59

5.1 PREPARATION OF GREEN TEA AQUEOUS EXTRACT

Green tea leaves were collected (Photograph 5.1) in the season from

Ooty, Tamil Nadu and authenticated by Dr. K. Madhava Chetty,

Department of Botany, S.V. University, Tirupathi, Andhra Pradesh.

Leaves were cleaned and shade dried completely as showed in

Photograph 5.2. The dried leaves were milled with hammer mill and

passed through 1mm mesh screen. One hundred grams of leaves boiled

with 1 liter of distilled water for 10 min at 70ºC. The heated solution was

filtered, evaporated under vacuum and freeze dried.86,87 Resulted green,

dry mass was used to prepare the tablet.

5.2 STANDARDIZATION OF GREEN TEA AQUEOUS EXTRACT

The prepared green tea aqueous extract was standardized as per

WHO guidelines and reported as total ash, water soluble ash, acid

insoluble ash, water soluble extractive, methanol soluble extractive and

loss on drying.

5.2.1 Description

Dark Green colored powder with characteristic odor.

5.2.2 Ash Content 88-92

The ash remaining following ignition of medicinal plant extract was

determined by three different methods which measure 1) Total ash

2) Acid Insoluble ash 3) Water soluble ash. The total ash method is

designed to measure the total amount of material remaining after

ignition. This includes both Physiological ash, which is derived from the

plant tissue itself, and non-physiological ash, which is the residue of the

60

extraneous matter (e.g. sand and soil) adhering to the plant surface. Acid

insoluble ash is the residue obtained after boiling the total ash with

dilute hydrochloric acid, and igniting the remaining insoluble matter.

This measures the amount of silica present, especially as sand and

siliceous earth. Water soluble ash is the difference between the total ash

and residue after treatment of the total ash with water.

Total Ash

Three grams of the air dried material was accurately weighed and

packed in ash less filter paper. This pack was kept in silica crucible and

ignited at 700°C for 15 minutes. The crucible was removed from oven

and cooled to room temperature. A white powder was obtained indicating

absence of carbon. The total ash content was estimated by using the

following formula.

Acid Insoluble Ash

One gram of ash was added to 25 ml of 2 molar hydrochloric acid

and boiled gently at 70-90°C for 5 minutes. It was filtered through filter

paper and packed in ash less filter paper. This pack was kept in crucible

and ignited for 15 minutes in oven at 700°C. The crucible was removed

and cooled to room temperature. The acid insoluble ash was calculated

by using following formula.

61

Water Soluble Ash

One gram of ash was added to 25 ml of water and boiled at 100°C

for 5 minutes. It was filtered through filter paper and packed in ash less

filter paper. This pack was kept in crucible and ignited for 15 minutes in

oven at 700°C. The crucible was removed and cooled to room

temperature. The water soluble ash was calculated by using fallowing

formula. All the calculated ash content values are showed in Table 5.6

5.2.3 Extractive Values88-92

Four grams of leaf aqueous extract was accurately weighed and

transferred into a glass stoppard conical flask. It was macerated with

100 ml of specific solvent for 6 hours, shaking frequently. It was allowed

to stand for 18 hours. It was filtered carefully, and 25 ml of the filtrate

was transferred to a flat bottomed dish and evaporated the solvent to

dryness on a water bath. It was dried at 105°C for 6 hours, and cooled in

desiccators for 30 minutes and weighed without delay. The content of

extractable matter in mg per g of extract was calculated. The results were

showed in Table 5.6.

5.2.4 Loss on Drying88-92

Four grams of leaf aqueous extract was accurately weighed and

transferred into a flat weighing bottle. The sample was dried in hot air

oven at 100°C until two consecutive weighing are almost constant. The

results are showed in Table 5.6.

62

5.2.5 Microbial Contamination93-95

Herbal Medicinal Products have the potential of contamination

with different microorganisms, which can adversely affect health status

of consumers as well as the stability of the products. Recently, microbial

contamination on crude drugs has become an issue and certain quality

assurances have been sought from the good manufacturing practices

stand point. According to WHO & EMEA guidelines, determination of

microbial contamination is a part of standardization and is necessary for

any herbal products. The Green tea aqueous extract was subjected to the

following examinations:

1. Presence or Absence of

Staphylococcus aureus

Pseudomonus aeruginosa

Salmonella typhimurium

Escherichia coli

2. Total Viable Count for Bacteria

3. Presence or Absence of Moulds

Microorganisms and Media

The standard microorganisms used in this study were purchased

from the National Collection of Industrial Microorganisms (NCIM),

National Chemical Laboratory, Pune – 411 008, India. The NCIM number

of each organism and corresponding suggested culture media were used

(Table 5.1).

