results & discussion - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/62401/7/17...9....

Development and Preclinical Evaluation of Solid Oral Dosage

Form Using a Natural Bioenhancing Agent for RA

Results & Discussion



CHAPTER VII

7.0. RESULTS AND DISCUSSION

According to the materials and methods mentioned earlier the mucilage, drug

and the formulation mixture were evaluated separately for their physicochemical and

pharmaceutical properties.

7.1. PHYSICAL PROPERTIES OF THE MUCILAGE

The average particle size of dried mucilage was found to be uniform. LOD was

within the limits. Since it is a mucilage, swelling ratio was performed to find out the

swelling behaviour of the mucilage and was found to be good. pH was at neutral

(Table 5).

Table 5: Physical properties of the mucilage

Values are reported as mean ± Standard Deviation, n=3

S.No. Physical properties Observations

1. Color Grey

2. Percentage Yield 9 ± 3%w/w

3. Average Particle size 191.32 ± 1.75μm

4. Loss on drying 2.51 ± 0.34%w/w

5. Swelling Ratio 23.26 ± 0.814%w/v

6. Total Ash 4.2 ± 0.178%w/w

7. Acid insoluble ash 1.4 ± 0.215%w/w

8. pH 7.6 ± q0.1

9. Charring 242.3 ± 2.516 o C

10. Density 1.076 ± 0.016g/ml

11. Viscosity 1.229 ± 0.028 Poise



7.2. CHEMICAL PROPERTIES OF THE MUCILAGE

Table 6: Chemical properties of the mucilage

S.No. Chemical properties Observation

1. Mucilage mounted with water Swells & forms mucilaginous mass

2. Mucilage mounted with Ruthenium red Particles stained red colour

3. Mucilage mounted with Corallin Soda Particles stained pink colour

4. Mucilage mounted with Iodine solution Particles stained crimson red colour

5. Molisch’s test (for carbohydrates) +ve

6. Silver Nitrate test (for chlorides) -ve

7. Barium Chloride test (for sulphates) -ve

8. Lead Acetate test (for tannins) -ve

9. Ferric chloride test (for phenols) -ve

10. Foaming test (for saponins) -ve

It is confirmed from the identification tests that the isolated compound is a

mucilage and Molisch’s test confirmed the presence of carbohydrates and with the

test for chlorides, sulphates, tannins, saponins and phenols the sample resulted

negative. (Table 6)



7.3. PREFORMULATION PARAMETERS OF THE DRUG

Table 7: Preformulation parameters of the drug

S.No. Characteristics Results

1. Physical appearance White fine powder

2. Solubility Sparingly soluble in water, methanol and

practically insoluble in methylene chloride

3. Bulk density 0.2742 ± 0.19 g/ml

4. Tapped density 0.2931 ± 0.13 g/ml

5. Compressibility index 27.34 ± 0.58%

6. Hausner’s ratio 1.41 ± 0. 24

7. Angle of repose 46°44 ’± 0 °46’

8. Loss on Drying 1.81 ± 0.51% w/w

9. Melting Point 166°C ± 3°C


The drug was analysed for its appearance, solubility, flow properties, LOD

and melting point. All were found within the limits and the flow was found to be

poor thus a suitable glidant was decided to be added in the formulation to improve

the flow thereby prepare better formulation. The melting point was found to be

166°C ± 3°C which helps in confirming the purity of the drug. The reported melting

point of leflunomide was 168°C and the observed melting point was 166°C hence

confirms the purity of the sample. ( Table 7).



7.4. DRUG EXCIPIENT COMPATIBILITY STUDY

Table 8: Drug excipient compatibility study

S. No.

Composition Details

Observations - Storage Condition /

Duration

Initial

40°C/75%RH

1 Month

1. Leflunomide A white colour powder NCC

2. Leflunomide and Lactose A white colour powder NCC

3. Leflunomide and Mucilage A dull white colour powder NCC

4. Leflunomide and Starch A white colour powder NCC

5. Leflunomide and Aerosil A white colour powder NCC

6. Leflunomide and Magnesium

stearate

A White colour Powder NCC

NCC: No Characteristic Change

To study the compatibility of the drug with the excipients, the above test was

performed under accelerated condition and was found that there was no

characteristic change between drug and excipients in case of its physical nature

(Table 8). Hence, it confirms the physical compatibility of the excipients including

the mucilage with leflunomide.

Further, the compatibility studies were analysed by FTIR and DSC studies.

DSC and FTIR result (Graph I-VI) shows that the drug and mucilage were

compatible.



FTIR SPECTRUM

Graph -I: FTIR Spectrum of Leflunomide

4000 3500 3000 2500 2000 1500 1000 500

0.75

0.80

0.85

0.90

0.95

1.00

Tran

smitt

ance

(%)

Wavenumber cm-1

S1

Legend: 3350cm-1 -N-H stretching vibrations of amide group, 1750 cm-1 and 1680 cm-1 - C=O, C=N stretching, 1600 cm-1 -C=C

weak bond, 1403 cm-1 shows C-O, C-N of bending vibrations, 1205,1168 and 1035 cm-1 shows CF3 bending vibrations



Graph -II: FTIR Spectrum of Mucilage

4000 3500 3000 2500 2000 1500 1000 500

0.965

0.970

0.975

0.980

0.985

0.990

0.995

1.000

1.005

Wavenumber cm-1

Tran

smitt

ance

(%)

S2

Legend: 3262 cm-1 O-H stretching vibrations, 2930 and 2790cm-1 C-H stretching vibrations, 1601 and 1360 cm-1 C=C, N=O

stretching vibrations, 1003 cm-1 shows the possibility of C-O and C-N bending vibrations



Graph -III: FTIR Spectrum of Formulation

4000 3500 3000 2500 2000 1500 1000 500

0.88

0.90

0.92

0.94

0.96

0.98

1.00

1.02

Tran

smitt

ance

(%)

Wavenumber cm-1

S3

Legend: 3335 cm-1 N-H stretching vibrations of amide group, C=O, C=N stretching at 1750 cm-1 and 1680 cm-1, 1603 cm-1 C=C weak

bond, 1403 cm-1 C-O, C-N of bending vibrations. CF3 (1205, 1168 and 1035 cm-1) of bending vibrations.



