Animals And Materials Used For Present Study Biology Essay

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The animals used in present study such as swiss albino mice weight (20-25 gm) were kept in colony cages at 2520C,relative humidity of 45-55% under 12h light and dark cycle. All animals were feed with standard animal feed, water and labium. The test compounds were administered orally using intra gastric tube in the form of suspension using 1%CMC as suspending agent. The experimental dose was selected between minimum effective dose and maximum non lethal non lethal dose. All animal experimentation was performed according to protocols and recommendation of animal ethics committee.

IAEC Reference No: IAEC/XXX/13CLBMCP/2010

ANOVA followed by donnet test was performed to ascertain the significance of exhibited analgesic and antipsychotic activities of synthesized compounds.

Following activities were carried out to the synthesized compounds by bellow mentioned methods.

Acute oral toxicity (acute toxic class method in mice)

Antipsychotic activity (actophotometer Method)

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Analgesic activity (Tail immersion Method)

5.1 Evaluation of Acute Oral Toxicity

Introduction

Acute oral toxicity defines to those adverse effects occurring following oral administration of a single dose of a substances or multipal dose given within 24 h. The various methods used to evaluate the acute oral toxicity as follows.

Fixed dose procedure (OECD guideline-420)

Acute toxic class method (OECD guideline -423)

Up and down procedure (OECD guideline -425)

OECD guideline -42363

a) OECD Guidelines for the Testing of Chemicals are periodically reviewed in the light of scientific progress or changing assessment practices. The original Guideline 423 was adopted in March 1996 as the second alternative to the conventional acute toxicity test, described in Test Guideline 401.

i) International agreement has been reached on harmonized LD50 cut-off values for the classification of chemical substances, which differ from the cut-offs recommended in the 1996 version of the Guideline. ii) Testing in single sex (generally females) is now considered sufficient.

b) The acute toxic class method set out in this Guideline is a stepwise procedure with the use of 3 animals of a single sex per step. Depending on the mortality and/or the moribity status of the animals, and on average 2-4 steps may be necessary to judge the acute toxicity of the test substance. This procedure is reproducible, uses less number of animals and also able to rank substances in a similar manner as that of the other acute toxicity test guidelines. (Test Guidelines 420 and 425). It is based on biometric evaluations with fixed doses, which are adequately separated so that it should be enable for a substance to be ranked for classification purposes and hazard assessment. In 1996 The method was adopted and it was extensively validated in vivo against LD50 data obtained from the literature, both nationally and internationally.

c) Guidance on the selection of the most appropriate test method for a given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing.

This Guidance Document also contains additional information on the conduct and interpretation of Test Guideline 423.

Experimental Protocol (acute toxic class method in mice)

In the present study acute oral toxicity of the synthesized compounds were performed by acute toxic class method. In this method the toxicity of synthesized compounds were tested using astep wise procedure, each step using three mice of single sex (female). The mice were fasted prior to dosing (food but water should be with held) for three to four hours. Following the period of fasting the animal should be weighted and synthesized compound were administered orally at a dose 2000mgkg body weight. Animals were observed individually after dosing at least once during the first 30min;periodically during the first 24 h with special attention giving during the first 4 h and daily there after, for total of 14 days. As know mortality observed with the above dose. Test compound dose reduced by specific intervals. The mortality not observed at the dose 1000 mgKg. So a series of doses 100 and 200 mgKg body weight were selected for there pharmacological evaluation. The test procedure with the starting dose of 2000mgKg body weight as per OECD-423 guidelines was shown as follows.

Flow chart for Acute toxic class method (OECD guideline 423) starting dose of 2000 mg Kg body weight p.o

Table: 5.1

Dose Selection by acute toxic method

Sr. No.

Treatment group

Dose mg/kg

Sign of toxicity

Onset of toxicity

Duration

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1

4a

100

200

No

No

14 days

2

4b

100

200

No

No

14 days

3

4c

100

200

No

No

14 days

4

4d

100

200

No

No

14 days

5

4e

100

200

No

No

14 days

6

4f

100

200

No

No

14 days

Results and discussion

Acute oral toxicity studies were performed according to the OECD guideline 423 method.

This method has been designed to evaluate the substance at the fixed doses and provide information both for hazard assessment and substance to be ranked for hazard classification purposes.

The synthesized compounds were administered initially at a dose of 2000mg/kg b.w n 1% CMC (p.o) and observed 14 days mortality due to acute toxicity.

