Pharmacological Screening Materials And Method Biology Essay

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Male albino rats of Wistar strain weighing about 150 - 200 gm were used for the study. The animals were got from Nandha College of Pharmacy and Research Institute, Erode, and were approved by (IAEC) Ethical Committee and approval no. is

8.1.2 Maintenance of animals

The animal house was well ventilated and animals had 20-25 ± 2 °C. The animals were housed in large spacious hygienic cages during the course of the experimental period. The animals were fed with rat pellets feed supplied by M/s Hindustan Lever Limited, Bangalore, India and filtered water ad libitum. Animals described as fasted were deprived of food for 16 hr but allowed free access to water. The place where the experiments were conducted was kept very hygienic by cleaning with antiseptic solution.

8.2 Anti-diabetic activity

8.2.1 Experimental Grouping of Animals

The experimental rats were divided into five groups of five animals in each group. The animals were fasted overnight before the experimental schedule began but allowed free access to tap water.

Group I: The rats received 2 ml Normal Saline. These animals serve as normal controls.

Group II: The rats were made diabetic by an intraperitonial injection of Single dose of (150 mg/kg body weight) Alloxan monohydrate in normal saline and served as diabetic control.

Group III: The diabetic rat given Glibenclimide, 0.5 mg/kg, i.p. once a day

Group IV: The diabetic rat given Scutia myrtina methanolic extract (SCME) 400 mg/kg orally once a day

8.2 .2 Induction of diabetes

Alloxan monohydrate induced diabetes mellitus was induced in the normoglycaemic male albino rats. Animals were allowed to fast 24 hrs and were injected intraperitonially with freshly prepared alloxan monohydrate in Sterile Normal Saline in dose of 150 mg/kg body weight. Blood glucose was measured after 24 hr of alloxanisation and it was confirmed that the given dose was sufficient for inducing diabetes in the animals. The animals were maintained in the diabetic state over a period of 21 days. Rats showing fasting blood glucose levels (>250 mg/dl) were selected for the study. Mortality rate of the animals were Nil.

8.2.3 Chemistry of Alloxan and its derivatives

Alloxan derivatives were much more toxic than alloxan itself. The toxicity increased with the lipophilicity. Dialuric acid, the reduction product of alloxan was more toxic than alloxan. Alloxantin obtained by reducing alloxan (one molecule) with half a molecule of hydrogen sulfide was even more toxic than alloxan. Alloxantin dissociates into Alloxan and Dialuric Acid but the toxicity of Alloxantin was greater than that of an equimolar solution of the dissociation product.

Alloxan

8.2.4 Collection of Blood Sample

A small amount of blood without sacrificing the animals was collected from the tail vein by snipping off the tip of the tail.

8.2.5 Determination of Blood Glucose

The blood from the tail vein was used to determine the glucose level. As bleeding starts, the animals were held close to the One Touch select simple blood glucose test strip and allowed the drop to fall on the strip. The One Touch select simple Glucometer was switched on and the test was allowed to react with the blood. After few seconds the blood glucose level was displayed on the screen.

8.2.6 Collection of Blood and Centrifugation

The drugs were dissolved in CMC ant it was administered orally via a standard orogastric cannula, Anti-diabetic activity in diabetic rats was assessed by fall in fasting blood glucose level. Blood samples were collected from the tip of the tail on 0th, 7th, 14th and 21st day. By without sacrificing the animals, from the tail vein by snipping off the tip of the tail and blood glucose were checked by one Touch select simple Blood glucose monitoring system.

8.2.7 ESTIMATION OF BIOCHEMICAL PARAMETERS IN BLOOD SERUM

After the experimental regimen, the bloods were collected through the retro-orbital puncture of eye of animals under mild chloroform anesthesia in Eppendorff's tube (1 ml) containing 50μl of anticoagulant (10% trisodium citrate) and serum was separated by centrifugation at 3000rpm for 15 min. The biochemical parameters Cholesterol, Triglycerides, Serum protein, SGPT and SGOT are determined by using the commercial kit available (Ecoline, Manufactured by Merck specialties, Private limited, Ambernath).52,53

8.3 CNS Stimulant activity

8.3.1 Locomotor activity using actophotometer

Albino rats of either sex (20 - 25 g) were randomly divided into three groups of six animals. The rats were placed individually inside the chamber of actophotometer for 10 min and basal activity score was noted. Group I was treated with vehicle (0.5% sod. CMC) and standard drug caffeine (10 mg/kg, i.p.) administered to group II. The animals of the group III were treated with Scutia myrtina extract (400 mg/kg) respectively and after 30 min of mice are placed again in actophotometer for 10 min and the activity was monitored. Percentage increases in activities were calculated.54,55