Interpretation of FTIR Spectrum analysis

The molecular vibrational characteristics of drug and mucilage have been

determined by FTIR spectrum analysis. The normalised FTIR spectra of drug,

mucilage and formulation are shown in the graph I-III. The modes are assigned

based on the Colthup’s table of IR absorption bands.

From the graph I, the drug shows the band at higher wave number side

(3350 cm-1) corresponding to N-H stretching vibrations of amide group. At the

lower wave number region, C=O, C=N stretching at 1750 cm-1 and 1680 cm-1. The

characteristic peak at lower wave number 1600 cm-1 shows C=C weak bond and the

wave number 1403 cm-1 shows C-O, C-N of bending vibrations. The peaks showed

the presence of fluoride groups, CF3 at the wave number (1205, 1168 and 1035cm-1)

of bending vibrations.

From the graph II, the mucilage shows the characteristic peaks at wave

number (3262 cm-1) O-H stretching vibrations, the characteristic peak at wave

number (2930 and 2790 cm-1) shows C-H stretching vibrations. At the lower wave

number region, C=C, N=O stretching vibrations are seen at 1601 and 1360 cm-1. The

sharp characteristic peaks at wave number 1003 cm-1 shows the possibility of C-O

and C-N bending vibrations.

Overall, from the graph III, the formulation shows the band at higher wave

number side 3335 cm-1 corresponding to N-H stretching vibrations of amide group.

At the lower wave number region, C=O, C=N stretching at 1750 cm-1 and 1680 cm-1.

The characteristic peak at lower wave number 1603 cm-1 shows C=C weak bond and

the wave number 1403 cm-1 shows C-O, C-N of bending vibrations. The peaks



showed the presence of fluoride groups, CF3 at the wave number (1205, 1168 and

1035 cm-1) of bending vibrations.

Hence, the FTIR spectra of pure drug, mucilage and the formulation showed

corresponding characteristic peaks which indicate that there is no interaction

between the drug and the mucilage.



DIFFERENTIAL SCANNING COLORIMETRY

Graph - IV: DSC thermogram of Leflunomide

Legend: showing sharp endotherm of leflunomide at 168°C



Graph - V: DSC thermogram of Mucilage

Legend: showing endotherm of mucilage at 155°C



Graph- VI: DSC thermogram of Formulation

Legend: showing endotherm of formulation at 165°C



Differential Scanning Colorimetry

DSC was carried out for the drug, mucilage and the formulation. The pure drug

showed a sharp endotherm at 168°C. (The reported melting point of leflunomide is

165°C - 170°C) The mucilage shows an endotherm at 155°C. The formulation

shows an endotherm at 165°C. There was no significant change in the melting

endotherms of the formulation compared to the pure drug. The slight variation in the

endotherms of the mucilage and formulation confirmed that there was no proof of

chemical reaction taken place between the mucilage and the drug.

7.5. FLOW PROPERTIES OF THE BLEND

Depending on the bulk density, tapped density, Compressibility Index, Hausner's

ratio and angle of repose (Table 9), the flow of the dried mucilage and all the

formulation mixture were listed under poor flow. Thus, before it is being formulated

as a dosage form, suitable glidant has to be added to increase the flow of the blend.



Table 9: Flow properties of the mucilage and formulation mixture

S.No. Flow Properties Observations - Formulations

Mucilage LI LII LIII LIV LV

1. Bulk Density (g/cm3) 0.2612±0.04 0.2522±0.02 0.2402±0.02 0.1985±0.03 0.2197±0.03 0.2057±0.02

2. Tapped Density (g/cm3) 0.2730±0.02 0.245±0.02 0.2645±0.04 0.2586±0.01 0.2964±0.01 0.252±0.02

3. Compressibility Index (%) 23.48±0.45 22.18±0.51 21.88±0.50 25.58±0.80 24.91±0.45 25.35±0.31

4. Hausner’s Ratio 1.3060±0.05 1.290±0.01 1.310±0.020 1.450±0.025 1.200±0.02 1.410±0.04

5. Angle of Repose(θ°) 43.44±0.2 41.34±0.5 42.74±0.3 41.95±0.5 43.56±0.4 43.98±0.2

Values are reported as three evaluations of mean ± Standard deviation, n=3

LI : Mucilage alone – only mucilage at the same concentration of the drug

LII : Leflunomide alone - only leflunomide at the therapeutic dose.

LIII : 1:1 - equal concentration of leflunomide and mucilage

LIV : 0.5:1.5 - half the concentration of leflunomide and mucilage at three times higher the concentration

LV : 1:2 - half the concentration of leflunomide and mucilage at double the concentration



Table 10: Sieve Analysis of the Formulation

S.No. Sieve No.

Empty

sieve(gm)

Sample

sieve(gm)

Difference

(gm)

%Retained %Cumulative

retained

1. #20 321.4 321.4 0 0 0

2. #30 328.6 328.8 0.02 0.2 0.2

3. #40 299.0 300.0 0.10 1.0 1.2

4. #60 287.2 297.4 1.02 10.2 11.4

5. #100 255.0 275.0 2.00 20.0 31.4

6. #120 274.0 299.0 2.50 25.0 56.4

7. #200 270.0 303.2 3.32 33.2 89.6

8. Receiver 348.8 359.0 1.02 10.2 99.8

Weight of sample=10 gm

Through sieve analysis it is known that large quantity of powder was

retained on sieve no. 200, which indicates poor flow of the drug (Table 10). Flow

property and particle size are inversely proportional to each other. As the

formulation has fine grade of particles, it had poor flow and thereby to increase the

flow of the powder, glidant was added to the formulation.

