105

6.1 CHARACTERIZATION OF TABLETS

All the prepared formulations were subjected for following

evaluation parameters and obtained results were showed in Tables 6.3 &

6.4.

6.1.1 Description (Size, Shape, Color)

The tablets were looking good and non sticky. The color, shape of

tablets was analyzed with naked eye. The diameter and thickness of

tablets was performed on 20 tablets from each formulation. Digital

vernier caliper was used for the study, which permits accurate

measurements and provides information of the variation between tablets.

6.2 WEIGHT VARIATION 99,100,102

Weight variation was carried out to ensure that, each of tablets

contains the proper amount of drug. The test was carried out by

weighing the 20 tablets individually using analytical balance, then

calculating the average weight, and comparing the individual tablet

weights to the average.

The percentage of weight variation is calculated by using the

following formula.

Table 6.1 Weight Variation Limits for Tablets (I.P)

S. No. Average weight of tablets (mg) Maximum percentage

difference allowed
1 130 or less ±10.0
2 130-324 ±7.50
3 More than 324 ±5.0
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6.3 HARDNESS 99,100,102

The resistance of tablets to capping, abrasion or breakage under

conditions of storage, transportation and handling before usage depends

on its hardness. Tablet hardness is defined as the load required crushing

or fracture a tablet placed on its edge. Sometime it is also termed as

tablet crushing strength. The hardness test was performed using

Monsant type (Make: Singhla) hardness tester. The instrument measures

the force required to break the tablet when the force generated by anvils

to the tablet. The tablet was placed between two anvils; force applied to

the anvils, and the crushing strength that just causes the tablet to break

was recorded. The crushing strength test was performed on 20 tablets

from each formulation.

6.4 FRIABILITY 99,100,102

For each formulation, the friability of 20 tablets was determined

using Roche type friabilator. 20 tablets from each formulation were

weighed and tested at a speed of 25 rpm for 4 min. After removing of

dusts, tablets were re-weighed and friability percentage was calculated

using the following equation.

6.5 DISINTEGRATION 99,100,118

The disintegration apparatus, described in I.P was used for the

study. It contains 2 basket rack assembly. Each basket rack assembly

consists of 6 glass tubes that are 3 inches long, open at the top and held
 107

against 10 mesh screen at the bottom. Each tablet was placed in each

tube, and the basket rack was positioned in 1-L beaker of distilled water.

The 37±2°C temperature was maintained throughout the study.

6.6 DISSOLUTION 99,100,102

The In-vitro dissolution study of tablets for best formulation was

determined by using USP Type 2 (paddle type) dissolution apparatus. The

test was performed in 200 ml of distilled water at 50 rpm maintained at

37± 0.5°C. The specific amount of samples were withdrawn at

predetermined time intervals for period of 2 hours (15, 30, 45, 60, 90,

120 min.) and replaced with the equal volume of the same dissolution

medium. The samples were filtered through 0.2μm membrane filter.

Amount of catechins, sodium selenite released from the tablets were

analyzed by HPTLC and LC ICP MS methods respectively.

6.6.1 Methodology of Analysis of Sodium Selenite Compound103

LC-ICP-MS technique is used to qualitate and quantitates sodium

selenite. The coupling of LC to ICP-MS is a relatively straight forward

task, and gives an accurate result. LC column is simply connected to the

nebulizer of the ICP-MS by a piece of PEEK™ tubing. When using the

Varian 820-MS equipped with the collisional reaction interface (CRI), it is

possible to measure the sodium selenate at m/z 80.

The LC-ICP-MS system used for this study consisted of a Varian

ProStar 230 Tertiary Solvent Delivery Module, a Varian ProStar 410

biocompatible auto sampler with a 200 µL sample loop, and a Hamilton

 108

PRP-X100 (4.1 mm x 250 mm, 10 µm) anion exchange column and the

Varian 820-MS. The Varian 820-MS setup, inducing the CRI gas flow,

was optimized using the ICP-MS Expert software auto optimization

routine. The Varian 820-MS was operated in high sensitivity mode, and

was connected to the LC column using a 20 cm length of PEEK tubing

(0.25 mm ID). Table 6.2 provides detailed listings of the ICP-MS and LC

conditions.