8.3.2 Motor coordination using Rota-rod

Albino rats of either sex (20 - 25 g) were randomly divided into three groups of six animals. Rota rod apparatus (Dolphin make) is a four panel techno device with timer. Animals (4 at a time) were placed on rod rotating at 20-25 rpm speed. Group I was treated with vehicle (0.5% sod. CMC) and standard drug caffeine (10 mg/kg, i.p.) administered to group II. The animals of the group III were treated with Scutia myrtina extract (400 mg/kg) respectively. The fall off time was recorded in all the groups before and 30 min after drug administration.56,57

8.3.3 Elevated plus maze (EPM)

This test has been widely validated to measure anxiety in rodents. This apparatus

was made of Plexiglas and consisted of two open arms (30cm Ã- 5cm) and two closed

arms (30cm Ã- 5cm)with 25cm walls. The arms extended from a central platform (5cm Ã-5cm). The maze was elevated 38.5cm from the room floor. Albino rats of either sex (20 - 25 g) were randomly divided into three groups of six animals. Group I was treated with vehicle (0.5% sod. CMC) and standard drug caffeine (10 mg/kg, i.p.) administered to group II. The animals of the group III were treated with Scutia myrtina extract (400 mg/kg) respectively. Each animal was placed at the center of the maze, facing one of the enclosed arms. Number of entries and the time spent in enclosed and open arms was recorded for 5 min test. Entry into an arm was defined as the animal placing all four paws onto the arm. All tests were taped by a video camera. After each test, the maze was carefully cleaned up with a wet tissue paper (10% ethanol solution).58, 59

8.3.4 Exploratory behavior using Y maze

8.3.4. a Runaway test

This test is used to study the effect of a drug on spontaneous activity and motor coordination. Albino rats of either sex (20 - 25 g) were randomly divided into three groups of six animals. Group I was treated with vehicle (0.5% sod. CMC) and standard drug caffeine (10 mg/kg, i.p.) administered to group II. The animals of the group III were treated with Scutia myrtina extract (400 mg/kg) respectively. The mice were placed individually in a symmetrical Y-shaped runway (33 cm x 38 cm x 13 cm) for 3 min and the number of the maze with all 4 ft (an 'entry') were counted.16, 60

8.3.5 Modified forced swimming test

Albino rats of either sex (20 - 25 g) were randomly divided into three groups of six animals. Rats were placed individually in a transparent glass cylinder (12 cm in diameter, height 25 cm), which was filled with water to a height of 15 cm. Two swim sessions were conducted. An initial 15-min pre-test followed 24 hr later by a 6 min test. In the pre test session, the mice which have not yet treated were forced to swim in a glass cylinder for 15 min. In the second session, each mouse received a respective dose of sample 1 hour prior to test, and placed in the cylinders again for 6 min. The following behaviors were recorded during the last 4 min.

1. Immobility: floating in water without swimming.

2. Swimming: active movements of extremities and circling in the container.

3. Climbing: active movements of forelimbs on the container wall.

Group I was treated with vehicle (0.5% sod. CMC) and standard drug caffeine (10 mg/kg, i.p.) administered to group II. The animals of the group III were treated with Scutia myrtina extract (400 mg/kg) respectively.61,62

8.4 STATISTICAL EVALUATION

Statistical evaluation was done using one way analysis of variance (ANOVA) followed by Dunnet' T-test. Statistical significance was set as p<0.001, p<0.01, p<0.05.

Table no 8.1 Effect of methanolic extract of Scutia myrtina on blood glucose level in Alloxan induced Diabetic rats

Group

Treatment

Dose

0th day

7th day

14th day

21st day

I

Normal control

2 ml normal saline

111 ± 3.05**

110.5 ± 2.9**

109.1 ± 2.7**

110 ± 1.8**

II

Diabetic control

150 mg/kg Alloxan & 2ml NS

274.8 ± 3.4

277.3 ± 3.8

271.8 ± 2.9

266.1 ± 3.0

III

Diabetic rat give GLB

0.5 mg/kg

287.8 ± 3.2*

258.6 ± 1.8**

218 ± 3.3**

181.1 ± 3.2**

IV

Diabetic rat given SCME

400 mg/kg

289.6 ±2.6*

269.1 ± 3.0ns

213.8 ± 2.5**

184.5 ± 2.4**

Data represents mean ± S.D. (n=6), *p< 0.05 Significant as compared to alloxan control.

