determined using the glucose oxidase method. Pure Glimepiride and matrix tablets Glimepiride were administered orally to each group. A dose of 160 μg/kg of Glimepiride was administrated in a form of suspension for each rabbit. Blood samples were collected at regular intervals of time (at 0, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24h) and analyzed for blood glucose levels with glucose oxidase method.

Accelerated Stability Studies of optimized matrix tablets

The promising formulation was tested for a period of 3 months at different temperatures of 400C with 75% RH, for their drug content (22).

RESULTS AND DISCUSSION

Characterization of Mucilage

The extracted mucilage was in brownish yellow in colour with a characteristic odour. The mucilage was slowly soluble in water produces hage viscous solution. The percentage yield was 23 ±2.173%. The Average particle size was found to be 165.15±10.265µm. The weight loss on drying was 4.20±2.573, the mucilage was charred at 220±4.520C and the bio burden was minimal. All the physical properties were represented in Table 1. The mucilage gave all positive reactions as a polysaccharide and the heavy metals present in the mucilage were within the limits. The chemical properties of the mucilage were shown in Table 2. The angle of repose of extracted mucilage was found to be 27.0±0.050 indicates the powdered mucilage has excellent flow properties. The bulk densities of the dried mucilage were utilized for calculating the compressibility index which was 21.6±0.02%, which indicates fair compression properties which were improved by the addition of magnesium stearate in the formulation. The flow properties of Aloe barbadensis miller leaves mucilage was shown in Table 3.

Drug-excipient compatibility studies

The DSC scan of Glimepiride showed a short endothermic peak at 215.500C. The thermo gram of formulated matrix tablets with Aloe barbadensis miller leaves mucilage and Povidone showed an endothermic peak of drug at 215.680C indicating a slight change in terms of shifting towards the lower temperature. It has been reported that the quantity of material used effects the peak shape and enthalpy. Thus these minor changes in the melting endotherm in the drug could be due to the mixing of the drug and excipients which lower the purity of each component in the mixture and may not necessarily indicate potential incompatibility. The DSC thermo gram of Glimepiride and the formulated matrix tablet blend were shown in Figure 1 and 2.

The IR spectrum of Glimepiride and the matrix tablets of Glimepiride were showed in Figure 3, 4 and 5. The characteristic bands 3344.3, 2900.7, 1627.8, 1427.2, 1342.4 and 1072.3 were observed both in pure Glimepiride and Glimepiride Aloe barbadensis miller leaves mucilage and Povidone mixture. This indicates that there is no chemical incompatibility between Glimepiride and the polymers (Aloe barbadensis miller leaves mucilage and Povidone) used.

Swelling Index of the tablets

The formulated tablets were shown uniformity of swelling in controlled manner. The swelling indexes of formulated tablets were shown in Figure 6.

The formulated tablets pass uniformity in weight (less than ±7.5%) which was within the limits as per Indian Pharmacopoeia. The thickness of formulated tablets was ranged from 4.3±0.21 to 4.9±0.15 mm indicating the uniformity in weight. The hardness of the tablets was ranged from 5.10±1.40 to 6.50±1.45 kg/cm2, which were more than 5 kg/cm2 and passes hardness test. The loss on friability was more than 1% which was within the limits. The amount of Glimepiride in formulated tablets was ranged from 99.84±2.56 to 100.62±5.25%. All these physical properties of formulated matrix tablets were shown in Table 5.

In vitro drug release and Kinetic modeling of in vitro dissolution data

The formulated tablets released more than 30% of the drug in first 1 h and further release was in controlled manner in zero order. Since these plots did not yield a straight line, the dissolution data was subjected to linear regression analysis (r) in zero order kinetics was 0.006705 (for F-5) and the 'r' values obtained for first order kinetics was found to be 0.004053(for F-5). Since greater degree of association best fitted with zero order kinetic models. It can be concluded that, all the matrix tablets followed zero order kinetics as the release pattern of the drug. The 'r' values for remaining formulations were represented in Table 6 and 7 respectively and these values were shown in Figure 7 and 8.

