Post on 23-Apr-2020
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
Scale: X-Axis – Concentration (µg/ml)
Y-Axis – Absorbance (nm)
123
Table 5.3 Standard graph for the estimation of ampicillin
Graph5.3 Calibration curve for the estimation of Ampicillin
Scale: X-Axis – Concentration (µg/ml)
Y-Axis – Absorbance (nm)
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
nanoparticles (Amoxicillin)
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
nanoparticles (Amoxicillin)
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
Table 5.14 In-vitro drug release studies of prepared sepia
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
Table 5.15 In-vitro drug release studies of prepared sepia
nanoparticles (Ampicillin)
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
nanoparticles (Ampicillin)
135
Table 5.16 In-vitro drug release studies of prepared sepia
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
BIOPHARMACEUTICAL PARAMETERS:
Table 5.20 Biopharmaceutical parameters of Amoxicillin-loaded
sepia nanoparticles
S.NO
Parameters
Values
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
- 0.0069
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
BIOPHARMACEUTICAL PARAMETERS:
.
Table 5.22 Biopharmaceutical parameters of Ampicillin-loaded
sepia nanoparticles
S.NO
Parameters
Values
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
0.0133
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.
Absorption Constant (Ka)
Elimination Constant (Ke)
Half – Life (t ½)
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
Table 5.26 Stability testing studies of Ciprofloxacin nanoparticles at
4ºC
Week
% of Drug Remaining (4ºC)
0
1st Week
2nd Week
3rd Week
4th Week
100
100
99.56
98.26
98.20
Table 5.27 Stability testing studies of Ciprofloxacin nanoparticles at
45ºC
Weeks
% of Drug Remaining (45ºC)
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
Table 5.29 Stability testing studies of Amoxicillin nanoparticles at
4ºC
Week
% of Drug Remaining (Room Temp.)
0
1st Week
2nd Week
3rd Week
4th Week
100
97.43
95.56
94.32
93.65
Week
% of Drug Remaining (4ºC)
0
1st Week
2nd Week
3rd Week
4th Week
100
98.56
97.54
93.42
92.65
148
Table 5.30 Stability testing studies of Amoxicillin nanoparticles at
45ºC
STABILITY TESTING (AMPICILLIN)
Table 5.31 Stability testing studies of Ampicillin nanoparticles at
room temperature
Week
% of Drug Remaining (45ºC)
0
1st Week
2nd Week
3rd Week
4th Week
100
94.75
93.54
92.34
88.86
Week
% of Drug Remaining (Room Temp.)
0
1st Week
2nd Week
3rd Week
4th Week
100
100
99
97
96.1
149
Table 5.32 Stability testing studies of Ampicillin nanoparticles at
4ºC
Table 5.33 Stability testing studies of Ampicillin nanoparticles at
45ºC
Week
% of Drug Remaining (4ºC)
0
1st Week
2nd Week
3rd Week
4th Week
100
100
99.5
98
97.7
Week
% of Drug Remaining (45ºC)
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
% of Drug Remaining (Room Temp.)
0
1st Week
2nd Week
3rd Week
4th Week
100
100
98
98.6
97
Week
% of Drug Remaining (4ºC)
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
% of Drug Remaining (45ºC)
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