5.Experimental Results Table 5.1 Calibration curve for the...

121

5.Experimental Results

Table 5.1 Calibration curve for the estimation of Ciprofloxacin

Sl.No.

Conc.(μg/ml)

Absorbance(nm)

1.

2.

3.

4.

5.

6.

10

12

14

16

18

20

0.102

0.280

0.420

0.651

0.840

1.092

Graph5.1 Calibration curve for the estimation of Ciprofloxacin

Scale: X-Axis – Concentration (µg/ml)

Y-Axis – Absorbance (nm)

122

Table 5.2 Calibration curve for the estimation of Amoxicillin

Sl.No

Conc.(μg/ml)

Absorbance(nm)

1.

2.

3.

4.

5.

10

20

40

80

100

0.012

0.02

0.04

0.074

0.078

Graph5.2 Calibration curve for the estimation of Amoxicillin



123

Table 5.3 Standard graph for the estimation of ampicillin

Graph5.3 Calibration curve for the estimation of Ampicillin



Concentration(µg/ml) Absorbance

10 0.012

20 0.019

40 0.032

80 0.06

100 0.073

124

Table 5.4 Standard graph for the estimation of ofloxacin

Graph5.4 Calibration curve for the estimation of Ofloxacin

Concentration(µg/ml) Absorbance (nm)

10 0.07

12 0.24

14 0.419

16 0.597

18 0.789

20 0.99

125

Table 5.5 Microbiological method of the estimation of Amoxicillin

Amoxicillin conc.(μg/ml)

Average Zone of inhibition (mm)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

13.9

19.6

25.0

28.7

36.2

39.8

43.6

48.0

50.2

53.4

Graph5.5 Microbiological method of the estimation of Amoxicillin

126

Table 5.6 Microbiological method of the estimation of Ciprofloxacin

Ciprofloxacin conc. (μg/ml)

Average Zone of inhibition (mm)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

14.5

21.0

27.0

32.5

37.0

41.5

45.0

49.0

54.0

57.5

Graph5.6 Microbiological method of the estimation of Ciprofloxacin

127

Table 5.7 Microbiological method of the estimation of ampicillin

Graph5.7 Microbiological method of the estimation of Ampicillin

Ampicillin Concentration(µg/ml)

Average Zone of Inhibition(mm)

0.5

13.7

1.0

17.5

1.5

21.3

2.0

25.0

3.0

32.6

3.5

36.38

4.0

40.2

4.5

43.9

5.0

47.7

128

Table 5.8 Microbiological method of the estimation of Ofloxacin

Graph5.8 Microbiological method of the estimation of Ofloxacin

Concentration(µg/ml)

Average Zone of Inhibition(mm)

0.5

14.2

1.0

18.4

1.5

22.7

2.0

26.9

2.5

31.2

3.0

35.4

3.5

39.6

4.0

43.8

4.5

48.0

5 52.3

129

Table 5.9 Determination of the drug content of prepared sepia

nanoparticles (Amoxicillin)

S.No Batch Code Drug content (%)

1 AMN1 59.25 ± 0.13

2 AMN2 62.34 ± 0.09

3 AMN3 65.33 ± 0.11

4 AMN4 64.13 ± 0.12

5 AMN5 61.34 ± 0.13

6 AMN6 59.24 ± 0.09

7 AMN7 57.34 ± 0.10

* Average of five preparations ± S.D

Table 5.10 Determination of the drug content of prepared sepia

nanoparticles (Ciprofloxacin)

S.NO

BATCHCODE DRUG CONTENT (%)

1 CN1 55 + 0.13

2 CN2 59 + 0.12

3 CN3 67 + 0.10

4 CN4 63 + 0.11

5 CN5 61 + 0.09

6 CN6 60 + 0.12

7 CN7 57 + 0.11

130

Table 5.11 Determination of the drug content of the prepared sepia

nanoparticles (Ampicillin)

SNO

BATCH CODE DRUG CONTENT PERCENTAGE

1.

AP1 58.17±0.11

2.

AP2 61.73±0.14

3.