The in Vitro drug dissolution data when treated according to Higuchi's diffusion equation (Q=Kt½) indicated that the formulations released the drug by diffusion which was shown in Figure 9 and represented in Table 8. Further to know the release pattern of Glimepiride from the matrix tablets, the results were analyzed according to Korsmeyer Peppa's exponential equation (q = Ktn ). The slope 'n' was computed to know whether the release was Fickian or non-Fickian. For non-Fickian release ('n' values = 0.5to 1.0), while for Fickian diffusion ('n' value = £0.5). The slope value for optimized Glimepiride matrix tablets (F-5) was 3.308515. The values of 'n' were more than 0.5. So, the F-5 matrix tablets follow the non-Fickian release. These values were tabulated in Table 9 and shown in Figure 10. The in-vitro drug release data was further plotted as (1-mt/mÂ¥) 1/3 verses time proposed by Hixson Crowell's to verify whether the drug release is by erosion mechanism. Figure 11 shows the plots of (1-mt/mÂ¥)1/3 vs. Time and shown in Table 10. The 'r' value was found to be -0.99214 for the formulation F-5. This observation showed that the drug release from the formulated matrix tablets was fitted well to the erosion mechanism. The slope of line indicated that the rate of disappearance of the tablets by erosion. The slope was calculated and it was found to be -0.00092 for matrix tablets.

Scanning Electron Microscopic analysis

The surface morphology of optimized matrix tablets (F-5) at time intervals of 0, 1st, 2nd and 3rd h of dissolution showed that the release of drug form the dosage form was by diffusion controlled and shown in Figure 12.

In vivo drug release studies

The mean % Reduced blood glucose levels with Glimepiride matrix tablets (F-5) was represented in Table 11 and shown in Figure 13. And the F-5 shown very highly significant values (P***<0.001) compared with orally given Glimepiride pure drug.

Accelerated Stability Studies of optimized matrix tablets

The promising formulation was tested for a period of 3 months at accelerated storage conditions of temperatures 400C and the relative humidity of 75% RH. The parameters viz., Thickness, diameter, weight of the tablet, hardness, friability and drug content showed no much variation even after accelerated stability studies. The results of stability data was represented in Table 12.

CONCLUSIONS

The present study revealed that Aloe barbadensis miller leaves mucilage and Povidone combination appears to be suitable for use as a release retardant in the manufacture of controlled release matrix tablets because of its good swelling, good flow and suitability for matrix formulations. From the dissolution study, it was concluded that dried Aloe barbadensis miller leaves mucilage can be used as an excipient for making controlled release matrix tablets.

Acknowledgements

The authors are thankful to Dr. Reddy's Laboratories, Hyderabad, India for providing the pure drug sample.

Table 1: Physical characterization of Aloe barbadensis miller leaves mucilage

Physical Properties

Observation

Appearance

Brownish yellow powder

Odour

Characteristic

Solubility

Slowly soluble in water produces hage viscous solution

Percent yield (g /kg)

23 ±2.173

Average particle size (µm)

165.15±10.265

Weight loss on drying (mg)

4.20±2.573

Swelling Index (%)

45±3.841

pH

7.0±0.56

Charring (0C)

220±4.52

Density of liquid (0.5% w/v)

0.997±0.055

Microbial count (cfu/g)

Bacteria:5 ; Fungi: 2

Cfu = Colony forming units

Table 2: Chemical characterization of Aloe barbadensis miller leaves fruit mucilage

Chemical properties

Observation

Mounted in 96% ethanol

Transparent angular masses

Mounted in ruthenium red

Particles stained red

Mounted in Iodine solution

Particles stained blue

Test for Carbohydrate (Mollish test)

+ve

Test for Tannins (Ferric chloride test)

-ve

Test for chloride (Silver-nitrate test)

-ve

Test for Sulphate (Barium chloride test)

-ve

Test for Uronic acid

+ve

Test for foreign matter (%)

NMT 0.1

Test for heavy metal (lead)

23 ppm

Test for Arsenic

<1 ppm

Ppm = Parts per million; NMT = Not more than; +ve = Positive; -ve= Negative

Table 3: Flow properties of Aloe barbadensis miller leaves mucilage

Flow properties

Observation

Angle of repose (q°)

27.0±0.05

Loose Bulk density (g/cm3)

0.55±0.04

Tapped bulk density(g/cm3)

0.74±0.04

Carr's Index (%)

21.6±0.02

Hausner's ratio

1.36±0.01

Number of trials (n)=5

Table 4: Formulae of matrix tablets

Ingredients (mg)

Formulations

F-1

F-2

F-3

F-4

F-5

Glimepiride

2

2

2

2

2

Aloe barbadensis miller leaves

dried mucilage

2.5

5

7.5

10

12.5

Povidone

2.5

5

7.5

10

12.5

Micro crystalline cellulose (Avicel)

188

183

178

173

168

Magnesium stearate

5

5

5

5

5

Total weight of tablet

200

200

200

200

200

Table 5: Physical properties of formulated matrix tablets

Formulation

Thickness

(mm)

Hardness

(kg/cm2)