Careful observation were made at at least twice a day for the effect on CNS, ANS, motor activity, salivation, skin coloration and other general signs of toxicity were also observed and recorded.

Since no sign of toxicity observed at 2000 mg/kg b.w. to the group of animals, the LD50 value of the title compounds (4a-4f) expected to exceed 2000 mg/kg b. w. and represented as class 5 (2000 mg/kg < LD50 < 2500 mg/kg)

From the toxicity studies the data revealed that all the synthesized compounds proved to be non toxic at tested dose levels and well tolerated by the experimental animals as there LD50 cut of values > 2000 mg/kg b. w.

5.2 Evaluation of Analgesic Activity

Introduction64

Definition: Analgesics are substances which relieve or decrease pain sensation by increasing threshold to painful stimuli without causing loss of consciousness.

Pain is a symptom of many diseases requiring treatment with analgesics. Many approaches have been used to differentiate the various actions of strong analgesics by developing animal models not only for analgesic activity. Analgesic effects in animals are comparable with the therapeutic effects in man. Painful stimuli can consist of direct stimulation of the sensory nerves or stimulation of pain receptors by various means such as heat or pressure. The role of endogenous peptides such as enkephalins and endorphins gives more insight into brain processes and the action of central analgesics.

Mechanism of action65

Cellular action

Action site is G-protein coupled opoid receptors. The inhibition of adenyly cyclase. Reducing the intra cellucelar c-AMP content affecting protein phosphorylation pathways.

Exert effects on ion channels through a direct G-protein coupling to the channel. Promote the opening of potassium channels and inhibit the opening of voltage-gated calcium channels.

These membrane effects reduce both neuronal excitability (because the increased K+ conductance causes hyperpolarisation of the membrane) and transmitter release (due to inhibition of Ca2+ entry).

The overall effect is therefore inhibitory at the cellular level. Increase activity in some neuronal pathways by suppressing the firing of inhibitory interneurons.

The other analgesic mechanism produces by effect on nociceptive pathway.

Evaluation methods

The various methods used to evaluate anticonvulsant activity are as follows,

Hoppners tail clip method

Radiant heat method

Hot plate method

Tail immersion method

Electric stimulation method

Grid shock test

Formalin test in rats

Experimental protocol (Tail immersion method) 66

The analgesic activity was determined by tail-immersion method. Swiss mice (n=6) of either sex selected by random sampling technique was used for the study. Diazepam at the dose of 10 mg/kg (i.p.) was administered as standard drug for comparison. The test compounds at 2 dose levels (100 and 200 mg/kg) were administered orally. The animals were held in position by a suitable restrainer with the tail extending out and the tail (up to 5 cm) was taken dipped in a beaker of water maintained at 55 + 0.50C. The time in sec taken to withdraw the tail clearly out of water was taken as the reaction time. The first reading (0 min) was taken immediately after the administration of the test compound and subsequent reaction time was recorded at 0,15,30,60 and120 min after the administration of compounds. A cut off point of 15 sec was observed to prevent the tail damage. The percentage analgesic activity was calculated using the following formula and the results are presented in table 5.3.3.

PAA = [B-A / B-100]

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Where,

A is the reaction time (in sec) after treatment

B is the reaction time (in sec) before treatment

PAA is the percentage analgesic activity

EVALUATION OF ANALGESIC ACTIVITY

Introduction

Analgesics are substances which relieve or decrease pain sensation by increasing threshold to painful stimuli without causing loss of consciousness.

Evaluation methods

Analgesics can be evaluated in three different ways as follows:

Prevention or relief of artificially induced pain in experimental animals.

Relief of experimental pain in human volunteers, and

Relief of pathological or incisional pain in patients.

In human beings, the analgesics are evaluated either against experimentally induced pain (radiant heat, ischemia induced with sphygmomanometer cuff, intraperitonial bradykinin ) or against ' endogenous' pain ( post-puerperal pain, post operative pain and pain due to malignancy).

