IV. RESULTS 4.1 Ultraviolet spectroscopy 4.1.1 Ultraviolet visible spectrum of diclofenac sodium The ultraviolet visible spectrum (200 – 350 nm) of diclofenac sodium in acidic (0.1 N HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems are shown in Fig. 1. The maximum ultraviolet absorption of diclofenac was found at 273, 275, 279 nm in acidic (0.1 N HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems, respectively. The obtained spectra and maximum absorption wavelength for diclofenac sodium in acidic and alkaline solvent system was compared with the reference spectra of diclofenac sodium. Fig. 1. UV-visible spectrum of diclofenac sodium 200 225 250 275 300 325 350 0.0 0.5 1.0 1.5 2.0 Acidic Solvent Alkaline Solvent Neutral Solvent 279 275 273 Wavelength in nm Absorbance
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IV. RESULTS 4.1 Ultraviolet spectroscopy 4.1.1 Ultraviolet visible spectrum of diclofenac sodium The ultraviolet visible spectrum (200 – 350 nm) of diclofenac sodium in acidic
(0.1 N HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems are shown in
Fig. 1. The maximum ultraviolet absorption of diclofenac was found at 273, 275, 279 nm
in acidic (0.1 N HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems,
respectively. The obtained spectra and maximum absorption wavelength for diclofenac
sodium in acidic and alkaline solvent system was compared with the reference spectra of
diclofenac sodium.
Fig. 1. UV-visible spectrum of diclofenac sodium
200 225 250 275 300 325 3500.0
0.5
1.0
1.5
2.0
Acidic SolventAlkaline SolventNeutral Solvent
279
275
273
Wavelength in nm
Abs
orba
nce
61
4.1.2 Ultraviolet visible spectrum of aspirin The ultraviolet visible spectrum (200 - 350 nm) of aspirin in acidic (0.1 N HCl),
and neutral (methanol) solvent systems are shown in Fig. 2. The maximum ultraviolet
absorption of aspirin was found at 230 and 278 nm in acidic (0.1 N HCl) and 225 and 276
nm in neutral (methanol) solvent systems, respectively. The obtained spectra and
maximum absorption wavelength for aspirin in acidic solvent system was compared with
the reference spectra of aspirin.
Fig. 2. UV-visible spectrum of aspirin
200 225 250 275 3000.0
0.5
1.0
1.5
Acidic SolventNeutral Solvent
276
278
225
230
Wavelength in nm
Abs
orba
nce
62
4.1.3 Ultraviolet visible spectrum of paracetamol The ultraviolet visible spectrum (200 - 325 nm) of parcetamol in acidic (0.1 N
HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems are shown in Fig. 3.
The maximum ultraviolet absorption of paracetamol was found at 245, 256 and 250 nm
in acidic (0.1 N HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems,
respectively. The obtained spectra and maximum absorption wavelength for paracetamol
in acidic and alkaline solvent system was compared with the reference spectra of
paracetamol.
Fig. 3. UV- visible spectrum of paracetamol
200 225 250 275 300 3250.0
0.5
1.0
1.5
2.0
Acidic SolventAlkaline SolventNeutral Solvent
245 256
250
Wavelength in nm
Abs
orba
nce
63
4.1.4 Ultraviolet visible spectrum of nimesulide The ultraviolet visible spectrum (200 - 550 nm) of nimesulide in acidic (0.1 N
HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems are shown in Fig. 4.
The maximum ultraviolet absorption of nimesulide was found at 301, 394 and 400 nm in
acidic (0.1 N HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems,
respectively. The obtained spectra and maximum absorption wavelength for nimesulide
in acidic and alkaline solvent system was compared with the reference spectra of
nimesulide.
Fig. 4. UV- visible spectrum of nimesulide
200 250 300 350 400 450 500 5500.0
0.5
1.0
1.5
2.0
Acidic SolventAlkaline SolventNeutral Solvent
301 400
394
Wavelength in nm
Abs
orba
nce
64
4.1.5 Ultraviolet visible spectrum of ketoprofen The ultraviolet visible spectrum (200 - 350 nm) of ketoprofen in acidic (0.1 N
HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems are shown in Fig. 5.
The maximum ultraviolet absorption of ketoprofen was found at 260, 262 and 257 nm in
acidic (0.1 N HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems,
respectively. The obtained spectra and maximum absorption wavelength for ketoprofen
in acidic and alkaline solvent system was compared with the reference spectra of
ketoprofen.
