experimental studies on okra gum -...

35

EXPERIMENTAL STUDIES ON OKRA GUM

The main aim of the present investigation is to provide new sustained release

excipient which, when incorporated into a final product, produces controlled release of

active ingredient over an extended period of 12 hrs or more when the dosage form is

exposed to G.I fluids in gastric environment. Many naturally available polymers were

being investigated for their applications in the design of sustained drug delivery systems.

Batch to batch variations were found in the dosage forms with the use of natural

polymers as excipients. These problems occur due to change in their properties like

swelling, viscosity etc. These properties vary based upon the source, purity and microbial

contamination. Hence the main objective of the present investigation is to evaluate the

properties of Okra gum and its applicability in the design of floating tablets.

Gums79, which for the most part are water soluble polysaccharides, have enormously

large and broad application in both food and non food industries. All applications depend

on the properties provided by very large molecules in various order of hydration, but

mostly depend on the properties they impart to solutions and gels. Gums commonly used

in foods are Starches, Cellulose derivatives, Guar gum, Locust bean gum, Pectin, Algin,

Xanthan gum. Gums are continuously being examined for commercial introductions to

provide broader uses. The principal property of polysaccharide gums is their easy

hydration to produce aqueous solutions possessing high viscosities of low gum

concentration, in case of Okra gum at 25 parts/million in water lower friction in fluid

flow by 80%.

The Okra plants are fast growing, heat loving, annual or perennial species, and

can grow up to 2 meters tall, usually not branched. The leaves are coarse, palmately

lobed with 5 to 7 lobes, and ranges from 10 to 20 cm long. Flowers are 4 to 8 cm in

36

diameter, cream colored and have the shape and form of a hibiscus blossom but do not

splay flat. Okra pods are capsule-like that are up to 18 cm long. Most are green, some

are red pigmented. Pods also mature quickly, regardless of shape or color.

Okra plants are cultivated throughout the warm and temperate regions of the

world for its fibrous pods containing round, white seeds. The seeds are soaked overnight

prior to planting. Germination occurs between 6 days and 3 weeks. The seed pods

rapidly become fibrous and woody and must be harvested within a week of the fruit

being pollinated to be edible. After that, the walls of the pod quickly lignified and

become inedible. It is an easy vegetable to grow in any average garden soil. Its seeds can

be saved from late season pods because it is self-pollinated.

5.1 Origin and Distribution

Okra originated somewhere around Ethiopia, and was cultivated by the ancient

Egyptians by the 12th century B.C. Its cultivation spread throughout North Africa and the

Middle East. The seed pods were eaten cooked, and the seeds were toasted and ground,

used as a coffee substitute. Okra Abelmoschus esculentus L. (Moench), it is grown in

different parts of the world, especially in tropical and sub-tropical countries. This crop

can be grown on large commercial farm or could be a garden crop. It is grown

commercially in India, Turkey, Iran, Western Africa, Yugoslavia, Bangladesh,

Afghanistan, Pakistan, Burma, Japan, Malaysia, Brazil, Ghana, Ethiopian and the

Southern United States. 70% of total production came from India with 3.5 tonnes. There

are Eight Abelmoschus species occur in India. Out of these, esculentus is the only known

cultivated species. Abelmoschus moschatus is cultivated for aromatic seeds and also

occur as wild species, while the rest six are truly wild types. The wild species occupy

diverse habitats. The species Abelmoschus ficulneus and Abelmoschus tuberculatus is

37

spread over the semi-arid areas in northwestern India and north India; Abelmoschus

crinitus and Abelmoschus manihot (tetraphyllus and pungens types) in tarai range and

lower Himalayas; Abelmoschus. manihot (tetraphyllus types), Abelmoschus angulosus,

and Abelmoschus moschatus in western and eastern ghats; and Abelmoschus crinitus and

Abelmoschus manihot (mostly pungens types) in the northeastern region depicts their

broad range of distribution in different regions of the country.

5.2 Isolation of mucilage from Lady’s finger by conventional procedure 80, 81, 82

Okra gum is not commercially available. Pods permitted to mature 4-8 days give a

gum product having ropiness values approximately 3 times greater than those of pods

permitted to mature 11-12 days. Therefore, use of pods of not more than 8 days maturity

of use directly after picking is preferred. The Lady’s finger/bhindi is shade dried and then

powdered by crushing and grinding. Lady’s finger/bhindi was dried in an oven at 37°C

for drying and it was powdered for 5 min in a mechanical blender and passed through

sieve no. 120 to get fine powder and adsorbed plant material was extracted successively

with pet-ether (60-80oC) (3 lit), chloroform (3 lit), ethyl acetate (3 lit) and methanol

(3 lit) in a soxhlet extractor. The extracts then concentrated under reduced pressure and

separately examined as described.

5.2.1 Petroleum ether extract: The petroleum ether extract on concentration under

reduced pressure yielded a yellow semi-solid (2 gm). It gave negative response to

Salkowski reaction 83 (No red color was developed when the compound (10 mg)

dissolved in chloroform (2 ml) was treated with Conc. Sulphuric acid). Hence it was

confirmed that the extract does not contain steroids.

5.2.2 Chloroform extract: The chloroform extract on concentration yielded a light

yellow semi solid (2 gm). No response was shown to Salkowski reaction and Liberman-

38

Burchard test 84 (No color change from green to red color when compound (10mg)

dissolved in chloroform (2ml) was treated with acetic anhydride (few drops) and Conc.