Friability

(%)

Drug content

(%)

F-1

4.3±0.21

6.50±1.25

0.80±0.02

99.85±3.95

F-2

4.9±0.15

5.10±1.40

0.55±0.05

100.62±5.25

F-3

4.4±0.41

5.80±1.35

0.64±0.03

99.84±2.56

F-4

4.4±0.39

6.50±1.45

0.69±0.06

99.96±5.68

F-5

4.5±0.58

6.40±1.30

0.84±0.07

99.97±3.94

Table 6: Zero order modeling of matrix tablets

Formulation

Zero Order

Slope

Regression coefficient(r)

k value

F-1

0.003559

0.003559

0.990392

F-2

0.002955

0.002955

0.992511

F-3

0.005966

0.004966

0.996615

F-4

0.006498

0.006498

0.988149

F-5

0.006705

0.006705

0.995252

Table 7: First order modeling of matrix tablets

Formulation

First Order

Slope

Regression coefficient (r)

k value

F-1

-0.00075

0.001727

-0.97846

F-2

-0.00049

0.001128

-0.99684

F-3

-0.00156

0.003593

-0.97261

F-4

-0.00153

0.003524

-0.99259

F-5

-0.00176

0.004053

-0.98231

Table 8: Higuchi modeling of matrix tablets

Formulation

Higuchi's values

Slope (n)

Regression co-efficient (r)

F-1

1.725046

0.971738

F-2

1.865816

0.996448

F-3

3.103433

0.985042

F-4

3.227632

0.993489

F-5

3.308515

0.993936

Table 9: Korsmeyer Peppa's modeling of matrix tablets

Formulation

Korsmeyer Peppa's values

Slope (n)

Regression co-efficient (r)

F-1

0.162456

0.930212

F-2

0.171559

0.955678

F-3

0.287578

0.947332

F-4

0.313169

0.974429

F-5

0.304558

0.968565

Table 10: Hixson-Crowell's modeling of matrix tablets

Formulation

Hixson-Crowell's values

Slope (n)

Regression co-efficient (r)

F-1

-0.00043

-0.98355

F-2

-0.00032

-0.99574

F-3

-0.00064

-0.99517

F-4

-0.00083

-0.99441

F-5

-0.00092

-0.99214

Table 11: Mean % Reduced blood glucose levels with F-5 matrix tablets

Time (h)

Mean Reduced blood glucose levels (%)

Group-I (Control)

Glimepiride(p.0)

Group-II

F-5

0 (basal BGL)

0.00±0.00

0.00±0.00

0.5

18.21±2.65**

25.41±2.10***

1

26.42±2.48**

27.89±1.49***

2

42.89±1.98**

33.01±2.15***

3

40.52±2.54**

36.38±3.01***

4

21.51±2.68**

40.69±2.78***

5

16.53±1.26**

39.81±0.91***

8

0.01±0.00**

38.89±1.54***

10

0.00±0.00**

33.88±2.54***

12

0.02±0.00**

36.39±0.36***

Number of animals (n) = 6; BGL- Blood Glucose Level

P**<0.01=highly significant and P***<0.001= very highly significant when compared with normal control group.

Table 12: Summary of physicochemical properties of F-5 matrix tablets before and after accelerated stability studies

Parameter

Before

stability studies

After

stability studies (90 days)

Thickness (mm)

4.5±0.58

4.5±0.52

Diameter (mm)

8.01±0.318

8.01±0.318

Weight of the tablet (mg)

199.85±5.6

199.85±5.4

Hardness (kg/cm2)

6.40±1.30

6.40±0.19

Friability (%)

0.84±0.07

0.85±0.06

Drug content (%)

99.97±3.94

99.97±4.52

Fig. 1: The DSC thermo gram of Glimepiride

Fig. 2: The DSC thermo gram of matrix tablets

Fig. 3: IR Spectrum of Glimepiride Pure drug

Fig. 4: IR Spectrum of Placebo tablets

Fig. 5: IR Spectrum of formulated matrix tablets

Fig. 6: Swelling Index of matrix tablets

Fig. 7: Zero order release Plots

Fig. 8: First order release Plots

Fig. 9: Higuchi Plots

Fig. 10: Korsmeyer Peppa's Plots

Fig. 11: Hixson-Crowell's Plots

Fig. 12: Surface morphology of matrix tablet (F-5) at time intervals of 0, 1st, 2nd and 3rd h of dissolution

Fig. 13: Reduced blood glucose levels (%) of optimized Glimepiride matrix tablets

(F-5) vs. Glimepiride oral control

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