Fig. 5. UV- visible spectrum of ketoprofen
200 250 300 3500.0
0.5
1.0
1.5
2.0
Acidic SolventAlkaline SolventNeutral Solvent
260
262
Wavelength in nm
Abs
orba
nce
257
65
4.1.6 Ultraviolet visible spectrum of meloxicam The ultraviolet visible spectrum (200 - 400 nm) of meloxicam in acidic (0.1 N
HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems are shown in Fig. 6.
The maximum ultraviolet absorption of meloxicam was found at 345, 362 and 356 nm in
acidic (0.1 N HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems,
respectively. The obtained spectra and maximum absorption wavelength for meloxicam
in acidic and alkaline solvent system was compared with the reference spectra of
meloxicam.
Fig. 6. UV- visible spectrum of meloxicam
200 250 300 350 4000.0
0.5
1.0
1.5
2.0
Acidic SolventAlkaline SolventNeutral Solvent
345
362
356
Wavelength in nm
Abs
orba
nce
66
4.1.6 Ultraviolet visible spectrum of celecoxib The ultraviolet visible spectrum (200 - 350 nm) of celecoxib in acidic (0.1 N
HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems are shown in Fig. 7.
The maximum ultraviolet absorption of celecoxib was found at 250, 252 and 254 nm in
acidic (0.1 N HCl), alkaline (0.1 N NaOH) and neutral (methanol) solvent systems,
respectively. The obtained spectra and maximum absorption wavelength for celecoxib in
acidic and alkaline solvent system was compared with the reference spectra of celecoxib.
Fig. 7. UV- visible spectrum of celecoxib
200 225 250 275 300 325 3500.0
0.5
1.0
1.5
2.0
Acidic SolventAlkaline SolventNeutral Solvent
250
252
254
Wavelength in nm
Abs
orba
nce
67
4.2 Infrared spectroscopy
4.2.1 Infrared spectrum of diclofenac sodium
The infrared spectrum of diclofenac sodium in KBr and ATR is shown in Fig. 8
and Fig. 9, respectively. The principal wave numbers obtained in infrared spectrum and
their corresponding assignment (bond, compound type and functional groups) were
characteristic for diclofenac sodium as mentioned below.
One of the most effective screening methods is the thin-layer chromatography
(TLC), simplest of all the widely used chromatographic methods to perform. The TLC
procedure was included to test the purity and for characterization of NSAIDs. Unlike in a
conventional method, Camag HPTLC system offers the advantages of automatic
application under the pressure of nitrogen gas and scanning in situ, where the conditions
can be more easily controlled. Beside, several samples can be run simultaneously using a
small quantity of mobile phase and the substances are permanently stored on the plate.
The mobile phase consisting of methanol: toluene (1:1 v/v) for aspirin and isopropyl
alcohol: n-hexane (4.9: 5.1 v/v) for other NSAIDs gave good resolution and sharp peaks
(Fig. 22 and 23). Also, the spots were compact and not diffused (Plate 1). It was observed
that pre-washing of TLC plates with methanol followed by drying and pre-saturation of
TLC chamber with mobile phase for 10 min ensured good reproducibility for peak shapes
of drugs. The chromatogramn in Fig. 22 and 23 outlines a single prominent peak in each
track represented by NSAIDs which is indicative of presence of drug or absence of
impurities. The densitogram obtained for NSAIDs is shown in the Plate 2.
The uv-absorbance spectra for NSAIDs diclofenac, paracetamol, ketoprofen,
celecoxib, nimesulide, meloxicam and aspirin are shown in Fig. 24, 25, 26, 27, 28, 29,
and 30, respectively. The maximum absorption wavelength for diclofenac, paracetamol,
ketoprofen, celecoxib, nimesulide, meloxicam and aspirin was recorded at 281, 246, 258,
307, 361 and 225, respectively.