AP3 60.17±0.09

4.

AP4 58.13±0.11

5.

AP5 62.13±0.12

6.

AP6 53.12±0.11

7.

AP7 59.15±0.13

Table 5.12 Determination of the drug content of the prepared sepia

Ofloxacin nanoparticles

SNO BATCH CODE

DRUG CONTENT PERCENTAGE

1. OF1

59.13±0.12

2. OF2

57.15±0.09

3. OF3

61±0.12

4. OF4

60±0.09

5. OF5

52±0.13

6. OF6

63.12±0.11

7. OF7

59.17±0.10

131

Table 5.13 In-vitro drug release studies of prepared sepia


S.No

Time Cumulative % drug Release

AMN1 AMN2 AMN3 AMN4 AMN5 AMN6 AMN7

1 15minutes 41.58

42. 8

47.04

42.18

39.72

38.52

32.24

2 30minutes 42.72

44.1

51.44

47.84

43.56

40. 8

36.41

3 1 hour 46.92

45.4

55.76

50.16

50.76

44. 1

38.24

4 2hours 50.88

47.2

68.52

55.68

52.04

46. 4

41.06

5 4hours 54.92 47.5

77.16

61.38

60.52

49.44

45.32

6 8 hours 59.88

48.2

83.52

65.64

62.34

54.71

49.24

7 12hours 63.24

72.3

85.44

71.76

70.44

63. 6

59.96

8 24hours

68.52 72.5 90.36 85.44 81.36 75.44 62.24

Graph 5.9 In-vitro drug release studies of prepared sepia


In-vitro drug release studies for the Sepia

Nanoparticles containing amoxycillin

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30

Time in hours

Cumu

lative

% dr

ug re

lease

AMN1

AMN2

AMN3

AMN4

AMN5

AMN6

AMN7

132


nanoparticles (Ciprofloxacin)

S.No Time

(hrs)

Cumulative Percentage Drug Release

CN-I CN-II CN-III CN-IV CN-V CN-VI CN-VII

1 0.25 38.5 45.88 39.25 40.74 39.52 37.44 36.16

2 0.5 41.88 46.92 42.14 40.92 41.52 38.16 38.64

3 1 42.36 50.88 44.52 42.18 42.78 41.04 40.04

4 2 43.44 54.92 53.76 44.7 43.44 42.36 40.29

5 4 53.64 59.92 63.6 51.72 48.84 44.55 42.72

6 8 55.92 60.58 69.02 59.04 53.76 53.76 43.56

7 12 68.88 67.28 74.6 67.38 63.62 63.6 53.76

8 24 75.42 80.04 85.0 72.83 67.44 67.2 63.0

Graph5.10 In-vitro drug release studies of prepared sepia nanoparticles

(Ciprofloxacin)

In-Vitro drug release profile for

Ciprofloxacin nanoparticles

0

10

20

30

40

50

60

70

80

90

0 10 20 30Time in hours

Cum

ulativ

e per

cent

age

drug

relea

se

CF-I CF-II CF-III CF-IVCF-V CF-VI CF-VII

133



SNO TIME

CUMULATIVE PERCENTAGE DRUG RELEASE

AP1 AP2 AP3 AP4 AP5

AP7

AP6

1. 15 mins 36.49 37.16 39.91 42.44 46.07 43.07 41.67

2. 30 mins 39.96 42.12 43.01 49.71 51.56 50.12 47.36

3. 1 hr

46.06 48.11 50.66 53.49 56.12 54.60 53.19

4. 2 hrs

51.55 56.61 59.11 61.44 62.34 59.96 59.12

5. 4 hrs

57.99 59.19 61.19 63.61 64.69 61.39 63.19

6. 8 hrs

65.67 67.91 69.66 70.16 72.12 70.12 69.96

7. 12 hrs 72.11 73.02 74.17 75.99 81.09 76.47 74.12

8. 24 hrs 77.14 79.49 80.19 84.05 88.00 79.01 80.71

134

Graph5.11 In-vitro drug release studies of prepared sepia


135


nanoparticles (Ofloxacin)