Sulphuric acid (2 drops). Hence it was confirmed that the extract does not contain

triterpenoids and steroids.

The chloroform extract when dissolved in α-napthol and treated with Sulphuric

acid pale violet color was developed therefore it indicates the presence of traces of

carbohydrates.

5.2.3 Ethyl acetate extract: The ethyl acetate extract on concentration yielded a light

yellow (2 gm). No response was shown to Salkowski reaction and Liberman-Burchard

test.

The ethyl acetate extract when dissolved in α-napthol and treated with Sulphuric

acid pale violet color was developed therefore it indicates the presence of traces of

carbohydrates.

5.2.4 Methanol extract: The methanol extract on concentration yielded light yellow

semi-solid (20 gm). It gave positive Molish test 85 (Violet color developed when

compound was dissolved in α-napthol and treated with Sulphuric acid) therefore it

indicates the presence of carbohydrates.

This extract when treated with Iodine solution produced blue colour. This

indicates the presence of polysaccharides.

5.3 Physico-Chemical characterization of Okra gum

5.3.1 Identification tests for gums

Tests for gums were carried out as recommended by FAO (1991)86. The samples

were subjected to spot identification tests recommended by AOAC (1984)87. To one gram

39

of Okra gum 5ml of reagents mentioned and results were summarized in the

Tables 5.1 to 5.3.

During this study, the physiochemical properties of Okra gum such as particle

Size distribution, Surface characteristics, Bulk density, Tapped density, Compressibility,

Moisture content, pH, Volatile acidity, Swelling and Water absorption properties,

Rheological properties were evaluated. Microbial study was carried out on the Okra gum.

Differential scanning calorimetry was used to characterize the physical state of Okra gum

powder. Infrared spectroscopic studies were also done to identify the functional groups

present in the polysaccharide chain.

Table 5.1: Identification tests as per FAO

Test Result

Swelling by Ethanol solution Positive

Colour reaction with Conc.HCL Negative

Colour Reaction With 5N NaOH Negative

Table 5.2: Spot identification tests for food hydrocolloids

S. No Reagent Okra gum

1

RGI Positive

2 RGII Positive, swells. Pink colored granular mass is obtained

40

Table 5.3: Chemical confirmatory tests

S. No Reagent Result

1 HCl test Negative

2 Aq. Methylene blue stain Stained

3 RG III reagent Negative

RGI- 3% Iodine in Alcohol

RGII-Ruthenium red solution

RGIII-Sulphuric acid (concentrated)

From the above tests it was confirmed the presence of Polysaccharide. In terms of pH, the

viscosity of the Okra mucilage is at maximum in 3-5 pH range 88. The appearance of the

Okra mucilage was cloudy and opaque at neutral or acid pH conditions. Okra extracts has

the mucilage property89 that is used as a pharmaceutical adjuvant and other

pharmaceutical applications such as a gelling and emulsifying agent, furthermore, it has a

bulk laxative qualities which is mainly manifested by its ability to lubricate90 and

adhere91, 92 .Okra gum was analyzed by taking 100gm of Okra fruits and it consists of

water 88.6g, protein 2.1g, fat 0.02g, carbohydrates 8.2g as major constituents.

Carbohydrates are present mainly in the form of mucilage. In concentration of 1-2% the

gum was found to be as effective as sodium carboxy methyl cellulose and better than

both Acacia and Tragacanth. When evaluated as an emulsifying agent only 0.4 or 0.5%

weight by volume of Okra mucilage was required to produce stable oil in water emulsion

of olive oil or liquid paraffin, indicating high levels of efficiency of the gum as an

emulgent. Dispersion containing Okra gum mucilage produced viscous, slightly cloudy

41

pseudoplastic, viscoelastic aqueous dispersions that exhibit pituity. Whereas granulating

agent in conventional tablets 93, the gum induced a slow release of the drug, signaling that

Okra gum could be a polymer with sustained release effects. It was concluded that Okra

gum could be used as a matrix material for producing tablets that would sustain drug

release for more than 6 hours. Okra gum has also proved to have bio adhesive potential

when formulated as matrix tablets 94, 95.

5.4 Past work on Okra gum

A thorough literature survey showed that there were no reports available with regards to

usage of Okra gum in the preparation of floating tablets. Okra pods were at cheaper price

and easily available can be extracted easily. Utilizing this gum in the tablet preparations

drastically reduces the prices of existing and upcoming pharmaceutical formulations.

5.5 Determination of particle size distribution

Okra gum was dispersed in glycerin and a smear of the dispersion was made and

examined under microscope. The size of 500 particles was measured using a calibrated

eyepiece micrometer. The size distribution of Okra gum particles was estimated. The

results are given in Table 5.4 and shown in Fig. 5.1

Table 5.4: Particle size distribution of Okra gum powder

Size range (µm) Number of Particles

0-30 20

30-60 40

60-90 230

90-120 180

>120 30

42

Fig. 5.1: Particle size distribution of Okra gum

5.6 Determination of Bulk density, Bulkiness, Tapped density and Compressibility

index

The bulk density of Okra gum was determined by tapping method96. Weighed

quantity of Okra gum was carefully introduced into a 100ml graduated cylinder .The

cylinder was dropped onto a hard wood surface 3 times from a height of 3 cm at an

interval of 2seconds. The bulk density was calculated by dividing the weight of the

sample by volume of the sample contained in the cylinder. Reciprocal of bulk density

gives the bulkiness.