82
Fig. 22: TLC chromatogram of NSAIDs
Fig. 23: TLC chromatogram of aspirin
83
Plate 1: Spotted plate post Plate 2: Densitogram of meloxicam, ketoprofen, diclofenac, paracetamol, nimesulide development under TLC visualizer. and celecoxib
84
Fig 24:HPTLC-UV absorption spectra of diclofenac Fig 25: HPTLC-UV absorption spectra of paracetamol
Fig 26: HPTLC-UV absorption spectra of ketoprofen Fig 27: HPTLC-UV absorption spectra of celecoxib
200 250 300 350 4000
25
50
75
100 281
Wavelength in nm
Abs
orpt
ion
Uni
t
200 250 300 350 4000
25
50
75
100 246
Wavelength in nm
Abs
orpt
ion
Uni
t
200 250 300 350 4000
25
50
75
100 263
Wavelength in nm
Abs
orpt
ion
Uni
t
200 250 300 350 4000
25
50
75
100 258
Wavelength in nm
Abs
orpt
ion
Uni
t
85
Fig 28: HPTLC-UV absorption spectra of nimesulide Fig 29: HPTLC-UV absorption spectra of meloxicam Fig 30: HPTLC-UV absorption spectra of aspirin
200 250 300 350 400 450 5000
25
50
75
100 307
Wavelength in nm
Abs
orpt
ion
Uni
t
200 250 300 350 400 4500
25
50
75
100 361
Wavelength in nm
Abs
orpt
ion
Uni
t
200 250 300 350 4000
25
50
75
100
225
Wavelength in nm
Abs
orpt
ion
Uni
t
86
4.4 Clinical signs of toxicity
In the present study, the diclofenac received birds showed clinical manifestation
such as anorexia, dullness, ruffled feathers, disinclination to move within the cage,
lethargy, depression, recumbence (Plate 3), shrunken eyes and occult blood in the faecal
droppings. All these clinical signs were observed on day 2 onwards and was continued to
be so until the end of experiment.
The birds which had received aspirin and paracetamol showed clinical signs of
toxicity such as dullness, ruffled feathers and disinclination to move. These clinical signs
were observed 2 to 3 h after daily dosing. But, over a period of time (6-7 h after dosing),
the observed clinical signs subsided indicating the observed clinical signs of toxicity were
transitory. In addition, these birds showed watery droppings accompanied with blood
mixed mucous. On the contrary to the diclofenac group, aspirin and paracetamol received
birds did not show signs of anorexia and in fact these birds consumed feed very well.
The birds which received ketoprofen, nimesulide, meloxicam and celecoxib
showed dullness and occasionally mucous mixed watery droppings otherwise these birds
did not show any clinical signs of toxicity.
4.5 Mortality
The diclofenac (Group II) received birds showed mortality on day 3, 4 and 5. Four
out of 6 birds died during the experimental period, whereas, no mortality was observed in
the birds, which received aspirin, paracetamol, nimesulide, ketoprofen, meloxicam and
celecoxib.
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4.6 Hematology Parameters
4.6.1 RBC count (×106/l)
The RBC count (×106/l) in Group II birds administered with diclofenac (2.5
mg/kg, PO) on days 1, 2, 3, 4, 5 and 6 was 2.91±0.15, 2.70±0.20, 2.15±0.12, 2.04±0.14
1.84±0.12 and 1.68±0.21, respectively. Except for day 1 and 2, RBC count for all other
days (3, 4, 5 and 6) was significantly (P<0.01) lower compared to control group values of
2.80±0.10, 2.80±0.08, 2.78±0.09 and 2.74±0.10, respectively (Table 2 and Fig. 31).
The RBC count (×106/l) in Group III birds administered with aspirin (10 mg/kg,
PO) on days 1, 2, 3, 4, 5 and 6 was 2.91±0.15, 2.70±0.20, 2.15±0.12, 2.04±0.14,
1.84±0.12 and 1.68±0.21, respectively. The RBC count on day 4, 5 and 6 was
significantly (P<0.01) lower compared to control group value of 2.80±0.08, 2.78±0.09
and 2.74±0.10, respectively (Table 2 and Fig. 31).
The RBC count (×106/l) in Group V birds administered with nimesulide (2
mg/kg, PO) on days 1, 2, 3, 4, 5 and 6 was 2.94±0.05, 2.86±0.04, 2.74±0.15, 2.60±0.09,
2.25±0.09 and 2.20±0.20, respectively. The blood RBC count on day 5 and 6 was
significantly (P<0.05) lower compared to control group value of 2.78±0.09 and
2.74±0.10, respectively (Table 2 and Fig. 31).
Other treated groups did not show any significant (P>0.05) alterations in the
values of RBC count compared to the control group values.
88 88
Table 2: Effect of oral administration of NSAIDs on RBC count (106/l) in broiler chickens
Values are Mean±SE; For each group n=6 unless otherwise mentioned *P<0.05, *P<0.01, ***P<0.001 in relation to control.
RBC count (106/l) Groups
Day
1 2 3 4 5 6
Group I (Control) 2.94±0.05 2.84±0.06 2.80±0.10 2.80±0.08 2.78±0.09 2.74±0.10
Post mortem examination of the birds treated with diclofenac revealed congested
musculature along with deposition of chalky white urates on the visceral organs (Plate 4).
The heart and pericardium showed diffuse deposition of chalky white material (Plate 5).