63

Standard and Test Preparation

Standard Preparation: The standard micro organisms were

aseptically transferred with the help of loop into test tube containing

sterile slant media. After incubation, one ml of sterile water was poured

into slant and swabbed. This suspension was collected carefully and

diluted with sterile water to make up the volume up to 10 ml. Serial

dilutions were made up to 1 x 105 dilutions and viability was assessed

using pour plate method. The morphological characters of these standard

cultures were used to compare with the cultures made from test

samples.

Test Preparation: One gram of green tea aqueous extract was taken

for the study. In case of finished dosage form, randomly selected twenty

tablets were converted into powder in sterile area and one gram of this

tablet powder was taken for the study. One g of the sample was

dissolved in sterile distilled water to make up the volume up to 10 ml.

Serial dilutions were made up to 1 x 105 dilutions and viability was

assessed using the pour plate method. The plates were incubated for

suitable period and at their suitable temperature. The plate was placed

on a colony counter and the number of colony forming units was

calculated by using the following formula. In case of moulds, number of

mycelia arises from each fungal spore is counted. The results of

microbial test were showed in Table5.7.

64

Table 5.1 Experimental Protocol for Microbial Load Determination

S.

No. Organism

NCIM

No.

Media Used Media Composition

Incubation

Name pH Ingredient Quantity

1. Staphylococcus aureus

2602 Nutrient Agar

7.0±0.2

Beef Extract

10.0 g

37±0.2°C & 24 hours

2. Salmonella typhimurium

2501 NaCl 05.0 g

Peptone 10.0 g

3. Pseudomonus aeruginosa

2053 Distilled water 01.0 L

Agar 20.0 g

4. Escherichia coli 2981 LB

(Luria

Broth)

7.0±0.2

Tryptone 10.0 g

Yeast Extract 5.0 g

NaCl 10.0 g

Distilled water 01.0 L

Agar 20.0 g

5. Fungi -- PDA

(Potato

Dextrose

Agar)

5.4±0.2

Potato 200.0 g 25±0.2°C &

36 hours Dextrose 20.0 g

Agar 15.0 g

Diss. Water,

Q.S

01.0 L

65

5.2.6 Quantification of Catechin Contents by HPTLC

The method used to analyze the green tea catechin compound is

described as follows.

5.2.6.1Methodology of Analysis of Green Tea Catechin Compounds28

Principle:

HPTLC fingerprinting technique is used to standardize the

prepared pure green tea aqueous extract and tablets.

In qualitative analysis, Rf values obtained for marker compounds

were compared with that of pure green tea aqueous extract and tablets.

In quantitative analysis, the area of the peaks obtained for marker

compounds were compared with that of pure green tea aqueous extract

and tablets.

Instrumentation:

The solutions were applied in triplicate on TLC plate using CAMAG

LINOMAT IV automatic spotter.

The plate was developed with mobile phase comprised of

1-propanol: water: acetic acid (20:80:1 v/v).

After development the plate was first scanned in UV in scanner III

and detection and quantification was performed by densitometry at λ =

333 nm.

The peak Rf values and corresponding areas were recorded.

HPTLC operating conditions are showed in Table 5.2.

66

Table5.2 HPTLC Instrument Operating Details

S. No. Parameters Details

1 Instrument HPTLC

2 Software winCATS Planar Chromatography Manager

3 Stationary Phase

Material: TLC Aluminium Sheets, Cellulose coated

Manufacturer: E. MERCK KGaA Plate Size (X x Y): 10 X 20 cm, 20 X 20 cm, (Merck No.