Tapped density is the ratio of weight of dry powder to its tapped volume. The

weighed quantity of dry powder was taken in graduated cylinder. The cylinder was

placed on the tap density apparatus. The results are showed in Table 5.5

The percent compressibility index (I) 97 of Okra gum powder was calculated using

the following formula and the results are given in Table 5.5

0

50

100

150

200

250

Num

ber

of p

artic

les

Size range(µm)

0-30

30-60

60-90

90-120

>120

43

I= (1-Vf/Vo) 100

Vo =Initial volume

Vf =Final volume after tapping

Hausner index was calculated using the formula given below.

Hausner index= (Vo/Vf)

Vo =initial volume, Vf=Final volume after tapping

5.7 Determination of flow properties

Flow properties of powders were determined by the Angle of repose,

Compressibility index and Hausner index and the results are given in Table 5.5. The

frictional forces in a loose powder can be measured by the Angle of repose98 (θ).This is

the maximum angle possible between the surface of a pile and the horizontal plane. It can

be calculated by,

Tan θ=h/r

h=Height of the pile

r=Radius of the pile

Hausner index was calculated using the formula given below.

Hausner index= (Vo/Vf)

Vo =initial volume, Vf=Final volume after tapping

44

5.8 Determination of Moisture content

Moisture content was determined by using Karl-Fischer auto titrator M/s.

Systronics, Model No.349 and the results are given in Table 5.5

5.9 Determination of pH value

The pH of 1%w/v aqueous solution of Okra gum was determined by using pH

meter (Systronics, Model no.361) and the results are given in Table 5.5.

5.10 Determination of swelling index99

The Swelling index of Okra gum was determined by placing one gram of powder

in a measuring cylinder. The initial volume of the powder in a measuring cylinder was

noted. The volume was made up to 100ml mark with 0.1N HCl (pH 1.2) at room

temperature. The cylinder was stoppered shaken gently and set aside for 24hrs and the

results are given in Table 5.5. The volume occupied by the gum sediment was noted after

24hrs.

The swelling index of the gum was calculated by the formula,

S.I= {wt-wo/wo}

Where, S.I. = Swelling index

Wt = Height occupied by swollen gum after 24hrs

Wo = Initial height of the powder in graduated cylinder

45

5.11 Determination of Volatile acidity100

About 1gm of the gum was accurately weighed, transferred to a 700ml long

necked flask, 100ml of water and 5ml of Orthophosphoric acid were added and allowed

to stand until the gum was completely swollen (app 24 hrs).Then, it was boiled for 2hrs

under a reflux condenser, steam distilled until 800ml of the distillate was obtained.

The distillate was titrated with 0.1N sodium hydroxide using phenolphthalein as

indicator and the results are given in Table 5.5. The procedure was repeated omitting the

sample. The difference between the two titrations represented the amount of alkali

required to neutralize the volatile acid.

Each ml of 0.1N NaOH =0.006005 g of C2H4O2

5.12 Differential scanning calorimetry (DSC)

DSC curve of Okra gum powder was obtained by a Differential scanning

calorimeter (Shimadzu DSC-50) at a heating rate of 10oC /min from 30 to 300oC in

nitrogen atmosphere (30ml/min). The DSC thermogram of Okra gum was shown in

Fig. 5.2

5.13 Infrared spectroscopy

Using Perkin-Elmer 841, IR, spectrophotometer, obtained an IR spectrum of Okra

gum. The sample was prepared into a pellet with one gram of KBR. The IR spectrum of

Okra gum was shown in Fig. 5.3

46

Table 5.5: Characterization of Okra gum

Property Results Obtained

Tapped density (gm/cc) 0.702±0.02

Bulk density (gm/cc) 0.632±0.04

Bulkiness (cc/gm) 1.58±0.04

Angle of repose(0) 28.20±1.28

Compressibility index (%) 10.42±1.34

Hausner’s ratio 1.2±1.54

pH 4.8±0.20

Water retention Capacity(ml) 19±1.67

Swelling index (%) 120±10.00

Volatile acidity (%) 17.2±2.98

Moisture Content 14.96±1.12

47

100.00 200.00 300.00Temp [C]

-10.00

-5.00

0.00

mWDSC Ch1 1-Okra Gum 12-42 2012-09-07.tad DSC

Fig. 5.2: DSC of Okra gum

48

Fig. 5.3: IR spectra of Okra gum

49

5.14 Microbiological properties of Okra gum

Fresh samples of Okra gum were analysed for the microbial growth including pathogens

by agar plate method and results were given in the Table 5.6. Further the samples of one year

old stored gum at room temperature in airtight polypropylene container were also analysed for

microbial growth. The results were shown in Table 5.7.

5.15 Acute toxicity study101:

Three male wistar rats were selected for the study. The overnight fasted animal

(with water ad libitum) was administered with Okra gum extract at a single dose of 2000 mg/kg

body weight by orally. The dose volume is 0.2ml per 100gm body weight. Food was withheld

for a further 3-4 hours after administration of test extract and was observed for signs for

toxicity. The body weight of the rats before and after administration were noted that changes in

skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous

system and motor activity and behavior pattern will be observed and also sign of tremors,

convulsions, salivation, diarrhea, lethargy, sleep and coma was noted. The onset of toxicity and

signs of toxicity were also noted for a period of 14 days. For, further confirmation the above

procedure was repeated on another set of three male wistar rats.