When all the results were within the acceptable limits, 200 mg of the

formulation mixture was filled into zero sized capsules. The filled in capsules were

then evaluated and the results have been tabulated.



7.6. EVALUATION OF CAPSULES

i) Weight variation

The uniformity of dosage units may be demonstrated by determining weight

variation and/or content uniformity.

Table 11: Weight variation test for capsules

S.No. Parameter Observations - Formulations

LI LII LIII LIV LV

1. Weight

variation (%)

3.11±0.69 2.76±0.74 2.32±0.13 2.42±0.42 2.60±0.08





LIV : 0.5:1.5 - half the concentration of leflunomide and mucilage at three times

higher the concentration

LV : 1:2 - half the concentration of leflunomide and mucilage at double the

concentration

(Normal Limit as per USP = 7.5% difference in weight)

The weight variation revealed that the capsules were within the prescribed limit for

difference in weight of capsules.

ii) Locked length of capsules

This test ensures whether the capsules are properly locked after the drug is being

filled.



Table 12: Locked length of capsules

S.No.

Parameter

Observations - Formulations

LI LII LIII LIV LV

1. Locked Length

(mm)

21.7±0.12 21.6±0.15 21.8±0.36 21.7±0.19 21.7±0.58








concentration

(Normal Limit = 21-23 for 0 size capsule)

All the capsules were within the limit prescribed ensuring perfect locking of the

capsules so that they do not get unlocked.

iii) Assay of capsules

This test ensures the percentage purity of the drug in the dosage form

Table 13: Calibration curve of leflunomide

S.No. Concentration (μg/ml) Absorbance (nm)

1 0 0

2 5 0.2316

3 10 0.4016

4 15 0.5730

5 20 0.7665

6 25 0.9508



Graph VII – Standard Curve of Leflunomide

Table 14: Assay of capsules

S.No.

Parameter


LI LII LIII LIV LV

1. Assay (%) 0 100.01±0.25 100.8±0.65 99.94±0.55 101.6±0.48








concentration

The amount of drug present in each capsule was found by performing assay

by UV method. All the groups resulted within the limits. The normal limit for assay

of leflunomide is, it should contain not less than 99% and not more than 110% of

r2 0.997

Slope 0.036

Intercept 0.044



labeled amount of leflunomide. Thus, from the results it can be seen that that the

formulations contains the prescribed amount of the drug.

iv) Disintegration time of capsules

This test ensures the time taken for the drug to get disintegrated from the capsule

into the medium.

Table 15: Disintegration time for capsules

S.No.

Parameter


LI LII LIII LIV LV

1. Disintegration

time(min)

4.30±0.0124 4.50±0.086 4.40±0.075 4.50±0.056 5.10±0.014








concentration

The disintegration time for a hard gelatin capsule as per USP is 30 minutes. From

the observations, it is seen that all the formulation lies within the prescribed limit for

disintegration test.



Table 16: Dissolution test for capsule

Values are reported as mean ± Standard Error Mean, n=3

Graph – VIII: Percentage drug release of capsule








concentration

Formulation Time (Minutes)

10 20 30 45 60

LI 0.08 ±0.04 0.01±0.01 0.08±0.05 0.05±0.01 0.01±0.01

LII 0.05±0.02 31.42±0.21 59.37±1.23 72.76±5.23 85.87±3.22

LIII 0.17±0.03 29.87±0.35 53.82±5.11 65.84±4.21 77.39±1.77

LIV 0.52±0.12 64.7±2.12 73.21±2.36 77.72±5.96 89.14±4.11

LV 0.32±0.11 41.46±1.45 65.28±1.56 76.92±2.14 88.41±2.64

0 20 40 60 800

20

40

60

80

100LI

LII

LIII

LIV

LV

Time (min)

Dru

g R

ele

ase (

%)



7.6.1. EVALUATION OF CAPSULES ON STABILITY

Stability studies were carried out as per ICH guidelines for all the batches of

this product at accelerated storage conditions. The stability test was conducted by

placing the capsules in the stability chamber at 40°C / 75% RH for six months and

the capsules were found to be stable.

Stability data are used for evaluating the formulation (Table 17) and there

was no change in the assay, disintegration time, dissolution profiles were observed.



Table 17: Evaluation of capsules on stability

Parameters LI LII LIII LIV LV

0M 3M 6M 0M 3M 6M 0M 3M 6M 0M 3M 6M 0M 3M 6M

Weight

Variation(%)

3.11±

0.69

3.10±

0.61

2.21±

0.13

2.76±0.

74

2.40±0

.09

3.81±0.

65

2.32±0

.13

2.12±0.

14

2.82±0

.18

2.42±0

.42

2.11±0

.56

2.26±0

.54

2.60±0.

08

2.82±0

.46

2.24±0.

12

Lock Length

(mm)

21.7±

0.025

21.7±

0.025

21.7±

0.025

21.6±0.

0158

21.6±0

.0158

21.6±0.

0152

21.8±0

.0368

21.8±0.

0368

21.8±0

.0364

21.7±0

.0198

21.7±0

.0198

21.7±0

.0264

21.7±0.

058

21.7±0

.0596

21.7±0.

0584

Assay (%) 0 0 0 100.0±0

.259

99.92±

0.892

99.56±0

.452

100.8±

0.657

100.1±0

.259

99.95±

0.368

99.94±

0.557

99.02±

0.912

98.98±

0.568

101.6±0

.488

100.8±

0.566

100.1±

0.672

DT

(Mins)

4.30±

0.012

4.35±

0.051

5.1±0.

0367

4.50±0.

0869

4.55±0

.0548

4.30±0.

0116

5.10±0

.759

4.55±0.