6.6.2 Construction of Standard Plots For Sodium Selenite Marker

Compound

Preparation of Stock solution: Ten mg of marker sodium selenite

was weighed carefully and transferred into 100 ml standard flask. This is

diluted up to the mark with de ionized water. 10 ml of this solution was

diluted to 100 ml with de ionized water, by using standard flask. This

solution was considered as stock solution and its concentration is

10µg/ml.

Preparation of serial dilutions: One ml of stock solution is diluted

up to 1 L with water to give concentration 10µg/l. In the same passion, a

series of solutions, having 20, 30, 40, 50, 60, 70, 80, 90 & 100 µg

sodium selenite per L, were prepared. All the solutions were injected into

LC, and obtained curves were recorded. A 10 point calibration curve of

the purchased sodium selenite was constructed, by taking

concentrations on X axis, and peak areas on Y axis. The prepared

calibration curve was used to determine the amount of sodium selenite

 109

present in the dissolution samples. Table 6.5 gives the data of Sodium

Selenite calibration curve. Fig. 6.1 shows calibration curve of Sodium

Selenite.

Table 6.2 Details of LC-ICP-MS Conditions

S. No. Parameter Value

Flow Parameters (L/min)
1. Plasma flow 18.0
2. Auxiliary flow 1.65
3. Sheath gas 0.24
4. Nebulizer flow 0.98
Other
5. RF power (kW) 1.40
6. Pump rate (rpm) 25
Ion optics (volts)
7. First extraction lens -500
8. Second extraction lens -879
9. Third extraction lens -643
10. Corner lens -763
11. Mirror lens left 40
12. Mirror lens right 37
13. Mirror lens bottom 40
14. Entrance lens 1
15. Fringe bias -3.9
16. Entrance plate -30
CRI (mL/min)
17. Skimmer gas source H2
18. Skimmer flow 70
LC Operating Conditions
19. Parameter Settings
20. Mobile Phase 60 m M Ammonium nitrate
21. Flow rate 1.0 mL/min
22. Run time 7 min
Column Anion exchange, Hamilton PRP-X 100,
4.1 mm ID x 250 mm, 10 µm
23. Column Temperature Ambient
24. Sample injection volume 200 µL
25. Detection Varian 820-MS
 110

6.6.3 Determination of Amount of Catechins Released

From Tablet 96, 97, 104, 105

The 2 ml sample of dissolution media was withdrawn and replaced

with the equal volume of the fresh dissolution medium. The collected

samples were filtered through 0.2μm membrane filter. 5µl of this filtered

solution was applied per each spot. This test was done in triplicate.

The amount of catechins released from tablet was analyzed with

help of standard plots. The following formula, generated from calibration

curve was used to calculate the amount of catechins. Fig. 6.2 to 6.7

shows HPTLC chromatograms of tablet dissolution samples. The amount

of all the catechins released from tablet was given in Table 6.6 to 6.12
 111

6.6.4 Determination of Amount of Sodium Selenite Released From

Tablet

The amount of Sodium Selenite released at corresponding time

intervals was calculated by using the following formula.

Where,

CCC = Concentration obtained from Calibration Curve

DF = Dilution Factor

VDM = Volume of Dissolution Medium

CF = Conversion Factor

The amount of Sodium Selenite released from tablet during

dissolution is showed in Table 6.13. Fig. 6.8 to 6.13 shows LC ICP MS

chromatograms of tablet dissolution samples.

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6.7 Results and Discussion

The results obtained from above tests were showed and described

below.