All the tests were performed by following the guidelines provided by CPCSEA and the

protocols were approved by Institutional animal ethical committee bearing Regd. No.

1263/bc/09/CPCSEA .

50

5.16 Determination of rheological properties of Okra gum powder

The processing, formulation changes, aging phenomena affects the rheological

properties102 of the gum. These play major role in the release of drug from the dosage form. It is

necessary in particular to the polysaccharide materials to check the viscosity.

The rheological properties of Okra gum were evaluated using Brookfield cone and plate

viscometer model LV DV-III. Okra gum concentration of 0.5, 1.0, 2.0, 3.0 % (w/v) with

distilled water were prepared, allowed to swell .0.5ml of sample was placed in plate of

viscometer and analysed for its viscosity, shear stress, rate of shear at various speeds. The

results are given in Tables 5.8 to 5.13 and Fig. 5.4 to 5.6.

The analysis of viscometer data may be analysed through the use of mathematical

models. Non-Newtonian behavior can be simply expressed through an equation, and in some

cases, the coefficients of a model can be used to infer performance of a fluid under conditions of

use. According to Newtonian systems there is a linear relationship between shearing stress and

rate of shear. In case of Non-Newtonian fluids will exhibit a nonlinear relationship. Some of the

more widely used equations include Bingham, Casson, and Power law equation.

Bingham equation: τ= τ0+ηD

Casson : √ τ=√τ0+√ηD

Power law: τ=kDn

Where τ=Shear stress

D=Shear rate

51

η= Viscosity

τ0=Yield value

n=Flow index

k=Consistency index

The above mathematical models were analysed by using Brookfield RHEOCALC

software version 2.4 (Serial No.RC001236) and shown in Table 5.16.

Table 5.6: Microbial load of Okra gum powder

Parameter Result General specification

Microbial limits

1.Total aerobic microbial count

20 CFU/g

Not more than 100CFU/g

2.Total fungal count 30 CFU/g Not more than 100CFU/g

3.Pathogens

a. Staphy. aureus

b. Pseudo. aeruginosa

c. E. coli

d.Salmonella

a. Absent/g

b. Absent/g

c. Absent/g

d. Absent/g

a.Shall be absent/g

b. Shall be absent/g

c. Shall be absent/g

d. Shall be absent/g

52

Table 5.7: Microbial load of Okra gum powder after storage

Parameter Result General specification

Microbial limits

1.Total aerobic microbial count

25 CFU/g

Not more than 100CFU/g

2.Total fungal count 33 CFU/g Not more than 100CFU/g

3. Pathogens

a. Staphy.aureus

b. Pseudo.aeruginosa

c. E.coli

d. Salmonella

a. Absent/g

b. Absent/g

c. Absent/g

d. Absent/g

a. Shall be absent/g

b. Shall be absent/g

c. Shall be absent/g

d. Shall be absent/g

53

Table 5.8: Rheological properties of 0.5% Okra gum

Speed (rpm) Shear stress (dynes/cm2)

Shear rate (sec-1)

Viscosity (cps)

10 25.28 20 130.2

20 36.62 40 93.96

30 46.12 60 76.42

40 53.72 80 66.48

50 62.14 100 61.84

60 68.22 120 56.68

70 76.20 140 54.12

80 80.36 160 50.10

90 88.20 180 49.02

100 92.75 200 47.2

110 98.88 220 46.22

120 104.68 240 45.09

130 108.22 260 42.36

140 111.88 280 41.32

150 116.95 300 40.33

160 121.46 320 38.26

170 125.32 340 36.22

180 130.34 360 35.89

190 134.66 380 35.26

200 138.14 400 34.12

210 140.68 420 33.78

220 146.56 440 32.12

230 150.28 460 31.23

54



Shear rate (sec-1)

Viscosity (cps)

220 146.48 440 32.10

210 140.68 420 33.68

200 138.14 400 34.08

190 134.58 380 66.48

180 130.32 360 61.84

170 125.29 340 56.68

160 121.44 320 54.12

150 116.94 300 50.10

140 111.86 280 49.02

130 108.20 260 47.2

120 104.62 240 46.22

110 98.46 220 45.09

100 92.60 200 42.36

90 88.12 180 41.32

80 80.34 160 40.33

70 76.18 140 38.26

60 68.18 120 36.22

50 62.12 100 35.89

40 53.68 80 35.26

30 46.10 60 34.12

20 36.56 40 33.78

10 25.20 20 32.12

55

Table 5.9: Rheological properties of 1% Okra gum

Speed (rpm) Shear stress

(dynes/cm2)

Shear rate

(sec-1)

Viscosity

(cps)

5 36.86 10 376.38

10 54.10 20 271.10

15 66.86 30 223.54

20 78.82 40 199.44

25 88.96 50 179.40

30 98.82 60 166.22

35 106.95 70 154.48

40 114.26 80 143.24

45 122.36 90 137.02

50 129.84 100 130.01

55 134.28 110 123.40

60 141.80 120 118.48

65 146.74 130 113.46

70 149.20 140 106.35

75 156.88 150 104.53

80 162.32 160 102.10

85 168.98 170 99.40

90 172.64 180 96.17

95 175.36 190 92.14

100 180.28 200 90.94

105 183.35 210 88.00

56

Table 5.9: Rheological properties of 1% Okra gum


(dynes/cm2)