0371

5.10±0

.0618

4.40±0

.0564

4.45±0

.0341

4.50±0

.0582

4.50±0.

0135

5.10.04

910±

5.00±0.

0211









Dissolution rate of capsules on stability

Table 18: Percentage drug release of capsules on stability

Formulation 10 minutes 20 minutes 30 minutes 45 minutes 60 minutes

0M 3M 6M 0M 3M 6M 0M 3M 6M 0M 3M 6M 0M 3M 6M

LI 0.08

±0.04

0.05±0

.01

0.05±0.

03

0.01±

0.01

0.05±0

.01

0±0.01 0.08±0

.05

0.02±

0.01

0.02±

0.01

0.05±0

.01

0±0.03 0.02±0.

01

0.01±0.

01

0.05±

0.02

0.06±

.0.03

LII 0.05±0.0

2

0.31±0

.09

0.30±0.

08

31.42

±0.21

40.79±

0.25

39.57±

0.49

59.37±

1.23

63.94

±2.15

62.71

±3.58

72.76±

5.23

73.95±

2.69

73.21±

3.12

85.87±

3.22

86.10

±3.94

85.23

±2.85

LIII 0.17±0.0

3

0.15±0

.11

0.14±0.

05

29.87

±0.35

25.91±

0.26

22.89±

0.51

53.82±

5.11

51.45

±2.84

50.91

±3.14

65.84±

4.21

64.97±

5.26

64.11±

6.15

77.39±

1.77

76.41

±6.45

76.11

±2.71

LIV 0.52±0.1

2

0.49±0

.92

0.41±0.

21

64.7±

2.12

63.81±

4.13

63.10±

2.64

73.21±

2.36

72.91

±2.85

72.10

±4.11

77.72±

5.96

76.86±

5.23

75.94±

2.65

89.14±

4.11

87.45

±4.23

85.51

±6.14

LV 0.32±0.1

1

0.05±0

.02

0.04±0.

01

41.46

±1.45

32.53±

3.27

32.45±

3.25

65.28±

1.56

58.41

±5.12

57.91

±2.15

74.92±

2.14

71.53±

4.62

70.25±

5.12

86.41±

2.64

87.45

±5.61

87.06

±4.59









Graph IX (a-e): DRUG RELEASE AT PRESCRIBED TIME INTERVALS

Graph IXa: Percentage drug release at 10 minutes








concentration

1 2 30.0

0.2

0.4

0.6

0.8LI

LII

LIII

LIV

LV

Time (month)

Dru

g R

ele

ase (

%)



Graph – IXb: Percentage drug release at 20 minutes








concentration

0 2 4 60

20

40

60

80LI

LII

LIII

LIV

LV

Time (month)

Dru

g R

ele

ase (

%)



Graph – IXc: Percentage drug release at 30 minutes








concentration

0 2 4 60

20

40

60

80LI

LII

LIII

LIV

LV

Time (month)

Dru

g R

ele

ase (

%)



Graph – IXd: Percentage drug release at 45 minutes








concentration

0 2 4 60

20

40

60

80LI

LII

LIII

LIV

LV

Time (month)

Dru

g R

ele

ase (

%)



Table 19: Percentage drug release at 60 minutes on stability


Graph IXe: Percentage drug release at 60 minutes








concentration

Period

(at 60 minutes)

Observations - Formulation

LI LII LIII LIV LV

0 Month 0.01±0.01 85.87±3.22 77.39±1.77 89.14±4.11 86.41±2.64

3 Month 0.05±0.02 86.10±3.94 76.41±6.45 87.45±4.23 87.45±5.61

6 Month 0.06±.0.03 85.23±2.85 76.11±2.71 85.51±6.14 87.06±4.59

0 2 4 6

0

20

40

60

80

100LI

LII

LIII

LIV

LV

Time (month)

Dru

g R

ele

ase (

%)



The dissolution test for the capsules were carried out on the initial day as

well as on stability after 3 and 6 months to ensure the percentage release of drug

during stability (Table 18, 19, Graph IX a-e). From the results, it was observed that

on the initial study the formulation LIV gave increased drug release compared with

the other formulations but on stability that is after six months of duration, LIV had a

decrease whereas LV showed increase in its dissolution rate. This LV which

contains double the ratio of mucilage with that of the drug when compared with LII,

the formulation containing leflunomide alone showed increased drug release. Thus,

it can be concluded that the presence of mucilage shows increase in the dissolution

rate of the drug thereby increasing the absorption as well as the bioavailability thus

acting as an effective bioenhancer.



7.7. IN-VITRO ANTI ARTHRITIC POTENTIAL OF THE MUCILAGE

The in-vitro anti arthritic potential of the mucilage was performed using protein

denaturation method. The results showed that the mucilage at different concentration

from 10-1000 µg/ml did not exhibit reliable in-vitro anti arthritic activity compared

to that of the standard drug (Table 20, Graph X).

Table 20: In-vitro anti arthritic potential of the mucilage

S. No

Concentration (µg/ml)

Percentage Inhibition

C. halicacabum mucilage Leflunomide

1. 10 0.1233±0.0095 22.17±0.7427

2. 50 0.0015±0.0083 42.11±0.2100

3. 100 0.0027±0.0038 53.88±0.3781

4. 200 0.0025±0.0029 61.27±0.2663

5. 400 0.0112±0.0028 67.43±0.5139

6. 800 0.0015±0.0019 75.90±0.0494

7. 1000 0.0053±0.0054 89.39±0.4392


Graph X: In-vitro anti arthritic potential of the mucilage



7.8. ACUTE ORAL TOXICITY TESTING

The acute oral toxicity (OECD 423) results showed that the mucilage did not

cause any apparent toxicity. No death or signs of toxicity were observed in rats

treated at dose of 2000 mg/kg body weight, thus establishing its safety in use.