1.05786) Pre-washing: No Modification: No

4 Mobile Phase 1-propanol: water: acetic acid (20:80:1 v/v)

5 Calibration Parameters

Calibration mode: Multi level

Statistics mode: CV Evaluation mode: Peak Height & Area

6 Sample Application

Parameters

Instrument: CAMAG Linomat 5 “Linomat5_100632” S/N 1

Spray gas: Inert gas Sample Solvent type: Methanol Dosage Speed: 150 nl/s

Predosage Volume: 0.2 ul Syringe Size: 100µl

Application Position Y: 10.0 mm Band Length: 6.0 mm

7 Detection Parameters

Instrument: CAMAG TLC Scanner 3

“Scanner3_100904” S/N Application Position: 10.0 mm

Solvent front Position: 84.0 mm Scanning Speed: 20 mm/s Data resolution: 100 µm/step

Wavelength: 333 Lamp: D2

Measurement Type: Remission Measurement Mode: Absorption

67

5.2.6.2 Construction of Standard Plots for Marker Catechin

Compounds 96-97

Preparation of Standard solutions of marker compounds for

calibration curve: It was prepared by dissolving 2.5mg of each marker

compound in 100ml of distilled water under sonicator. The solution was

filtered through 0.2µ. The resulted solution has the concentration

25µg/ml (25ng/µl).

Calibration Curve:

Standard solutions of each marker compound was applied in

triplicates 2, 4, 6, 8, 10 12µl, over silica gel plate to obtain amounts of

50, 100, 150, 200, 250, 300ng per spot respectively. The obtained HPTLC

chromatograms of all marker compounds showed in Fig‟s. 5.1 to 5.6. The

calibration data showed in Table 5.8 and calibration equations showed in

Table 5.9. Calibration curves were constructed for each marker

compound and showed in Fig‟s. 5.7 to 5.12.

5.2.6.3 Quantification of Catechins present in Prepared Green Tea

Extract

One hundred milligrams of prepared pure green tea aqueous

extract is weighed and transferred to 100ml volumetric flask carefully. A

small quantity of water is added and sonicated for solubility. It was

diluted with same up to the mark 100ml. 5µl of this solution is directly

applied as spot on HPTLC plate. Rf and peak area values were recorded.

68

Qualitative Analysis: The Rf values of extract peaks and marker

compound peaks were compared. This confirms that the prepared pure

extract contains all the desired catechins.

Quantitative Analysis: Peak area obtained, calibration equation

were used to quantification of compounds present in the extract.

Table 5.10, Shows the details of catechin contents in the prepared

sample. Fig. 5.13 shows the HPTLC chromatograms of the prepared

green tea aqueous extract. Photograph 5.7 shows the HPTLC density

grams of marker compounds, Green Tea aqueous extract, and prepared

formulations.

5.3 PRE FORMULATION STUDIES

The following preformulation studies were carried out.

5.3.1 Drug Incompatibility Studies98

Compatibility of the one active compound green tea with another

active compound sodium selenite and the compatibility of each active

compound with all other excipients were determined by FTIR spectral

69

analysis. This study was carried out to detect any changes on chemical

constitution of the drug after combined it with the excipients. All the

samples alone and in various combinations were taken for FTIR study. IR

spectra of samples in KBr pellets at moderate scanning speed between

400 to 4000 cm-1 wave numbers was carried out using FTIR. The FTIR

spectra of active compounds alone and in combination with various

excipients are showed in the Fig‟s. 5.14 to 5.25. The wave numbers of all

prominent peaks are shown in Table 5.11.

5.3.2 Derived Properties 99,100

Bulk density and tapped density of granules were determined.

5.3.2.1 Bulk Density

Bulk density is not an intrinsic property of a material; it can

change depending on how the material is handled. For example, a

powder poured in to a cylinder will have a particular bulk density, if the

cylinder is disturbed; the powder particles will move and usually settle

closer together, resulting in a higher bulk density. For this reason, the

bulk density of powders is usually reported both as "freely settled" and

"tapped" density. It is defined as the mass of a powder divided by the

bulk volume. Bulk density of a compound varies substantially with the

method of crystallization, milling or formulation. Bulk density depends

on particle size distribution, powder shape and tendency of the particles

to adhere to one another. Loose bulk density was determined using

graduated cylinder. Accurately weighed (5 gm) of sample was taken and

70

it was transferred in to 100 ml graduated cylinder. The volume of the

packing was recorded and the loose bulk density was calculated by the

following formula.

5.3.2.2 Tapped Density

It is defined as the mass of a powder divided by the tapped volume.

The tapped bulk density determined using graduated cylinder. The

graduated cylinder was tapped for at least 100 times and the tapped

volume of packing was recorded. The tapped bulk density was calculated

by the following formula.

5.3.3 Flow Properties 99,100

The prepared granules were subjected to following flow properties.