Shear rate

(sec-1)

Viscosity

(cps)

100 179.26 200 90.84

95 175.34 190 92.12

90 172.58 180 96.14

85 168.96 170 99.38

80 162.30 160 102.06

75 149.18 150 104.50

70 149.18 140 106.33

65 146.72 130 113.42

60 141.78 120 118.39

55 134.26 110 123.38

50 129.82 100 130.00

45 122.34 90 136.99

40 114.24 80 143.20

35 106.92 70 154.46

30 98.80 60 166.18

25 88.94 50 179.33

20 78.80 40 199.34

15 66.84 30 223.52

10 54.08 20 271.04

5 36.84 10 376.24

57



Shear rate (sec-1)

Viscosity (cps)

1 49.02 2 2458.62

2 70.24 4 1760.84 3 86.42 6 1447.20

4 100.34 8 1255.40

5 111.40 10 1116.08

6 120.36 12 1024.14 7 131.23 14 983.98

8 140.32 16 899.46

9 149.68 18 702.36 10 158.74 20 600.12

11 167.99 22 499.84

12 178.33 24 398.26

13 187.42 26 301.27 14 196.33 28 199.89

15 207.21 30 99.23

14 196.28 28 199.84 13 187.38 26 301.22

12 178.21 24 398.22

11 167.78 22 499.80 10 157.66 20 600.08 9 149.56 18 702.31

8 140.22 16 899.38

7 131.18 14 983.96

6 120.26 12 1024.12 5 111.34 10 1116.05 4 100.26 8 1225.38

3 86.38 6 1447.18 2 70.21 4 1760.82

1 49.01 2 2458.60

58

Table 5.11: Rheological properties of 2 % Okra gum


(dynes/cm2)

Shear rate

(sec-1)

Viscosity

(cps)

0.5 62.48 1 6296.23

1.0 89.84 2 4498.00

1.5 110.36 3 3688.44

2.0 127.96 4 3199.31

2.5 140.24 5 2805.98

3.0 151.08 6 2517.66

3.5 162.32 7 2320.20

4.0 171.66 8 2148.89

4.5 180.09 9 2003.76

5.0 189.10 10 1890.85

4.5 180.07 9 2003.74

4.0 171.64 8 2148.78

3.5 162.30 7 2320.18

3.0 151.06 6 2517.46

2.5 140.22 5 2805.73

2.0 127.90 4 3199.28

1.5 110.33 3 3688.41

1.0 89.78 2 4497.98

0.5 62.44 1 6295.20

59

Table 5.12: Rheological properties of 2.5 % Okra gum


Shear rate (sec-1)

Viscosity (cps)

0.1 52.10 0.2 26643.14

0.2 72.29 0.4 18443.16

0.3 87.55 0.6 14714.45

0.4 100.65 0.8 12645.45

0.5 110.75 1.0 11175.12

0.6 121.49 1.2 10201.65

0.7 131.49 1.4 9455.15

0.8 140.75 1.6 8860.49

0.9 147.51 1.8 8249.51

1.0 155.66 2.0 7871.64

1.1 161.84 2.2 7392.12

1.2 170.36 2.4 7115.65

1.3 177.29 2.6 6850.40

1.4 183.96 2.8 6587.21

1.3 180.32 2.6 6972.42

1.2 175.06 2.4 7291.11

1.1 167.03 2.2 7596.14

1.0 161.18 2 8055.45

0.9 153.15 1.8 8517.64

0.8 146.06 1.6 9128.41

0.7 137.25 1.4 9821.45

0.6 127.29 1.2 10712.25

0.5 117.39 1.0 11794.32

0.4 106.14 0.8 13390.54

0.3 93.14 0.6 15524.96

0.2 77.23 0.4 19330.27

0.1 55.63 0.2 27841.12

60

Table 5.13: Rheological properties of 3 % Okra gum


(dynes/cm2)

Shear rate

(sec-1)

Viscosity

(cps)

0.05 68.43 0.1 6870.34

0.10 94.54 0.2 47327.15

0.15 114.20 0.3 38073.52

0.2 130.17 0.4 31891.19

0.30 157.13 0.6 25891.54

0.35 168.24 0.7 24015.74

0.40 177.18 0.8 22406.96

0.45 185.21 0.9 20607.74

0.40 181.05 0.8 22576.19

0.35 172.26 0.7 24566.97

0.30 162.24 0.6 27136.00

0.25 148.18 0.5 29895.17

0.20 135.54 0.4 33988.96

0.15 119.12 0.3 39989.79

0.10 100.45 0.2 50745.53

0.05 75.09 0.10 75694.18

61

Fig. 5.4: Shear stress Vs Shear rate profiles of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%(w/v) Okra gum

050

100150200250300350400450500

0 50 100 150 200

Shea

r ra

te(s

ec-1 )

Shear Stress(dynes/sq.cm)

Okra gum 0.5%

0

50

100

150

200

250

0 50 100 150 200

Shea

r ra

te(s

ec-1 )

Shear Stress (dynes/sq.cm)

Okra gum 1.0%

0

5

10

15

20

25

30

35

0 50 100 150 200 250

Shea

r ra

te(s

ec-1 )

Shear stress(dynes/sq.cm)