Hence, the test drug falls in the category 5 in accordance to Globally Harmonized

System of classification of chemicals

In case of OECD 425, the formulation was found to be safe at 175mg/kg

body weight.

7.9. IN-VIVO ANTIARTHRITIC STUDY BY CFA INDUCED METHOD

7.9.1. ASSESSMENT OF BODY WEIGHT

The changes in body weight of the animals were observed on once in every

seven days. The results showed that there was not much significant changes in the

body weight of the animals in all the groups (Graph XI).

Group I = Normal Control

Group II = Arthritic Control

Group III = Leflunomide treated - only leflunomide at the therapeutic dose.

Group IV = Formulation treated 1:1, equal concentration of leflunomide and

mucilage

Group V = Formulation treated 1:2, half the concentration of leflunomide and

mucilage at double the concentration

Group VI = Formulation treated 0.5:1.5, half the concentration of leflunomide

and mucilage at three times higher the concentration

Group VII = Formulation treated 0:1, Mucilage alone – only mucilage at the

same concentration of the drug.



Graph XI: ASSESSMENT OF BODY WEIGHT


0 7 14 21 28

0

100

200

300

Control

CFA+ Veh

CFA+ Std

CFA+ 1:1

CFA+ 1:2

CFA+ 0.5:1.5

CFA+ Muc

Days

B.w

t(g

)



7.9.2. ASSESSMENT OF PAW VOLUME

The changes in paw volume (Graph XII, XIII) of the animals in all the

groups were measured using plethysmometer by water displacement method on once

in every seven days. The results showed that the animals, which received the

formulation with one part of leflunomide and two parts of mucilage, had significant

reduction in the paw volume of the animals compared with other groups. These

results revealed that the groups administered with mucilage proved that it could be

well used as a bioenhancer in formulations.

Thus, from this study it can be confirmed that the formulation in which the

mucilage was used at two times the ratio of drug showed much significant activity

thus proving its bioenhancing activity.



Graph XII: ASSESSMENT OF PAW VOLUME


0 7 14 21 28

0

1

2

3

4

5

Control

CFA+ Veh

CFA+ Std

CFA+ 1:1

CFA+ 1:2

*****

**

***

**

CFA+ 0.5:1.5

**

CFA+ Muc

**

###p<0.001 vs Control

*p<0.05, **p<0.01, ***p<0.001 vs CFA + Vehicle

$$p<0.01 vs Std

###

$$

###### ###

Days

Pa

w V

olu

me

(ml)





Group III = Leflunomide Treated - only leflunomide at the therapeutic dose.

Group IV = Formulation treated 1:1, equal concentration of leflunomide and mucilage

Group V = Formulation treated 1:2, half the concentration of leflunomide and mucilage at double the concentration

Group VI = Formulation treated 0.5:1.5, half the concentration of leflunomide and mucilage at three times higher the concentration

Group VII = Formulation treated 0:1, Mucilage alone – only mucilage at the same concentration of the drug

### - When compared with the normal control group, the group received CFA showed that the disease has been established

significantly.

*** - When compared with CFA treated group and other groups that received the formulations, on day 28 the group that received

mucilage two times the ratio of drug showed significant reduction in paw volume compared with the other groups.

$$$ - When compared with the group that received leflunomide alone with the other groups that received different ratios of mucilage

with drug, the group of animals that received mucilage two times the ratio of drug showed significant reduction in paw volume

compared with the other groups.



Graph XIII: Percentage reduction in paw volume


0 7 14 21 280

20

40

60

CFA + Std

CFA + 1:1

CFA + 1:2

CFA+ 0.5:1.5

CFA + Muc

Days

% re

duct

ion



The above graph represents the % reduction in the paw volume. On day 28, there is a

significant elevation in the % reduction of paw volume with the group administered with 1:2

ratio of the drug with the mucilage compared with that of the group administered with

leflunomide alone. Thus, it can be confirmed that the group which contains mucilage double

the ratio of the drug increased the bioavailability of the drug.



7.9.3. ANALYSIS OF BIO-CHEMICAL PARAMETERS

Table 21: BIO-CHEMICAL PARAMETERS

Group ALP(U/L) ALT(U/L) AST(U/L) CR(mg/dl) LDH (U/L) TP(g/dl) Ur( mg/dl) CRP-hs

(mg/dl)

I 477.67±51.26 82.67±9.84 102.5±4.37 0.66±0.04 158.17±28.47 6.98±0.28 27.67±1.99 3.19±0.17

II 716.00±71.01 *** 86.17±4.51 152.33±10.66 ** 0.62±0.02 525.67±39.79 *** 7.08±0.24 28.67±2.73 3.26±0.34

III 596.00±92.37 70.67±4.63 128.00±6.50 0.60±0.01 193.83±17.47 6.64±0.25 31.00±1.71 3.29±0.23

IV 578.00±54.27 74.83±4.66 129.17±7.54 0.61±0.04 148.50±33.74 6.55±0.26 28.33±2.12 3.09±0.1

V 578.50±45.92 66.00±5.34 123.67±9.10 0.60±0.04 169.00±68.06 6.04±0.29 28.50±1.45 3.27±0.32

VI 582.00±83.67 70.17±2.47 113.83±3.59 0.65±0.03 129.00±22.03 6.51±0.11 29.50±1.48 2.91±0.35

VII 532.00±71.01 75.17±4.51 114.33±10.66 0.62±0.02 125.67±39.79 6.08±0.24 28.67±2.73 3.26±0.34

Values are reported as mean ± Standard Error Mean, n=6. * p<0.05, **p <0.01, ***p <0.001 vs control





Group III = Leflunomide treated - only leflunomide at the therapeutic dose.

Group IV = Formulation treated 1:1, equal concentration of leflunomide and mucilage

Group V = Formulation treated 1:2, half the concentration of leflunomide and mucilage

at double the concentration

Group VI = Formulation treated 0.5:1.5, half the concentration of leflunomide and

mucilage at three times higher the concentration

Group VII = Formulation treated 0:1, Mucilage alone – only mucilage at the same

concentration of the drug.