SNO TIME CUMULATIVE PERCENTAGE DRUG RELEASE

OF1 OF2 OF3 OF4 OF5 OF6 OF7

1. 15 mins 32.24 34.56 37.32 39.12 42.18 45.05 41.06

2. 30 mins 33.12 34.97 38.12 42.21 46.96 50.31 44.82

3. 1 hr

34.92 39.54 44.16 48.32 52.12 56.18 53.24

4. 2 hrs

39.12 44.42 49.38 53.69 55.25 67.10 62.18

5. 4 hrs

43.02 46.52 54.54 57.12 60.50 75.18 67.23

6. 8 hrs

48.96 52.48 59.72 63.34 64.28 82.38 73.14

7. 12 hrs

53.08 55.71 64.76 68.12 70.16 84.15 77.21

8. 24 hrs

59.78 58.23 69.12 73.77 77.28 87.00 79.32

136

Graph5.12 In-vitro drug release studies of prepared sepia

nanoparticles (Ofloxacin)

137

Table 5.17 Serum concentration (µg/ml) of Ciprofloxacin

TIME

ANIMAL 1

ANIMAL 2

ANIMAL 3

ANIMAL 4

MEAN X ±

SEM

15 Min

1.90

1.7

0.9

1.6

1.5 ± 0.217

30 Min

2.25

2.12

1.5

2.0

1.9675±0.164

1 Hr

2.40

2.38

2.0

2.35

2.285±0.094

2 Hr

2.65

2.50

2.25

2.55

2.4875±0.008

4 Hr

2.77

2.65

2.30

2.70

2.6±0.104

8 Hr

3.05

2.90

2.45

2.85

2.812±0.1281

12 Hr

3.33

3.10

2.70

3.0

3.035±0.131

24 Hr

3.41

3.20

2.90

3.25

3.19±0.106

36 Hr

2.90

2.75

2.65

2.70

2.75±0.054

48 Hr

1.95

1.60

1.50

1.85

1.725±0.1051

72 Hr

0.90

0.85

0.70

0.65

0.775±0.059

138

BIOPHARMACEUTICAL PARAMETERS:

Table 5.18 Biopharmaceutical parameters of Ciprofloxacin loaded sepia nanoparticles

S.NO

Parameters

Value

1.

2.

3.

4.

5.

6.

7.

Absorption Constant (Ka)

Elimination Constant (Ke)

Half – Life (t ½)

Tmax

Apparent volume of distribution

(Vd)

C Max

AUC

- 0.04

0.0732

17.325 hours

36.6 hrs

3.62 liters

31.5 mg/ml

209.7 mg/ml

139

Table 5.19 Serum concentration (µg/ml) of Amoxicillin

TIME

ANIMAL 1

ANIMAL2

ANIMAL3

ANIMAL4

MEAN X ± SEM

15 Min

1.85

1.70

1.72

1.87

1.78± 0.0873

30 Min

1.93

1.87

1.90

1.90

1.90± 0.0122

1 Hr

1.97

1.92

1.93

1.83

1.91±0.02955

2 Hr

2.10

1.97

2.05

2.20

2.08±0.04813

4 Hr

2.25

2.20

2.15

2.27

2.21±0.02689

8 Hr

2.47

2.35

2.23

2.52

2.39±0.06486

12 Hr

2.69

2.66

2.72

2.70

2.69±0.01250

24 Hr

2.75

2.69

2.75

2.81

2.75±0.02449

36 Hr

2.50

2.31

2.60

2.60

2. 5±0.068

48 Hr

2.15

2.02

2.22

2.25

2.16±0.05115

72 Hr

1.90

1.92

2.0

1.89

1.92±0.02496

140


Table 5.20 Biopharmaceutical parameters of Amoxicillin-loaded

sepia nanoparticles

S.NO

Parameters

Values

1.

2.

3.

4.

5.

6.

7.