Okra gum 1.5%

0

2

4

6

8

10

12

0 50 100 150 200

Shea

r ra

te(s

ec-1 )


Okra gum 2%

0

0.5

1

1.5

2

2.5

3

0 50 100 150 200

Shea

r ra

te(s

ec-1 )


Okra gum 2.5%

00.10.20.30.40.50.60.70.80.9

1

0 50 100 150 200

Shea

r ra

te(s

ec-1 )

Shear stree(dynes/sq.cm)

Okra gum 3%

62

Fig. 5.5: Okra gum of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%(w/v) Concentrations-Test for Thixotrophy

050

100150200250300350400450500

0 50 100 150 200

Shea

r ra

te(s

ec-1 )


Okra gum 0.5%-Thixotrophy

Up curve

Down curve

0

50

100

150

200

250

0 50 100 150 200

Shea

r ra

te(s

ec-1 )



Up curve

Down curve

05

101520253035

0 100 200 300

Shea

r ra

te(s

ec-1 )



Up curve

Down curve

0

2

4

6

8

10

12

0 50 100 150 200

Shea

r ra

te(s

ec-1 )



Up curve

Down curve

0

0.5

1

1.5

2

2.5

3

0 50 100 150 200

Shea

r ra

te(s

ec-1 )



Up curve

Down curve

00.10.20.30.40.50.60.70.80.9

1

0 50 100 150 200

Shea

r ra

te(s

ec-1 )



Up curve

Down curve

63

Fig. 5.6: Shear rate Vs Viscosity profiles of 0.5%, 1%, 1.5%, 2%, 2.5%, 3% (w/v) Okra gum

0

20

40

60

80

100

120

140

0 200 400 600

Vis

cosi

ty(c

ps)

Shear rate(sec-1)

Okra gum 0.5%

050

100150200250300350400

0 50 100 150 200 250

Vis

cosi

ty(c

ps)

Shear rate(sec-1)

Okra gum 1%

0

500

1000

1500

2000

2500

3000

0 10 20 30 40

Vis

cosi

ty(c

ps)

Shear rate(sec-1)

Okra gum 1.5%

0

1000

2000

3000

4000

5000

6000

7000

0 5 10 15

Vis

cosi

ty(c

ps)

Shear rate(sec-1)

Okra gum 2%

0

5000

10000

15000

20000

25000

30000

0 1 2 3

Vis

cosi

ty(c

ps)

Shear rate(sec-1)

Okra gum2.5%

01000020000300004000050000600007000080000

0 0.5 1

Vis

cosi

ty (c

ps)

Shear rate(Sec-1)

Okra gum 3%

64

Table 5.16: Analysis of rheological properties of various concentrations of Okra gum

Concentration of Okra gum%

0.5 1.0 1.5 2 2.5 3.0

Bingham

η (cps) 25.5 67.8 454.6 1324 4627 14415

τ0(dynes/cm2) 34.0 51.2 56.0 64.8 58.5 64.8

Cof (%) 92.5 91.5 92.5 93.0 93.8 94.8

Casson

η (cps) 15.6 39.2 248.7 677.9 2403 7149

τ0(dynes/cm2) 13.0 22.5 26.2 32.7 29.9 33.94

Cof (%) 96.9 94.9 95.7 95.4 96.7 96.8

Power Law

Consistency

index,k(cps)

462.9 1140 3419 6458 11302 19852

Flow index,n 0.49 0.52 0.49 0.45 0.45 0.45

Cof (%) 98.0 99.2 98.7 97.9 98.7 98.9

65

5.17 Evaluation of variation in the gum between different sources

To evaluate the effect of geographical region of collection on the gum properties, bhindi (Okra gum) was

collected from the following three different regions in Andhra Pradesh. From Krishna District, Kurnool

District, Visakhapatnam District and evaluated for their properties. The swelling index, viscosity and

microbial load of all the three samples were analysed as per the methods described earlier. The results are

shown in Table 5.17, Table 5.18, Table 5.19, Table 5.20 and Fig. 5.7

5.18 Evaluation of variation in the gum collected at different times:

Season of collection may also influence the properties of the gum and hence the bhindi was collected in

summer and winter season from Krishna district. The swelling index, Viscosity and Microbial load of all

the two gum samples were analysed as per the methods described earlier. The values are shown in Table

5.18 to 5.20

5.19 Accelerated stability studies of Okra gum powder

Okra gum powder was subjected to accelerated stability studies according to ICH guidelines to predict the

stability of Okra gum and analysed the samples at regular intervals as per stability protocol Table 5.21 and

the results are shown in the Table 5.22 and Fig. 5.8. IR spectra of Okra gum powder of 400C+75%RH of 3

months sample is shown in Fig. 5.9.