ALP-Alkaline phosphatase , ALT-Alanine aminotransaminase, AST- Aspartate

aminotransaminase, CR-Creatinine, LDH- Lactate dehydrogenase, Ur- Urea, TP- Total

protein, CRP- hs- High sensitive C- reactive Protein

The above mentioned biochemical parameters were analyzed and it was observed that the

group that received the adjuvant had significant elevation in its ALP, AST and LDH levels

compared with that of the normal control. The other groups did not show much significant

change with the levels of the biochemical parameters (Table 21).



7.9.4. FIGURES SHOWING PAW OF CFA INDUCED ARTHRITIC RATS

Fig. 10: Paw of normal control group

Fig. 10a Fig. 10b

R L R L

Fig. 10c Fig. 10d

R L R L

Fig.10e

R L

Legend: Showing paw of normal control group without paw edema,

(a) on day 1, (b) on day 7, (c) on day 14, (d) on day 21, (e) on day 28



Fig. 11: Arthritic rats with severe paw edema

Fig. 11a Fig. 11b

R L R L

Fig. 11c Fig. 11d

R L R L

Fig. 11e

R L

Legend: (a-e) showing arthritic rats with severe paw edema on adjuvant induction

(1, 7, 14, 21, 28 days)



Fig. 12: Group III treated with leflunomide

Fig. 12a Fig. 12b

R L R L

Fig.12c Fig.12d

R L R L

Fig.12e

R L

Legend: (a) normal paw on day 1, (b) severe paw edema on day 7, (c) severe paw

edema on day 14, (d) & (e) reduction in edema on day 21 and day 28



Fig. 13: Group IV treated with 1:1 (leflunomide:mucilage)

Fig. 13a Fig. 13b

R L R L

Fig. 13c Fig. 13d

R L R L

Fig. 13e

R L

Legend: (a) normal paw on day 1, (b) severe paw edema on day 7, (c) severe paw

edema on day 14, (d) & (e) significant reduction in paw edema on day 21 and day 28



Fig. 14: Group V treated with 1:2 (leflunomide: mucilage)

Fig. 14a Fig. 14b

R L R L

Fig. 14c Fig. 14d

R L R L

Fig. 14e

R L

Legend: (a) normal paw on day 1, (b) & (c) severe paw edema on day 7 and day 14,

(d) & (e) significant reduction in paw edema on day 21 and day 28



Fig. 15: Group VI treated with 0.5:1.5 (leflunomide:mucilage)

Fig. 15a Fig. 15b

R L R L

Fig. 15c Fig. 15d

R L R L

Fig. 15e

R L

Legend: (a) normal paw on day 1, (b) & (c) severe paw edema on day 7 and day 14,

(d) & (e) significant reduction in paw edema on day 21 and day 28



Fig. 16: Group VII administered with only mucilage

Fig. 16a Fig. 16b

R L R L

Fig. 16c Fig. 16d

R L R L

Fig. 16e

R L

Legend: (a) normal paw on day 1, (b-e) severe edema on all days



It is concluded from the paw images that there was a significant decrease in

the groups (Fig.: 13, 14, 15) that received leflunomide with mucilage when

compared with that of the group (Fig.: 12) that received leflunomide alone. It can

also be seen that the group (Fig.: 16) that received mucilage alone did not show any

decrease in the paw edema of the rats confirming that the mucilage does not possess

anti-arthritic activity or therapeutic efficacy on the dose administered. Fig. 10 shows

the images of normal control group and Fig. 11 shows the group induced with the

phlogistic agent i.e Complete Freund’s Adjuvant. The reason for the groups that

received the formulations containing leflunomide with different ratios of mucilage

might be due to the increase in bioavailability of the drug which was not found in

case of the formulation that contain leflunomide alone.



7.9.5. CFA INDUCED ARTHRITIC MODEL- HISTOPATHOLOGY

Fig. 17: Group I normal control

Fig. 17a Fig. 17b

Fig.17c Fig. 17d

Fig. 17e

Legend: a) Muscle and Bone Marrow (20X), b) Ankle joint-joint cavity (20X), c)

Synovial membrane (20X), d) Interphalangeal joint (20X),

e) Ankle Joint (4X)



The hind limb tissues (from paw to ankle joint including skin, muscle,

cartilage, bones and joints) from rats were evaluated for general

histopathological changes. Tissues from this group revealed normal histology

with no signs of arthritis and associated lesions.



Fig. 18: Group II – Complete Freund’s Adjuvant induced

Fig. 18a Fig. 18b

Fig. 18c Fig. 18d

Fig. 18e Fig. 18f



Fig. 18g

Legend: a) Periarticular congested fibrovascular tissue (20X), b) Infiltration of

MNC in Joint capsule (40X), c) Exudate in ankle joint cavity (20X), d) Synovial

membrane cell proliferation interphalangeal Joint (20X), e) Vacuolated cells and

Giant cells (4X), f) Abcess formation (4X), g) Ankle joint (4X)

Joints: Mild degree of pannus formation was present with mononuclear cell

infiltration. Synovial membrane cells showed mild proliferation and

hypertrophy. Periarticular adipose tissue showed vascular congestion.

Articular cartilage and bones: were apparently normal.

Severe degrees of inflammatory changes were present from paw to ankle

region. The tissue was expanded with severe edema, infiltration by

polymorphs and mononuclear cells, necrotic debris and fibrosis. Multi-focal

abscess formation was evident. Occasional giant cells were also noticed.

Muscle necrosis and infiltration by inflammatory cells were also observed.