The liver was congested and friable with varying degrees of urate deposition on the
surface of liver (Plate 6). Kidneys were congested and considerably enlarged, bulging out
of the renal fossa exhibiting prominent lobulation. Multifocal chalky white urate crystals
were noticed on surface of the kidneys (Plate 7). Peticheal hemorrhages were observed on
proventricular mucosa (Plate 8) and on ceca (Plate 9). These birds also showed deposition
of chalky white urate crystals on condyles of hock joint (Plate 10).
Post mortem examination of the birds administered with aspirin revealed enlarged
papillae accompanied with varying degrees of erosions on the mucosa of proventriculus
(Plate 11). The intestine showed grossly severe congestion and hemorrhages on the
serosal surface (Plate 12). All other organs were apparently normal.
The birds administered with paracetamol grossly revealed presence of erosions or
formation of crater on the mucosa of proventriculus (Plate 13). Intestine showed severe
congestion (Plate 14). All other organs were apparently normal.
Post mortem examination of the birds administered with nimesulide revealed
slight erosions on the mucosa of proventriculus and intestine. All other organs were
apparently normal.
Other treated groups did not show gross morphological changes in any of the
organs examined.
133
Plate 3: Group II- birds (in the upper slot) showing anorexia, Plate 4: Group II- bird showing congested musculature with dullness, lethargy and recumbence. deposition of chalky white urates on the visceral organs. Plate 5: Group II- bird showing diffused urate Plate 6: Group II – pericardium of heart showing considerable
deposition on the surface of liver and pericardium. thickening with diffuse deposition of chalky white material.
134
Plate 7: Group II- bird showing peticheal hemorrhages in Plate 8: Group II- bird showing peticheal hemorrhages the proventricular mucosa. on ceca. Plate 9: Group II- bird showing enlarged kidneys with Plate 10: Group II- bird showing chalky white crystals prominent lobulation and deposition of urate crystals. on condyles of hock joint.
135
Plate 11: Group III- bird showing enlarged papillae and Plate 12: Group III- bird showing severe congestion and
erosions on the mucosa of proventriculus. hemorrhages on the serosal surface of intestine. Plate 13: Group IV- bird showing erosions on the mucosa Plate 14: Group IV- bird showing congestion of intestine.
of proventriculus.
136
4.9.2 Histopathology
On microscopic examination of diclofenac treated group; proventriculus section
showed degeneration and necrosis of villus epithelium, hemorrhages in the submucosa
and lamina propria, epithelial hyalinization occompanied with infiltration of
inflammatory cells (Plate 15). DeGalantha’s stained section of proventriculus tissue
showed deposition of uric acid on the serosal surface of the proventriculus (Plate 16).
Section of intestine showed shortening of villi, hemorrhage, degeneration and
necrosis of villus epithelium with infiltration of lymphoid cells in the lamina propria
(Plate 17 & 18).
Kidney section showed vascular and degenerative changes along with urate
deposition. Focal or multifocal areas of tubular epithelial degeneration and necrosis
accompanied with inflammatory changes (Plate 19). Tubular atrophy and presence of
proteinaceous material along with vacular degeneration was also observed in tubular
epithelial cells of some tubules. Black colour stained uric acid crystals were prominently
observed in kidney tubules in DeGalantha’s stain (Plate 20).
The liver section showed congestion of sinusoids and vessels. Hepatocyte
degeneration and necrosis accompanied with infiltrations of inflammatory cells (Plate
21). Deposition of black stained uric acid crystals in rosette pattern with occasional focal
irregular black spots were significantly observed in the DeGalantha’s stained sections of
liver (Plate 22).
Kidney sections under electron microscope showed dilatation of glomerular tufts
with RBCs, presence of electron dense granular area at proximal portion of glomerular
137
tufts (Plate 23). Tubular epithelial vaculation with intact nucleus, granular cytoplasm and
absence of endoplasmic reticulum (Plate 24).
Liver sections under electron microscope showed distortion of hepatocytes,
dilatation of sinusoidal areas and vaculation of cytoplasam (Plate 25). Degenerating
nucleus with hazy nuclear boundries, presence of fat globules and electron dense material
in cytoplasam was observed (Plate 26).
The section of heart showed congestion, hemorrhage, sub-epicardial edema and
urate deposition with infiltration of inflammatory cells in between cardiac muscle fibers
(Plate 27). In addition to these, infiltration of inflammatory cells in the pericardium,
congestion and oedema of myocardium was also noticed (Plate 29). Focal myocarditis
characterized by loss of cross striations, fragmentation of myocardial fibers with
infiltration of inflammatory cells were noticed occasionally. Deposition of black stained
uric acid crystals in rosette pattern with focal irregular black spots were significantly
observed in the DeGalantha’s stained heart sections (Plate 28 & 30).