5.3.3.1 Angle of Repose

Pharmaceutical powders may be broadly classified as free flowing

or cohesive. Most flow properties are significantly affected by changes in

particle size, density, shape, electrostatic charge and absorbed moisture

which may arise from processing or formulation. The frictional forces in a

loose powder can be measured by the angle of repose. It is defined as the

bulk powder materials are poured onto a horizontal surface, a conical

pile will form. The internal angle between the surface of the pile and the

horizontal surface is known as the angle of repose. Material with a low

71

angle of repose forms flatter piles than material with a high angle of

repose. In other words, the angle of repose is the angle a pile forms with

the ground.

Table 5.3 Relation between Angle of Repose and

Type of Flow

S. No. Angle of repose Type of flow

1 <25 Excellent

2 25-30 Good

3 31-40 Passable

4 >40 Very poor

The angle of repose of powder was determined by the fixed funnel

method. The glass funnel was fixed at a constant height. The accurately

weighed powdered blend was poured through a funnel that can be raised

vertically until a maximum cone height (h) was obtained. Radius of the

heap (r) was measured and the angle of repose (ө) was calculated by

using the following formula.

Where, h= height of cone in cm, r = radius of the base of heap in cm on

the graph paper.

5.3.3.2 Carr’s Index

The Carr‟s index is frequently used in pharmaceutics as an

indication of the flow ability of a powder. A Carr‟s index greater than 25%

is considered to be an indication of poor flow ability, and below 15%, of

good flow ability. The Carr‟s index is an indication of the compressibility

of a powder.

72

It is calculated using following formula.

Table 5.4 Relationship between % Compressibility and Flow Ability

S. No. % Compressibility Flow ability

1 05 – 12 Excellent

2 12 – 16 Good

3 18 – 21 Fair Passable

4 23 – 35 Poor

5 33 – 38 Very Poor

6 < 40 Very Very Poor

5.3.3.3 Hausner’s Ratio

The Hausner‟s ratio is used in a wide variety of industries as an

indication of the flowability of a powder. A Hausner‟s ratio greater than

1.25 is considered to be an indication of poor flowability. The Hausner‟s

ratio is a number that is correlated to the flow ability of a powder.

Table5.12 holds the results of all above tests.

5.4 PREPARATION OF TABLETS101

The chemo preventive green tea aqueous extract and sodium

selenite were developed into conventional tablet form. A protocol used to

develop the tablets was showed in Table 5.5. A total of 12 formulations

were planned. In all the formulations, amount of chemopreventive

agents, that is, Green tea and Sodium Selenite kept constant.

Formulations GST-1 to GST-6 were prepared by direct compression

method whereas, GST-7 to GST-12 were prepared by wet granulation

73

technique. In direct compression method, the disintegrants which acts as

directly compressible binder were used. Various quantities of sodium

starch glycollate and micro crystalline cellulose were added in this

connection. In wet granulation technique, starch powder was used as

disintegrant and starch paste is used as binder. Sufficient quantity of

lactose was used to make up the tablet weight up to 400 mg.

Wet Granulation Technique

All active compounds, and excipients (except starch paste, talc,

magnesium sterate) were mixed in geometrical ratio. A sufficient quantity

of starch paste is added little by little to make cohesive mass. The mass

was passed through sieve No. 22 and obtained granules were dried in

tray dryer at 40°C. The dried granules were again passed through sieve

No. 16. Then the resulted granules were allowed to mix with talc and

magnesium stearate. The granules were compressed by means of

spherical concave punches using tablet pilot press (Photograph.5.4).

(Machine No: 101/81, Make: Chamunda Pharma Machinery Pvt. Ltd.,

Ahmadabad, Model: PP-I (D, B & BB Combo Tooling), Type: GMP, Lab

Scale, 9 Stations).

Direct Compression Method

All active compounds and excipients were mixed in geometrical

ratio. The resulted mass was compressed by means of spherical concave

punches using laboratory 9 station single rotary punching machine.

74

5.5 PACKING OF TABLETS

The developed tablets (Photograph 5.5) were packed into blister

type packing by using blister packing machine (Medi Pack-300, Global

Packing Industries). Poly Vinyl Chloride polymer sheet having 0.250 mm

(250 µ), thickness is used to form blister. Water Vapor Transmission Rate

(WVTR), and Oxygen Transmission Rate (OTR) of the sheet is specified by

the supplier (WVTR = 3.0 g/m2/day at 38°C/90% RH; OTR = 20

cc/m2/day).