In animals, painful reactions can be artificially induced by the any one following methods:

Thermal method ( radiant heat as a source of pain)

Chemical method (irritants such as acetic acid and bradykinin)

Physical pressure method (Tail compression)

Experimental protocol (Tail immersion method in rats)

The analgesic activity was determined by tail-immersion method64, 65, 66. Rat (n=6) of either sex selected by random sampling technique was used for the study. Pentazocin at the dose of 10mg/kg (i.p.) was administered as standard drug for comparison. The test compounds at dose levels (200 and 400 mg/kg) were administered orally. The animals were held in position by a suitable restrainer with the tail extending out and the tail ( up to 5 cm ) was taken dipped in a beaker of water maintained at 55±0.5o C. the time in sec taken to withdraw the tail clearly out of water was taken as the reaction time. The first reading was taken immediately after the administration of the test compound and subsequent reaction time was recorded at 30, 60, 120, 180 min after the administration of compounds. A cut off point of 15 sec was observed to prevent the tail damage. The percentage analgesic activity was calculated using the following formula and the results are represented in table.

PAA = ((T2-T1)/T2) -100

Where,

T1 is the reaction time (in sec) before treatment

T2 is the reaction time (in sec) after treatment

PAA is the percentage analgesic activity.

Table. 5.2 ANALGESIC ACTIVITY BY TAIL IMMERSION METHOD THE SYNTHESIZED COMPOUNDS

Each value is mean pain reaction time (in sec)SEM using 6 animals in each groups. Significant differences with respect to 0 min time was evaluated by

COMPOUDS

DOSE

(mg/k)

0 min

15 min

30 min

60 min

120 min

MEAN + SEM

MEAN + SEM

%

MEAN + SEM

%

MEAN + SEM

%

MEAN + SEM

%

4a

100

4.660.20

4.710.23

16.29

6.600.20

18.70

9.340.24

48.48

5.970.38

29.78

200

4.900.22

5.170.20

27.76

7.670.31

37.94

11.570.27

83.94

7.540.25

63.91

4b

100

4.780.24

5.250.21*

29.62

8.090.20*

45.50

10.350.23*

64.54

7.410.33*

61.08

200

5.300.23

5.670.26*

40.00

8.500.28*

52.83

11.770.21*

87.12

8.000.22*

73.91

4c

100

4.910.24

5.150.22*

27.16

7.840.27*

41.00

10.120.27*

60.89

6.930.25*

50.65

200

5.340.16

5.630.37*

30.86

8.240.26*

48.20

11.740.19*

86.64

7.90.22*

71.73

4d

100

4.690.20

4.920.21

21.48

7.610.22

36.87

9.350.25

48.64

6.020.36

30.86

200

4.970.21

5.560.25

42.71

7.790.32

40.10

11.590.27

84.26

7.600.28

65.21

4e

100

5.040.23

5.780.23**

37.28

8.890.21**

59.89

11.210.26**

78.21

7.520.34**

63.47

200

5.370.10

6.400.19**

58.02

9.080.20**

63.30

11.830.27**

88.07

8.670.25**

88.47

4f

100

4.590.23

4.690.24

15.80

6.580.21

18.34

8.820.26

40.22

5.880.32

27.82

200

5.840.24

5.020.31

23.95

7.650.29

37.89

11.230.25

78.53

7.490.10

62.77

Control

-

3.570.15

4.050.46

-

5.560.78

-

6.290.51

-

4.600.15

-

Standard

Dipyrone

19.5

2.370.36

5.30.20

30.86

6.830.28

22.84

7.410.32

17.80

6.730.25

46.08

(ANOVA) Dunnet's t test *P< 0.05, **P<0.01, NS (Non significant), % (Percentage Analgesic activity).

5.4. Results and Discussion

Synthesized compounds were evaluated for analgesic activity by tail-immersion method. The activity was studied at 100 and 200 mg/kg b.w. (p.o) and effect was measured at the time interval of 15, 30, 60 and 120 min.

From the data shown in table 5.3 observations made as follows

Most of the synthesized compounds showed significant analgesic activity and also the graded dose response was observed.

Highest analgesic activity was observed at 60 min for all the compounds (100 and 200 mg/kg).

When compared with standard drug (of dipyrone (19.5 mg/kg). i.p) the compounds 4e exhibited comparable analgesic activity at 100 and 200 mg/kg b.w.

Compounds 4b, 4c. Exhibited moderate analgesic activity.

Among the compounds synthesized compound 4f exhibited lowest analgesic activity.

The order of analgesic activity of the synthesized compounds was shown

4e > 4b > 4c > 4d > 4a >4f

Compounds 4e exhibited a statistically significant analgesic activity in the tail immersion method test in the mice, being however less active than the equitoxic dose of dipyrone (19.5 mg/kg).

It appears reasonable to suggest that the presence of o-methoxy substituted phenyl group in 1, 5 benzodiazepine exhibited better activity.