On microscopic examination of aspirin treated group, proventriculus section
showed hemorrhages in the submucosa or lamina propria, hyalinization of villus
epithelium occompanied with infiltration of inflammatory cells (Plate 31).
Section of intestine showed erosion and desquamation of of villus epithelium into
the lumen with infiltration of inflammatory cells (Plate 32).
Microscopic examination of paracetamol treated group, proventriculus section
showed villus degeneration and infiltration of inflammatory cells (Plate 33).
138
Plate 15: Group II- proventriculus section showing Plate 16: Group II- black coloured uric acid on the serosal
degeneration and necrosis of villus epithelium. (H & E 100X) surface of the proventriculus (DeGalantha’s 100X) Plate 17: Group II-section of intestine showing shortening Plate 18: Group II-section of intestine showing hemorhage, of villi (H & E 100X) degeneration and necrosis of villus epithelium (H & E 100X)
139
Plate 19:Group II- kidney sections showing tubular Plate 20: Group II-kidney sections showing black stained uric
necrosis with inflammatory changes (H & E 200X) acid crystals in kidney tubules (DeGalantha’s 100X) Plate 21: Group II- liver section showing hepatocyte Plate 22: Group II-liver section showing deposition of black degeneration and necrosis with inflammatory cells (H & E 200X) stained uric acid crystals in rosette pattern (DeGalantha’s 200X)
140
Plate 23: Group II-Kidney sections under electron microscope showing dilatation of glomerular tufts with RBCs, presence of electron dense granular area at proximal portion of glomerular tufts (8950X)
Plate 24: Group II-Kidney section showing tubular epithelial vaculation with intact nucleus, granular cytoplasm and absence of endoplasmic reticulum (4475X)
141
Plate 25: Group II-Liver sections showing distortion of hepatocytes, presence of fat globule, dilatation of sinusoidal areas and vaculation of cytoplasam (4475X)
Plate 26: Group II-Liver section showing degenerating nucleus with hazy nuclear boundries, presence of electron dense material in cytoplasam (4320X)
142
Section of the intestine showed hyperplastic changes in the crypts, erosion and
desquamation of villus epithelium into the lumen with infiltration of inflammatory cells
(Plate 34).
Section of the liver showed slightly swollen hepatocyte with granular cytoplasm
and mild congestion of vessels as well as sinusoids. Periductular hepatocytes
degeneration and necrosis with massive infiltration of inflammatory cells were also
noticed (Plate 35).
Microscopic examination of nimesulide treated group; proventriculus section
showed increased secretory activity, desquamation and degeneration of villus epithelium
and infiltration of inflammatory cells in the sub mucosa. (Plate 36).
Section of the intestine showed hyperplastic changes in crypts with increased
goblet cell activity and infiltration of inflammatory cells (Plate 37).
Section of the liver showed degeneration and necrosis of hepatocytes around the
bile duct, hyperplasia of bile duct epithelium accompanied with massive infiltration of
inflammatory cells (Plate 38).
However, no apparent histopathological lesions were observed in any of the tissue
section of control birds and in birds treated with NSAIDs such as ketoprofen, meloxicam
and celecoxib.
143
Plate 27:Group II- heart showing sub-epicardial edema with Plate 28:Group II-heart showing deposition of black stained infiltration of inflammatory cells (H & E 100X) uric acid crystals (DeGalantha’s 100X) Plate 29: Group II- heart showing infiltration of inflammatory Plate 30: Group II-heart showing deposition of black stained
cells in the pericardium (H & E 100X) uric acid crystals on pericardium (DeGalantha’s 100X)
144
Plate 31: Group III- proventriculus section showing hemorrhages Plate 32: Group III- section of intestine showing erosion of
in the lamina propria with inflammatory cells (H & E 100X) villus epithelium with inflammatory cells (H & E 100X) Plate 33: Group IV- proventriculus section showing Plate 34: Group IV- intestine showing hyperplastic changes in
hemorrhage and villus degeneration (H & E 100X) crypts, erosion of villi with inflammatory cells (H & E 100X)
145
Plate 35: Group IV-section of liver showing periductular Plate 36: Group V- proventriculus section showing increased
hepatocyte degeneration and necrosis (H & E 100X) secretion with hemorrhage in the sub mucosa (H & E 200X) Plate 37: Group V- section of intestine showing hyper plastic Plate 38: Group V- section of liver showing degeneration and
changes in crypts with increased goblet cell activity(H & E 100X) necrosis of hepatocytes around the bile duct (H & E 100X)