Aluminum foil having thickness 0.025 mm, which is impermeable

to water and oxygen, is used as backing layer. The packed tablets were

showed in Photograph 5.6.

Blister Packing was made for the best formulation at Endoven

Pharmaceuticals Pvt. Ltd., Bala Nagar, Hyderabad.

75

Table 5.5 Protocol Used to Develop the Green Tea with Sodium Selenite Tablets (All quantities were in mg)

Ingredient Name Green Tea

Aqueous

Extract

Sodium

Selenite

Sodium

Starch

Glycollate

Microcrystalli

ne Cellulose

EMCOCEL®

SP15

Talk

powder

Magnesium

Sterate

Lactose

Filler cum

binder quality

Total

Weight

Ingredient Use Chemo

preventive

Chemo

preventive

Super

Disintegrant

Super

Disintegrant

Glidant Lubricant Filler/Binder

GST-1

Direct

Compression

Method

200 001 015 - 001 001 182 400

GST-2 200 001 020 - 001 001 177 400

GST-3 200 001 025 - 001 001 172 400

GST-4 200 001 - 04 001 001 193 400

GST-5 200 001 - 08 001 001 189 400

GST-6 200 001 - 12 001 001 185 400

Ingredient Name Green Tea

Aqueous

Extract

Sodium

Selenite

Starch

Powder Starch Paste

Talk

powder

Magnesium

Sterate

Lactose

Filler cum

binder quality

Total

Weight

Ingredient Use Chemo

preventive

Chemo

preventive Disintegrant

Granulating

Agent Glidant Lubricant

Filler/

Binder

GST-7

Wet Granulation

Technique

200 001 025 15 001 001 157 400

GST-8 200 001 050 15 001 001 132 400

GST-9 200 001 075 15 001 001 107 400

GST-10 200 001 100 15 001 001 082 400

GST-11 200 001 150 15 001 001 057 400

GST-12 200 001 175 15 001 001 032 400

76

5.6 RESULTS AND DISCUSSION

The results obtained for all above tests are showed and discussed

as follows.

Table 5.6 Results of Physicochemical Properties of Prepared Green Tea

Samples.

S. No. Physicochemical Test Obtained

Values

Limits*

*(As per Gazette published

by Indian Government)

A. Ash Values

1 % Total Ash 5.26 % 4-8%

2 % Acid Insoluble Ash 0.59 % Not More Than 1.0 %

3 % Water soluble Ash 3.08 % Not less than 45% of total

ash

B. Extractive Values

4 Water soluble extractive 18.4 % Not Less Than 32%

5 Alcohol soluble extractive 54 % Not Less Than 40%

C. Percentage LOD 1.0 % Not More Than 5 %

All the physicochemical tests performed for the green tea samples

are within the limits and hence it passes all the tests.

Table 5.7 Results of Microbial Test of Green Tea Aqueous Extract

S. No. Name of the Organism Green Tea Aqueous Extract

1 Staphylococcus aureus Absent

2 Salmonella typhimurium Absent

3 Pseudomonus aeruginosa Absent

4 Escherichia coli Absent

5 Fungi Absent

77

WHO and EMEA guidelines imposes the limit for the presence of

Total Viable Count and it should be less than 1 x 105 colony forming

units per gram. Furthermore, specific objectionable bacteria like

Salmonella typhimurium, Staphylococcus aureus, Pseudomonus

aeruginosa, Escherichia coli must be absent.

All the plates were examined. The presence of Staphylococcus

aureus, Pseudomonus aeruginosa, Escherichia coli, Salmonella

typhimuriu, and any type of mould were not observed in any of the

samples. The sample is free from microbial contamination.

Due to the absence of microorganisms like bacteria and moulds,

the TVC method is not required to count the number of organisms.

Calibration Curve of Green Tea Catechins

The linear regression data for the calibration curves (n = 3), (Tables

5.8 & 5.9), showed a good linear relationship over the concentration

range 50 to 300 ng per spot with respect to peak area.