66

Table 5.17: Comparative viscosities of 1.0% Okra gum collected at three different geographical regions

Speed (rpm) Viscosity of Krishna District

sample (cps)

Viscosity of Godavari District

sample (cps)

Viscosity of Visakhapatnam

sample (cps) 5 374.4 372.5 376.5

10 270.1 266.7 272.8

15 221.6 218.3 226.7

20 198.4 195.6 203.8

25 178.3 174.9 184.9

30 165.4 163.2 170.9

35 153.3 151.9 159.4

40 142.7 139.4 146.5

45 136.0 154.8 143.7

50 129.3 127.6 135.4

55 122.9 119.5 128.5

60 117.4 114.6 123.6

65 112.6 108.6 118.5

70 105.4 101.5 112.9

75 103.4 100.5 110.9

80 101.1 98.4 108.8

85 98.2 95.6 104.6

90 95.1 92.9 101.6

95 91.7 88.4 97.9

100 90.6 87.7 94.8

105 87.2 84.6 92.7

67

Table 5.18: Comparative viscosities of 1.0% Okra gum collected at different seasons

Speed (rpm) Viscosity of Krishna district sample collected in summer (cps)

Viscosity of Krishna district sample collected in winter (cps)

5 374.4 370.0

10 270.1 264.2

15 221.6 214.7

20 198.4 189.9

25 178.3 169.5

30 165.4 159.7

35 153.3 144.7

40 142.7 135.9

45 136.0 129.8

50 129.3 123.7

55 122.9 114.9

60 117.4 109.8

65 112.6 100.6

70 105.4 98.6

75 103.4 94.5

80 101.1 89.92

85 98.2 87.7

90 95.1 84.3

95 91.7 81.7

100 90.6 77.8

105 87.2 74.6

68

Fig. 5.7: Comparative viscosities of Okra gum collected at three different geographical regions

Table 5.19: Comparative swelling index of Okra gum collected at different geographical regions and seasons

Swelling index (%)

Krishna dist sample

Kurnool dist sample

Vishakhapatnamdist sample

Krishna dist sample collected in summer

Krishna dist sample collected in winter

Trail-I 120 120 120 122 124

Trail-II 117 118 119 118 120

Trail-III 119 118 118 120 122

Average 118.6 118.6 119 120 122

0

50

100

150

200

250

300

350

400

0 20 40 60 80 100 120

Vis

cosi

ty(c

ps)

Speed(rpm)

Krishna dist

Kurnool dist

Vishakapatnam dist

69

Table 5.20: Comparative microbial load of the Okra gum collected at different geographical regions

and seasons

Parameter Krishna dist

sample

Kurnool

Dist sample

Vishakhapatnam

Dist sample

Krishna dist

Sample

collected in

summer

Krishna dist

Sample

collected in

winter

Total aerobic

microbial

count

25 CFU/g 26 CFU/g 28 CFU/g 25 CFU/g 26 CFU/g

Total fungal

count

33 CFU/g 38 CFU/g 39 CFU/g 33 CFU/g 35 CFU/g

Pathogens

Staphy.aureus Absent/g Absent/g Absent/g Absent/g Absent/g

Pseudo.aeruginosa Absent/g Absent/g Absent/g Absent/g Absent/g

E.coli Absent/g Absent/g Absent/g Absent/g Absent/g

Salmonella Absent/g Absent/g Absent/g Absent/g Absent/g

70

Table 5.21: Stability protocol for Okra gum powder

Name of the powder material: Okra gum

Date of Starting: 09-10-11

Quantity Loaded: 15 g approx. in each Petri dish

Quantity sampled: 1g approx

Schedule 400C+75% RH 250C+60% RH Test

Initial 09-10-11 09-10-11 1.Description

1 Month 09-11-11 09-11-11 2.Rheological properties

2 Month 09-12-11 09-12-11 3.pH

3 Month 09-1-12 09-1-12 4.Volatile acidity

6 Month 09-04-12 09-04-12 5.Moisture content

71

Table 5.22: Accelerated stability data of Okra gum powder

Condition Period

(months)

Parameters

Description Moisture

content

Volatile

acidity

pH

Initial Initial Pale yellow

colored free

flowing

powder

14.96 17.2 4.8

400C+75%RH 1 Pale yellow

colored free

flowing

powder

14.00 17.0 4.9


colored free

flowing

powder

13.95 16.9 4.9


colored free

flowing

powder

13.75 16.7 4.9


colored free

flowing

powder

13.7 16.6 4.9

250c+60%RH 6 Pale yellow

colored free

flowing

powder

13.6 16.6 4.9

72

Fig. 5.8: Viscosity Vs Shear rate plots of Okra gum powder subjected to stability analysis

0

50

100

150

200

250

300

350

400

0 50 100 150 200 250

Visc

osity

(cps

)

Shear rate (sec-1)

Initial

40°C+75%RH,1M

40°C+75%RH,2M

40°C+75%RH,3M

25°C+60%RH,1M

25°C+60%RH,3M

73

Fig. 5.9: IR spectra of Okra gum powder of 400C+75%RH of 3 months sample

74

5.20 RESULTS AND DISCUSSION

Particle size distribution of excipients and drug play vital role on bulk properties of pharmaceutical

interest such as flow properties, packing densities, compressibility and segregation characteristics103 etc.

During the Preformulation studies the particle size distribution of excipients and active ingredients should

be finalized, follow the prescribed specifications and in the further studies the materials, which have met

the specifications, should be used. One of the most common methods for particle size determination is

optical microscopy104, because it gives direct measurement of the individual particle. The particle size

distribution of Okra gum was given in Table 5.4 and shown in Fig. 5.1 When examined under compound

microscope Okra gum granules were found to be irregular and 30-120 µm in size.

Powders normally flow under influence of gravity; dense substances are generally less cohesive

than lighter ones, as the weight of the particles for a given volume is increased105. Hence, differences in

densities of various ingredients of formulation may lead to improper mixing and filling during

manufacturing of formulation results in weight variation and variations in content uniformity of finished

products. Hence, determination of density of any ingredient will helpful in successful formulation

development.