Tmax


(Vd)

C Max

AUC

- 0.0069

0.634

10.43 hours

39.25 hrs

3.76 liters

8.6 mg/ml

130.58 mg/ml

141

Table 5.21 Serum concentration (µg/ml) of Ampicillin

TIME

ANIMAL 1

ANIMAL 2

ANIMAL 3

ANIMAL 4

MEAN X

± SEM

15 Min

1.90

1.80

1.72

1.60

1.75

30 Min

2.12

2.09

1.90

1.70

1.95

1 Hr

2.25

2.22

2.10

1.90

2.12

2 Hr

2.30

2.40

2.30

2.22

2.30

4 Hr

2.65

2.75

2.60

2.56

2.64

8 Hr

2.85

2.90

2.85

2.73

2.83

12 Hr

3.05

3.15

3.10

2.92

3.05

24 Hr

3.20

3.30

3.20

3.05

3.18

36 Hr

2.90

3.65

3.00

2.96

2.97

48 Hr

1.50

2.80

2.95

2.75

2.5

72 Hr

0.55

1.00

0.90

0.83

0.82

142


.

Table 5.22 Biopharmaceutical parameters of Ampicillin-loaded

sepia nanoparticles

S.NO

Parameters

Values

1.

2.

3.

4.

5.

6.

7.




Tmax


(Vd)

C Max

AUC

0.0133

0.0034

15.13 hours

52.10 hours

77 liters

95 mg/ml

18.70 mg/ml

143

Table 5.23 Serum concentration (µg/ml) of Ofloxacin

TIME

ANIMAL 1

ANIMAL 2

ANIMAL 3

ANIMAL 4

MEAN X

± SEM

15 Min

1.86

1.90

1.60

1.70

1.76

30 Min

1.92

2.03

1.83

1.90

1.92

1 Hr

2.09

2.18

2.05

2.10

2.10

2 Hr

2.18

2.40

2.30

2.40

2.32

4 Hr

2.30

2.68

2.70

2.63

2.57

8 Hr

2.60

2.89

2.82

2.91

2.80

12 Hr

2.90

3.03

3.10

3.22

3.06

24 Hr

3.10

3.28

3.30

3.36

3.26

36 Hr

2.96

3.00

3.10

3.28

3.08

48 Hr

2.30

2.53

2.90

2.95

2.67

72 Hr

1.6

1.2

1.90

1.95

1.912

144

BIOPHARMACEUTICAL PARAMETERS: .

Table 5.24 Biopharmaceutical parameters of Ofloxacin-loaded

sepia nanoparticles

S.NO

Parameters

Values

1.

2.

3.

4.

5.

6.

7.




Tmax

Apparent volume of distribution (Vd)

C Max

AUC

0.0098

0.0057

28.875 hours

32 hours

86.2 mg/ml

93 mg/ml

17.8060

145

STABILITY TESTINGS (CIPRO)

The formulated nanoparticles were divided into three portions and one

portion was kept at room temp, second at 45ºC, and third at 4ºC for one

month at weekly intervals, samples were analysed

spectrophotometrically.

Table 5.25 Stability testing studies of Ciprofloxacin nanoparticles at

room temperature

Week

% of Drug Remaining (Room Temp.)

0

1st Week

2nd Week

3rd Week

4th Week

100

100

99.37

96.24

94.38

146


4ºC

Week

% of Drug Remaining (4ºC)

0

1st Week

2nd Week

3rd Week

4th Week

100

100

99.56

98.26

98.20


45ºC

Weeks


0

1st Week

2nd Week

3rd Week

4th Week

100

90.72

89.42

89.12

88.93

147

STABILITY TESTING (AMOX)

Table 5.28 Stability testing studies of Amoxicillin nanoparticles at

room temperature


4ºC

Week


0

1st Week

2nd Week

3rd Week

4th Week

100

97.43

95.56

94.32

93.65

Week


0

1st Week

2nd Week

3rd Week

4th Week

100

98.56

97.54

93.42

92.65

148


45ºC

STABILITY TESTING (AMPICILLIN)