78

Fig. 5.1 (a–f) Showing Six HPTLC Chromatograms of Catechin Marker

Compound for Calibration Curve

79

80

Fig. 5.2 (a–f) Showing Six HPTLC Chromatograms of EC Marker

Compound for Calibration Curve

81

82

Fig. 5.3 (a–f) Showing Six HPTLC Chromatograms of GC Marker

Compound for Calibration Curve

83

84

Fig. 5.4 (a–f) Showing Six HPTLC Chromatograms of EGC Marker

Compound for Calibration Curve

85

86

Fig. 5.5 (a–f) Showing Six HPTLC Chromatograms of ECG Marker

Compound for Calibration Curve

87

88

Fig. 5.6 (a–f) Showing Six HPTLC Chromatograms of EGCG Marker

Compound for Calibration Curve

89

90

Table 5.8 Data of Green Tea Marker Catechin Compounds for Calibration

Curve

S. No. Amount

(ng) of

compound

Per spot

Peak Area

Catechin EC GC EGC ECG EGCG

1 50 989 1019 992 972 1021 965

2 100 1995 2101 1899 1967 2119 1982

3 150 3031 3128 2897 2893 3016 3012

4 200 3897 4109 3972 3976 4015 4036

5 250 5113 5023 4954 4984 5013 4978

6 300 6019 6012 6012 5893 6029 5995

Table 5.9 Details of Calibration Equation and Linearity of Standard Plots

of Catechin Marker Compound

S. No. Catechin Compound Calibration Equation R2 Value

1 Catechin y = 20.21x - 29.66 0.998

2 EC y = 20.21x + 12.01 0.998

3 GC y = 19.77x + 11.02 0.998

4 EGC y = 19.64x + 17.23 0.999

5 ECG y = 19.84x + 63.4 0.999

6 EGCG y = 20.09x -21.53 0.999

91

Fig. 5.7 Calibration Curve of Catechin Marker Compound

Fig. 5.8 Calibration Curve of EC Marker Compound

92

Fig. 5.9 Calibration Curve of GC Marker Compound

Fig. 5.10 Calibration Curve of EGC Marker Compound

93

Fig. 5.11 Calibration Curve of ECG Marker Compound

Fig. 5.12 Calibration Curve of EGCG Marker Compound

94

Fig. 5.13 HPTLC Chromatogram of the Prepared Green Tea Aqueous

Extract

Table 5.10 Quantity of Catechins Present in Prepared Green Tea Extract

S.

No.

Compound Rf Value of Peak

Area

Amount of

compound (ng)

Obtained from

standard plot

Amount of

compound (mg)

Present in 100

mg of extract

Marker Extract

1 Catechin 0.65 0.66 00621.2 032.5 00.65

2 EC 0.46 0.46 04215.7 208.0 04.16

3 GC 0.54 0.55 02067.1 104.0 02.08

4 EGC 0.29 0.29 10102.4 513.5 10.27

5 ECG 0.85 0.85 09467.5 472.0 09.48

6 EGCG 0.75 0.74 38501.0 1917.5 38.35

95

Fig. 5.14 FTIR Spectra of Green Tea Powder

Fig. 5.15 FTIR Spectra of Sodium Selenite

96

Fig. 5.16 FTIR Spectra of Lactose

Fig. 5.17 FTIR Spectra of Micro Crystalline Cellulose

97

Fig. 5.18 FTIR Spectra of Magnesium sterate

Fig. 5.19 FTIR Spectra of Talc Powder

98

Fig. 5.20 FTIR Spectra of Starch Powder

Fig. 5.21 FTIR Spectra of Sodium Starch Glycollate

99

Fig. 5.22 FTIR Spectra of Green Tea with Sodium

Selenite

Fig. 5.23 FTIR Spectra of Green Tea with Micro Crystalline Cellulose

100

Fig. 5.24 FTIR Spectra of Green Tea with Starch Powder

Fig. 5.25 FTIR Spectra of Green Tea Tablet Powder

101

Table 5.11 Compounds and Corresponding FTIR Peaks

S.

No.