Fig. 19: Group III treated with leflunomide

Fig. 19a Fig. 19b

Fig. 19c Fig. 19d

Fig. 19e



Legend: (a) Bone erosion (20X), b) Bone Erosion (40X), c) Articular Cartilage and

bone erosion (40X), d) Synovium hypertrophy and pannus infiltration (40X), e)

Giant Cells (20X)



hypertrophy.

Articular cartilage and bones: mild degree of bone erosion was present.

Severe degree of inflammatory changes was present. The tissue was expanded

with severe edema, infiltration by polymorphs and mononuclear cells,

necrotic debris and fibrosis. Multi-focal abscess formation was evident in the

sub-cutaneous tissue. Muscle necrosis and infiltration by inflammatory cells

were also observed.



Fig. 20: Group IV treated with formulation 1:1 (leflunomide: mucilage)

Fig. 20a Fig. 20b

Fig. 20c Fig. 20d

Legend: a) Interphalangeal joint synovial membrane infiltration (40X), b) Bone

Erosion (20X), c) Muscle necrosis and bone erosion (10X) d) Articular cartilage and

bone erosion (20X)

Joints: mild infiltration of mononuclear cells in synovial membrane of ankle

and interphalangeal joints were present.

Articular cartilage and bones: moderate degree of articular cartilage

degeneration and bone erosion was present.



Severe degree of inflammatory changes were present. The tissue was

expanded with severe edema, infiltration by polymorphs and mononuclear

cells, necrotic debris and fibrosis. Multi-focal abscess formation was evident

in the sub-cutaneous tissue. Muscle necrosis and infiltration by inflammatory

cells were also observed.



Fig. 21: Group V treated with Formulation 1:2 (leflunomide: mucilage)

Fig. 21a Fig. 21b

Fig. 21c

Legend: a) Ankle joint apparently normal (4X), b) Giant cells (4X), c) Bone

Erosion (20X)

Joints: mild infiltration of mononuclear cells in synovial membrane of ankle

and interphalangeal joints were present. Compared with other groups the

joints looked apparently normal.

Articular cartilage and bones: mild degree of bone erosion was present.



Fig. 22: Group VI treated with formulation 0.5:1.5 (leflunomide: mucilage)

Fig. 22a Fig. 22b

Fig. 22c Fig. 22d

Fig. 22e

Legend: a) Pannus interphalangeal joint (20X), b) Bone erosion (40X),

c) Ankle joint synovial proliferation and infiltration (20X), d) Articular cartilage

degeneration and bone erosion (20X), e) Osteolytic foci/Dissoluted bone cells (20X)





hypertrophy.

Articular cartilage and bones: moderate degree of articular cartilage

degeneration, bone erosion, and osteolytic foci were present



Fig. 23: Group VII treated with only mucilage

Fig. 23a Fig. 23b

Fig. 23c Fig. 23d

Legend: a) Giant Cells (40X), b) Synovial membrane and infiltration (20X),

c) Bone erosion (40X), d) Bone Erosion and Exudate (20X)

Joints: Exudate noticed in joint cavity and severe infiltration of mononuclear

cells in synovium and peri-articular regions.

Articular cartilage and bones: Marked degeneration and erosion of

articular cartilage and bone was observed.

Moderate degree of muscular necrosis with severe inflammatory cell

infiltration. The tissue was expanded with severe edema, infiltration by

polymorphs and mononuclear cells, necrotic debris and fibrosis. Giant cells

were observed frequently. Multi-focal abscess formation was evident.



Fig. 24: Histopathology of Spleen

Fig. 24a Fig. 24b

Fig. 24c Fig. 24d

Fig. 24e Fig. 24f



Fig. 24g

Legend: (a-g) Histopathology of spleen revealing no lesions in all the groups

From the histopathological examinations, it is revealed that the groups

treated with mucilage as a combination in the formulation showed enhanced activity

compared with the group treated with leflunomide alone (Fig. 19). Among the

groups treated with different ratios of mucilage and leflunomide (Fig. 20,21,22), the

formulation which had 1:2 ratio that is, the group which had one part of leflunomide

and two part of mucilage showed significant decrease in the severity of the disease

condition where as the group which received the mucilage alone (Fig. 23) did not

show any therapeutic activity. Thus, it can be concluded that the mucilage at double

the ratio of the drug increased the bioavailability of the drug at a significant level.

The nomal control group (Fig. 17) and the group induced with CFA (Fig. 18) are

used for comparing the pathological changes

Spleen from all groups did not reveal any treatment related lesions and

remained similar to those of normal control group (Fig. 24).



7.9.6. IMMUNOHISTOCHEMISTRY

Fig. 25: IMMUNOHISTOCHEMISTRY SHOWING PRESENCE OF CD 4 CELLS

Fig. 25a Fig. 25b

Fig. 25c Fig. 25d

Fig. 25e Fig. 25f



Fig. 25g

Legend: (a) normal control group with less CD 4 cells (b) majority of CD 4 cells in

the group induced with CFA (c) less number of CD 4 cells with leflunomide (d) less

number of CD 4 cells in 1:1(leflunomide: mucilage), (e) very less number of CD 4

cells in 1:2 (leflunomide: mucilage), (f) comparatively more CD 4 cells in 0.5:1.5

(leflunomide: mucilage), (g) majority of CD 4 cells in the group that received only

mucilage

The cells coloured brown indicates the number of CD 4 cells in the tissue.

From the results, it is observed that the positive cells of CD 4 are found more in

CFA induced group (Fig. 25b) compared with the normal group (Fig. 25a). When

the CFA induced group is compared with the other treatment group (Fig. 25c-g), the

number of cells is lesser in case of formulation 1:2 compared with the other group

even the group containing drug alone indicating the effect of mucilage as a

bioenhancer in the formulation. The mucilage is more effective when it is

administered as double the ratio to that of the drug.