The properties such as Bulk density, Tapped density, Compressibility index and Angle of repose

were often referred to as the derived properties of powders depend mainly on particle size distribution,

particle shape, tendency of the particles to adhere together.

Compressibility index (I) values up to 15% usually result in good to excellent flow properties and

indicate desirable packing characteristics. Compressibility Index 25% was often sources of poor tabletting

qualities. The bulk density and compressibility index of Okra gum were found to be 0.632 gm/cc and

10.42% respectively. The values of bulk density and compressibility index indicated that the Okra gum

powder has good flow properties and compressibility.

75

When the angle of repose is less than 300, it indicates that the material is free flowing and values

greater than 400 suggest a poorly flowing material. The static angle of repose value for Okra gum was

found to be 280.091 indicating good flow properties.

Moisture content of excipients used can influence the tabletting and stability properties of

formulations. Moisture content of Okra gun determined by Karl-Fischer auto titrator was found to be

14.96%

The swelling Index of Okra gum was found to be 120%. High value of swelling index revealed the

high swelling ability of Okra gum. The swelling ability of any polysaccharide depends upon its water

retention capacity. The water absorption capacity of okra gum was found to be 19ml.

The pH of the 1% w/v Okra gum solution was found to be 4.8 indicating the gum is weakly acidic

in nature. Acidic nature of Okra gum may be due to the presence of acetyl groups, which is confirmed by

the determination of volatile acidity of Okra gum.

The volatile acidity of Okra gum was found to be 17.2%. It was reported that the viscosity of gum

is directly proportional to the volatile acidity of gum. Hence determination of volatile acidity is a useful

tool in the evaluation of the quality of the gum with regard to its viscosity.

Differential scanning calorimetry (DSC) measures the heat or gain resulting from physical or

chemical changes within a sample as a function of temperature. A sharp symmetric melting endotherm can

indicate relative purity, whereas broad, asymmetric curve suggests impurities or more than one thermal

process. The endothermic peak usually indicates the loss of water present in the compound. The DSC

thermogram of Okra is shown in Fig. 5.2. Okra gum exhibited broad endothermic peak at 172oC. The

produced peak may be due to loss of free/bound water present in the Okra gum.

76

IR spectroscopy is a useful tool in identification as well as purity of a compound. The IR spectrum

of Okra gum was shown in Fig. 5.3. The principal absorption peaks of Okra gum at 1599 cm-1 (stretching

of ether group absorbance) and 3453 cm-1 (stretching of hydroxyl group of carboxylic acid) were

observed.

From the microbiological studies of Okra gum powder analyzed immediately after collection and

one year old sample, proved that the Okra gum does not support microbial growth and free from all the

pathogen organisms.

From the acute toxicity studies of Okra gum powder analyzed, proved that okra gum does not

show any signs of toxicity.

Viscosity is the main parameter to assess the quality of natural gums. The applications of any

natural gum are dependent on its viscosity and other rheological properties. For any polymer to be used in

slow release hydrophilic matrix systems it should possess certain characteristics like the fast hydration of

the polymer and a matrix having a high gel strength and should be stable during the shelf life of the

product. To determine the rheological properties okra gum, various concentrations were prepared and

evaluated by using Brookfield cone and plate viscometer LV DV III+ and the results (Table 5.8 to Table

5.13 and Figs. 5.4 to Fig. 5.6) showed that the Okra gum possess pseudo plasticity and it should not

possess thixotrophy. Viscosity data was analysed by using Bingham, Casson, Power law equations and the

results were shown in the Table 5.16. From the Table 5.16, it was clearly indicated, Okra gum in aqueous

solution obeys power law with correlation coefficient values ranging 97.9 to 99.2. Okra gums hydrates

quickly and swells rapidly and form a thick viscous layer around it. This is the most important criterion

required for hydrophilic matrix systems. The viscosity and other rheological properties confirmed for its

suitability in the development of a controlled release delivery systems.

77

Okra gum collected from three different geographical regions was studied for their uniformity in

their physical properties. The results of swelling index, viscosity showed that there is no significant

difference between these samples (Table 5.17, Table 5.19 and Fig. 5.7). The microbiological studies

revealed that all the three samples are free from pathogens as shown in Table 5.20. These studies confirm

the uniformity in the physical properties of the gum from different sources.

Okra gum collected from two different seasons was studied for their uniformity in their physical

properties. The results of swelling index, viscosity showed that there is no significant difference between

these two samples and free from pathogen organisms as shown in Table 5.18 to Table 5.20.

The results of accelerated stability studies on Okra gum powder showed that there is no significant

difference between the initial and the samples withdrawn at the different time intervals like 1, 2, 3 and 6

months (Table 5.22 and Fig. 5.8). The IR spectrum of Okra gum powder, which was subjected to stability

studies, was shown in Fig. 5.9. The principal absorption peaks of Okra gum at 1599 cm-1 (ether group

absorbance) 3453 cm-1 (stretching of hydroxyl group of carboxylic acid) were observed. These studies

proved that the Okra gum is stable over a long period of time.

The above results i.e. good flow & compactability, high swelling index, lack of variations in

properties of Okra gum due to source and stability of Okra gum suggested that in controlled release

floating drug delivery system in particular to hydrophilic formulations, Okra gum could be used as an

excipient.

experimental studies on okra gum -...

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