Name of the

Compound Wave numbers of prominent peaks

1 Green tea 3428.79, 1631.35, 1452.10, 1382.44, 1146.40,

1074.93, 1026.70, 577.73

2 Sodium Selenite 2852.63, 2427.01, 1235.65, 878.29, 841.25,

789.03, 740.96, 653.00, 615.18, 450.31

3 Lactose 3380.4, 2896.72, 1659.49, 1433.32 ,1387.48 ,

1340.25 , 1261.96, 1166.42 ,1091.01,

1033.59,899.90 ,875.29 , 772.98 ,672.60,

629.15, 604.54 ,548.46, 464.26

4 MCC 3402.98, 2924.38, 1650.25, 1461.45, 1430.34,

1381.94, 1242.53, 1205.87, 1160.08 , 1108.39,

1979.82, 1019.57, 861.76, 575.46

5 Magnesium

stearate

3446.53, 3275.27, 2919.50, 2956.65, 2851.59,

1637.10, 1572.98, 1542.77, 1467.32, 1417.81,

1382.80, 1112.96, 721.78, 667.08

6 Talc 3444.87, 1425.79, 1017.70, 875.87, 710.58,

670.23, 534.51, 466.14, 424.51

7 Starch powder 3434.15, 2928.03, 1659.56, 1464.01, 1381.23 ,

1162.28, 1086.84, 9888.01, 857.53, 765.26,

709.67, 574.32, 523.07

8 Sodium starch

glycollate

3430.65, 2930.16, 1634.18, 1424.47, 1380.59,

1158.50, 1082.55, 1015.5 7, 929.14, 857.88,

763.06, 709.59, 576.76, 527.58

9 Green tea &

Sodium Selenite

3394.01, 2926.93, 1629.93, 1519.07, 1452.38,

1383.15, 1239.13, 1155.76, 1079.96, 1022.83,

842.11, 787.77, 613.66, 447.31

10 Green tea and

MCC

3427.09, 2926.37, 1633.50, 1460.56, 1383.82,

1158.17, 1114.10, 1080.67, 1027.14, 474.87

11 Green tea and

starch

3429.75, 2929.09, 1632.67, 1382.63, 1156.73,

1081.04, 1025.41, 860.54, 765.26

12 Green tea

tablet powder

3386.90, 2898.45, 1632.20, 1432.25, 1384.31,

1260.60, 1140.92, 1033.36, 772.91, 604.40,

549.82

102

No additional peaks were observed in the IR spectra of formulation,

hence it is concluded that there is no drug-drug or drug-excipient

interactions.

Table 5.12 Comparative Study of Various Powder Characteristics for

Formulation

Formulation

Code

Loose Bulk

Density

Tapped Bulk

Density

Angle of

Repose

Carr’s

Index

Hausner’s

Ratio

GST-1 0.22±0.02 0.26 ±0.012 21.04±0.57 15.38 1.18

GST-2 0.22±0.08 0.24 ±0.042 23.08±0.59 08.33 1.09

GST-3 0.23±0.07 0.25 ±0.125 21.36±0.38 08.00 1.08

GST-4 0.23±0.02 0.24 ±0.063 25.32±0.61 04.16 1.04

GST-5 0.23±0.07 0.27 ±0.004 24.45±0.12 14.81 1.17

GST-6 0.27±0.01 0.31 ±0.021 22.79±0.21 12.90 1.14

GST-7 0.26±0.01 0.29 ±0.101 23.92±0.69 10.34 1.11

GST-8 0.30±0.07 0.33 ±0.011 22.47±0.09 09.09 1.10

GST-9 0.31±0.06 0.38 ±0.002 25.96±0.71 18.42 1.22

GST-10 0.33±0.03 0.41 ±0.045 26.10±0.65 19.51 1.24

GST-11 0.29±0.02 0.29 ±0.006 25.53±0.59 00.00 1.00

GST-12 0.27±0.12 0.28 ±0.126 21.38±0.08 03.57 1.03

The angle of repose of GST- 4, 9, 10, 11 granules shows good flow

properties as they got little higher from the values of 25. The remaining

shows excellent flow property. The percentage compressibility of GST- 2,

3, 4, 7, 8, 12 granules are excellent, GST- 1, 5, 6 granules are good and

that of remaining are fair passable. The Hausner‟s ratio value above 1.25

indicates poor flow properties. The Hausner‟s ratio of all the formulations

falls below 1.25, and hence shows very good flow ability.

103

Photograph 5.1 Fresh Green Tea Leaves Photograph 5.2 Dried Green Tea Leaves

Photograph 5.3 Granules of Green Tea Aqueous Extract and Sodium Selenite Photograph 5.4 Compressibility Machine

104

Photograph 5.5 Unpacked Tablets Photograph 5.6 Packed Tablets

Photograph 5.7 HPTLC Density gram of Marker catechins, Green Tea Extract

and Herbo Mineral Tablets