Table 5.31 Stability testing studies of Ampicillin nanoparticles at

room temperature

Week


0

1st Week

2nd Week

3rd Week

4th Week

100

94.75

93.54

92.34

88.86

Week


0

1st Week

2nd Week

3rd Week

4th Week

100

100

99

97

96.1

149


4ºC


45ºC

Week


0

1st Week

2nd Week

3rd Week

4th Week

100

100

99.5

98

97.7

Week


0

1st Week

2nd Week

3rd Week

4th Week

100

99.50

98.5

95.7

94.6

150

STABILITY TESTING (Ofloxacin)

Table 5.34 Stability testing studies of Ofloxacin nanoparticles at

room temperature

Table 5.35 Stability testing studies of Ofloxacin nanoparticles at 4ºC

Week


0

1st Week

2nd Week

3rd Week

4th Week

100

100

98

98.6

97

Week


0

1st Week

2nd Week

3rd Week

4th Week

100

100

100

99

98.7

151

Table 5.36 Stability testing studies of Ofloxacin nanoparticles at

45ºC

Week


0

1st Week

2nd Week

3rd Week

4th Week

100

98

97

95.7

93.9

152

Graph5.13 IR spectra of Amoxicillin Trihydrate

Graph5.14 IR spectra Amoxicillin+ sepia (Physical mixture)

153

Graph5.15 IR Spectra of Ciprofloxacin

3408.63, 34.05

3528.23, 15.99

3119.27, 37.78

3098.05, 34.48

3082.62, 33.94

3032.47, 35.87

3013.18, 32.72

2926.37, 35.58

2831.85, 43.57

2816.41, 47.52

2766.26, 35.8

2692.95, 21.05

2619.65, 22.21

2494.26, 33.54

2461.47, 28.79

1703.35, 4.449

1633.91, 11.99

1622.33, 4.274

1550.96, 34.93

1518.16, 12.61

1491.16, 5.232

1468.01, 5.078

1446.79, 4.322

1396.63, 23.99

1381.20, 18.78

1336.83, 23.83

1331.04, 25.04

1302.11, 24.71

1269.32, 5.416

1215.30, 41.82

1180.58, 40.15

1138.14, 39.14

1101.49, 45.34

1076.41, 55.3

1041.69, 43.42

981.89, 35.96

935.59, 38.36

534.35, 49.43

750.40, 38.61

542.06, 46.74

484.19, 47.5

470.69, 47.35

3379.70, 28.79

2970.74, 43.65

3204.15, 51.94

3242.73, 54.66

3225.37, 55.12

500100015002000250030003500

10

20

30

40

50

60

Graph5.16 IR Spectra of Ciprofloxacin+ Sepia (Physical mixture)

154

Graph5.17 IR Spectra of sepia (Polymer)

Graph5.18 IR-Graph of Ampicillin

155

Graph5.19 IR-Graph of Ampicillin and polymer physical mixture

Graph5.20 IR-Graph of ofloxacin

156

Graph5.21 IR-Graph of ofloxacin and polymer physical mixture

157

Graph 5.22 DSC Graph of sepia

158

Graph 5.23 DSC Graph of formulations (Ciprofloxacin HCl and sepia)

159

Graph 5.24 DSC Graph of drug Ciprofloxacin HCl

160

Graph 5.25 DSC Graph of formulations (Amoxicillin trihydrate and

sepia)

161

Graph 5.26 DSC Graph of drug Amoxicillin trihydrate

162

Graph 5.27 DSC Graph of drug,Ampicillin

163

Graph 5.28 DSC Graph of formulations (Ampiciliin and sepia )

164

Graph 5.29 DSC Graph of Ofloxacin

165

Graph 5.30 DSC Graph of formulation (Ofloxacin and sepia)

166

Fig 5.19 SEM photograph of Amoxicillin-loaded sepia nanoparticles

Fig 5.20 SEM photograph of Ciprofloxacin-loaded Sepia

Nanoparticles

167

Fig 5.21 SEM Photograph of Ampicillin -loaded sepia nanoparticles

Fig 5.22 SEM Photograph of Ofloxacin-loaded sepia nanoparticles

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