Table 6.3 Results of Description of Tablets

S. No. Parameter Result

1 Color Greenish Brown

2 Shape Round, Biconvex
3 Odor Characteristic
4 Size in mm
i. Thickness 5.15±0.12 mm
ii. Diameter 8.65±0.23 mm

Table 6.4 Results of Physico-Chemical Characterization of Tablets

Formulation Average Hardness Friability Disintegration Time

Code Weight (kg/cm2) (%)
(mg)
GST-1 400±0.20 3.10±0.08 0.69±0.05 30 min & 20 sec
GST-2 400±0.12 4.03±0.14 0.50±0.05 28 min & 24 sec
GST-3 400±0.11 4.21±0.09 0.53±0.03 25 min & 40 sec
GST-4 400±0.09 4.06±0.11 0.84±0.01 20 min & 12 sec
GST-5 400±0.12 4.13±0.12 0.98±0.00 16 min & 14 sec
GST-6 400±0.11 4.03±0.17 0.44±0.05 13 min & 35 sec
GST-7 400±0.07 3.17±0.04 0.84±0.07 110 min & 10 sec
GST-8 400±0.06 3.34±0.33 0.72±0.08 104 min & 12 sec
GST-9 400±0.06 4.13±0.12 0.98±0.11 100 min & 54 sec
GST-10 400±0.09 3.20±0.24 0.63±0.04 092 min & 33 sec
GST-11 400±0.12 3.17±0.07 0.84±0.05 082 min & 23 sec
GST-12 400±0.10 3.14±0.03 0.70±0.09 079 min & 43 sec
 113

The maximum weight variation obtained was ± 0.20%, which falls

within the acceptable weight variation range of ± 5%. Hence all the

tablets passed the weight variation test. Hardness for tablets was in the

range of 4.0 to 4.2 kg/cm2, which falls above the limit of not less than

3.0 kg/cm2. None of the tablets showed friability value more than 0.87%

which is less than ideal limit 1%. Out of all the formulations, formulation

GST-6 has passed the disintegration test by showing Thirteen minutes

and Thirty five seconds, which is less than ideal limit Fifteen minutes.

The acceptance criteria of dissolution rates of herbal medicinal product

are as follows.106, 107

1) Europian Pharmacopoeia Limits: 50-85% release within 30-60 min.,

and/or, Mean 75% release within 45 min.

2) F.I.P Limits: For rapid release = >80% within 20-30 min., for non-rapid

release = 20-30% within 60-120 min.

The best formulation has showed release of maximum amount

(around 100%) of catechins and sodium selenite within one hour. Hence

the formulation was passed the dissolution test.

 114

Table 6.5 Data of Sodium Selenite Calibration Curve

S. No. Concentration of Sodium Selenite (µg/L) Peak Area

1 0 0

2 10 1600

3 20 3298

4 30 4952

5 40 6621

6 50 8300

7 60 9950

8 70 11712

9 80 13343

10 90 14950

11 100 16519

Calibration Equation y = 166.0x + 0.0

Regression Coefficient R² = 0.999

Fig. 6.1 Calibration Curve of Sodium Selenite.

 115

Fig. 6.2 HPTLC Chromatogram of 15 min. Dissolution Sample for

Catechins
 116

Fig. 6.3 HPTLC Chromatogram of 30 min. Dissolution Sample for

Catechins
 117

Fig. 6.4 HPTLC Chromatogram of 45 min. Dissolution Sample for

Catechins
 118

Fig. 6.5 HPTLC Chromatogram of 60 min. Dissolution Sample for

Catechins
 119

Fig.6.6 HPTLC Chromatogram of 90 min. Dissolution Sample for

Catechins
 120

Fig. 6.7 HPTLC Chromatogram of 120 min. Dissolution Sample for

Catechins
 121

Fig. 6.8 LC ICP MS Chromatogram of 15 min. Dissolution Sample for

Sodium Selenite

Fig. 6.9 LC ICP MS Chromatogram of 30 min. Dissolution Sample for

Sodium Selenite
 122

Fig. 6.10 LC ICP MS Chromatogram of 45 min. Dissolution Sample for

Sodium Selenite

Fig. 6.11 LC ICP MS Chromatogram of 60 min. Dissolution Sample for

Sodium Selenite
 123

Fig. 6.12 LC ICP MS Chromatogram of 90 min. Dissolution Sample for

Sodium Selenite

Fig. 6.13 LC ICP MS Chromatogram of 120 min. Dissolution Sample for

Sodium Selenite
 124

Table 6.6 Amount of Catechin Released from Tablet during Dissolution of tablet.

S. No. Sampling Peak Concentration Amount of Catechin % of

Time Area obtained from Released in 200ml Catechin
(min) Calibration Curve of dissolution media Released
(mg)
1 15 285.6 15.60 0.624 48.00
2 30 399.8 21.25 0.850 65.40
3 45 500.3 26.22 1.049 80.72
4 60 601.3 31.22 1.249 96.09
5 90 611.0 31.70 1.268 97.56
6 120 619.1 32.10 1.284 98.79

Table 6.7 Amount of EC Released from Tablet during Dissolution of tablet.