Fig. 26: IMMUNOHISTOCHEMISTRY SHOWING LOCALISATION OF IL-2

Fig. 26a Fig. 26b

Fig. 26c Fig. 26d

Fig. 26e Fig. 26f



Fig. 26f

Legend: (a) normal control group with minimum localisation of IL-2 (b) high

expression of IL-2 in the group induced with CFA (c) less expression of IL-2 with

leflunomide (d) less expression of IL-2 with 1:1(leflunomide: mucilage), (e) very

less expression of IL-2 with 1:2 (leflunomide: mucilage), (f) comparatively high

expression of IL-2 with 0.5:1.5 (leflunomide: mucilage), (g) high expression of IL-2

with the group that received only mucilage

The cells coloured brown indicates the expression of IL-2 inflammatory

marker in the tissue. From the results, it is observed that the localisation of large

amounts of the proinflammatory cytokine IL-2 was seen in CFA induced group

(Fig. 26b) compared with the normal group (Fig. 26a). A reduction in the expression

of the cytokine was observed with the treated animals whereas the expression of the

same was intermediate in the other treatment groups (Fig. 26c-g). The formulation

1:2 reduced the expression of IL-2 to a greater extent than the other groups even the

group containing drug alone indicating the effect of mucilage as a bioenhancer in the

formulation. The mucilage was found to be more effective when administered as

double the ratio to that of the drug.



Fig. 27: IMMUNOHISTOCHEMISTRY SHOWING TGF-β SIGNALING

Fig. 27a Fig. 27b

Fig. 27c Fig. 27d

Fig. 27e Fig. 27f



Fig. 27g

Legend: (a) normal control group with minimum TGF-β signaling (b) active TGF-β

signaling in the group induced with CFA (c) less signaling of TGF-β with

leflunomide (d) less signaling of TGF-β with 1:1(leflunomide: mucilage), (e) very

less signaling of TGF-β with 1:2 (leflunomide: mucilage), (f) comparatively high

TGF-β signaling with 0.5:1.5 (leflunomide: mucilage), (g) high signaling of TGF-β

with the group that received only mucilage

The cells coloured brown indicates the presence of active TGF-β signaling in

the tissue. From the results, it was observed that the presence of active TGF-β

signaling was greater in CFA induced group (Fig. 27b) compared with the normal

group (Fig. 27a). When the CFA induced group was compared with the other

treatment group (Fig. 27c-g), the TGF-β signaling was lesser in case of formulation

1:2 compared with the other group even the group containing drug alone indicating

the effect of mucilage as a bioenhancer in the formulation. The mucilage is more

effective when it was administered as double the ratio to that of the drug.



Fig. 28: IMMUNOHISTOCHEMISTRY SHOWING EXPRESSION OF TNF-α

Fig. 28a Fig. 28b

Fig. 28c Fig. 28d

Fig. 28e Fig. 28f



Fig. 28g

Legend: (a) normal control group with minimum expression of TNF-α b) high

expression of TNF-α in the group induced with CFA (c) lesser expression of TNF-α

with leflunomide (d) lesser expression of TNF-α with 1:1(leflunomide: mucilage),

(e) very less expression of TNF-α with 1:2 (leflunomide: mucilage), (f)

comparatively high expression of TNF-α with 0.5:1.5 (leflunomide: mucilage), (g)

high expression of TNF-α with the group that received only mucilage

The cells coloured brown indicates the expression of TNF-α inflammatory

marker in the tissue. From the results, it is observed that the localisation of large

amounts of the proinflammatory cytokine TNF-α was seen in CFA induced group

compared (Fig. 28b) with the normal group (Fig. 28a). A reduction in the expression

of the cytokine was observed with the treated animals whereas the expression of the

same was intermediate in the other treatment groups (Fig. 28c-g). The formulation

1:2 reduced the expression of TNF-α to a greater extent that the other groups even

the group containing drug alone indicating the effect of mucilage as a bioenhancer in

the formulation. The mucilage was found to be more effective when administered as

double the ratio to that of the drug.



7.10. PERCENTAGE ABSORPTION OF LEFLUNOMIDE BY EVERTED

SAC TECHNIQUE

Table 22: Percentage absorption of leflunomide by everted sac technique

.Values are reported as mean ± Standard Error Mean, n=3



LIII : 1:1- equal concentration of leflunomide and mucilage

LIV : 0.5:1.5 - mucilage at three times higher the concentration of leflunomide.

LV : 1:2- half the concentration of leflunomide and mucilage at double the

concentration

Formulation 30 minutes 60 minutes 90 minutes

Outer (%) Inner (%) Outer (%) Inner (%) Outer (%) Inner (%)

LI 0.54±0.08 0.01±0.02 0.89±0.06 0.23±0.05 0.35±0.02 0.07±0.01

LII 43.85±3.21 21.28±1.23 32.12±1.52 64±3.12 53.75±2.85 41.7±3.11

LIII 52.0±5.23 44.25±2.12 61.7±3.12 32.51±2.41 52.3±3.46 45.1±1.56

LIV 81.97±6.14 10.75±2.65 69.1±5.64 30.32±3.12 69.7±4.15 8.5±1.56

LV 83.1±6.52 17.71±2.14 76.27±4.26 23.91±1.52 71.35±6.15 26.40±2.86




Graph XIV: Percentage absorption of leflunomide by everted sac technique

The formulation containing 2:1 of mucilage: leflunomide showed greater

release of leflunomide than the formulation, which has only leflunomide. This

proves that the rate of absorption through the sac is enhanced by the presence of

mucilage. The results of Everted sac technique (Table 22, Graph XIV) revealed that

the presence of mucilage in the formulation with the ratio 2:1 (mucilage:

leflunomide) increased the absorption of the drug.

30 60 90

0

20

40

60

80

100LI

LII

LIII

LIV

LV

*** ***

***

***

*** ***

*p< 0.05, **p<0.01, ***p<0.001 vs LII

Time (min)

Ab

so

rpti

on

(%

)

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