S. No. Sampling Peak Concentration Amount of EC % of EC

Time Area obtained from Released in 200ml Released
(min) Calibration Curve of dissolution
media (mg)
1 15 1977.4 097.25 3.89 46.72
2 30 2679.7 132.00 5.28 63.54
3 45 3649.8 180.00 7.20 86.49
4 60 4109.6 202.75 8.11 97.43
5 90 4129.8 203.75 8.15 97.99
6 120 4215.7 208.00 8.32 100.00

Table 6.8 Amount of GC Released from Tablet during Dissolution of tablet.

Sampling Peak Concentration Amount of GC % of GC

S. Time Area obtained from Released in 200ml Released
No. (min) Calibration Curve of dissolution
media (mg)
1 15 0544.8 027.00 1.80 43.28
2 30 1310.9 065.75 2.63 63.19
3 45 1765.6 088.75 3.55 85.33
4 60 2027.5 102.00 4.08 98.05
5 90 2042.4 102.75 4.11 98.73
6 120 2047.3 103.00 4.12 98.99
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Table 6.9 Amount of EGC Released from Tablet during Dissolution of tablet.
S. No. Sampling Peak Concentration Amount of EGC % of
Time Area obtained from Released in 200ml EGC
(min) Calibration Curve of dissolution Released
media (mg)
1 15 4701.4 238.50 09.54 44.08
2 30 6346.2 322.25 12.89 62.78
3 45 8575.4 435.75 17.43 84.90
4 60 9552.4 485.50 19.42 94.54
5 90 9925.6 504.50 20.18 98.28
6 120 10102.4 513.50 20.54 100.00

Table 6.10 Amount of ECG Released from Tablet during Dissolution of tablet.
S. No. Sampling Peak Concentration Amount of ECG % of
Time Area obtained from Released in 200ml ECG
(min) Calibration Curve of dissolution Released
media (mg)
1 15 4580.9 227.70 09.10 47.98
2 30 5715.8 284.90 11.40 60.04
3 45 7831.9 391.56 15.66 82.52
4 60 8662.1 433.40 17.34 91.34
5 90 9477.5 474.50 18.98 100.0
6 120 9477.5 474.50 18.98 100.0

Table 6.11 Amount of EGCG Released from Tablet during Dissolution of tablet.
S. No. Sampling Peak Concentration Amount of EGCG % of
Time Area obtained from Released in 200ml EGCG
(min) Calibration Curve of dissolution Released
media (mg)
1 15 19917.7 992.50 39.70 51.76
2 30 25834.8 1287.00 51.48 67.12
3 45 30692.5 1528.82 61.15 79.73
4 60 36413.1 1813.57 72.54 94.58
5 90 38501.5 1917.50 76.70 100.0
6 120 38501.5 1917.50 76.70 100.0
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Table 6.12 Dissolution Profile Data of all the Catechins Released from
Tablet

S. No. Sampling Time % of catechins released

(min) Cat EC GC EGC ECG EGCG
1 15 48.00 46.72 43.28 44.08 47.98 51.76
2 30 65.40 63.54 63.19 62.78 60.04 67.12
3 45 80.72 86.49 85.33 84.90 82.52 79.73
4 60 96.09 97.43 98.05 94.54 91.34 94.58
5 90 97.56 97.99 98.73 98.28 100.0 100.0
6 120 98.79 100.00 98.99 100.00 100.0 100.0

Table 6.13 Amount of Sodium Selenite Released from Tablet during

Dissolution of Tablet.

S. Sampling Peak Concentration Amount of Sodium % Amount of

No. Time (min) Area obtained from Selenite Released Sodium
Calibration Curve in 200ml of Selenite
(µg/L) dissolution media Released
(mg)
1 15 4067 24.5 0.49 49
2 30 5810 35.0 0.70 70
3 45 7387 44.5 0.89 89
4 60 8300 50.0 1.00 100
5 90 8300 50.0 1.00 100
6 120 8300 50.0 1.00 100