**p< 0.01 Significant as compared to alloxan control.

***p< 0.001 Significant as compared to alloxan control.

ns: non significant compared to normal control.

Table no 8.2 Effect of the methanolic extract of Scutia myrtina on biochemical parameters

Groups

Triglycerides (mg/dl)

Total protein (g/dl)

Cholesterol (mg/dl)

SGOT

(u/l)

SGPT

(u/l)

Normal control

(2 ml normal saline

95.3 ±0.8***

7.5 ± 0.1***

67.16 ± 1.4***

64 ± 1.29***

44.83 ± 1.35***

Diabetic control (150 mg/kg Alloxan & 2ml NS)

190 ± 1.9

4.5 ± 0.1

181.3 ± 2.4

125 ± 1.2

75 ± 1.39

Diabetic rat give GLB (0.5 mg/kg)

105.85 ± 1.3***

8.41 ±0.1***

76.5 ± 1.5***

64.6 ± 1.60***

45.66 ± 1.33***

Diabetic rat given SCME (400 mg/kg

114.4 ± 1.3***

5.5 ± 0.1***

84.16 ± 1.3***

83.5 ± 1.33***

45 ± 1.15***

Data represents mean ± S.D. (n=6).

*p< 0.05 Significant as compared to control.

**p< 0.01 Significant as compared to control.

***p< 0.001 Significant as compared to control.

ns: non significant compared to control.

Table no 8.3 Effect of methanolic extract of Scutia myrtina on locomotor activity

Group

Treatment

Dose

Mean score in 5 min

% increase in activity

Before drug administration

After drug administration

I

Control

0.5% sod. CMC

716±4.02

716.3±3.87

0.04

II

Caffeine (standard)

10 mg/kg

720.5±3.76

970.5±2.88***

34.69

III

SCME

400 mg/kg

711.5±5.01

854.6±1.33***

20.11

Data represents mean ± S.D. (n=6).

*p< 0.05 Significant as compared to control.

**p< 0.01 Significant as compared to control.

***p< 0.001 Significant as compared to control.

ns: non significant compared to normal control.

Table no 8.4 Effect of methanolic extract of Scutia myrtina on muscle coordination

Group

Treatment

Dose

Mean fall of time (sec)

Before drug administration

After drug administration

I

Control

0.5% sod. CMC

232.16±5.89

232.83±6.80

II

Caffeine (standard)

10 mg/kg

234±9.30

234.33±8.51

III

SCME

400 mg/kg

233.83±7.5

233.5±6.56

The extract and the standard had no significant effect on the mean times spent on the Rota rod.

Table no 8.5 Effect of methanolic extract of Scutia myrtina on Elevated plus maze

Group

Treatment

Dose

Time spent (sec)

No of entries on

Enclosed arm

Open arm

Enclosed arm

Open arm

I

Control

0.5% sod. CMC

8±0.44

4.16±0.47

238.16±3.58

61.8±3.58

II

Caffeine (standard)

10 mg/kg

5±0.73*

2.16±0.3**

283±2.3***

17±2.3***

III

SCME

400 mg/kg

7.16±0.7ns

2.6±0.33*

273.16±1.72***

26.8±1.7***

Data represents mean ± S.D. (n=6).

*p< 0.05 Significant as compared to control.

**p< 0.01 Significant as compared to control.

***p< 0.001 Significant as compared to control.

ns: non significant compared to normal control.

Table no 8.6 Effect of methanolic extract of Scutia myrtina on Y maze

Group

Treatment

Dose

No of entries after treatment (min)

30

60

90

120

I

Control

0.5% sod. CMC

11±0.36

11.1±0.4

10.8±0.4

11±0.36

II

Caffeine (standard)

10 mg/kg

18.3±0.66***

18.5±0.56***

18.8±0.47***

18.6±0.49***

III

SCME

400 mg/kg

15.6±0.49***

15.8±0.3***

16.5±0.42***

15.5±0.42***

Data represents mean ± S.D. (n=6).

*p< 0.05 Significant as compared to control.

**p< 0.01 Significant as compared to control.

***p< 0.001 Significant as compared to control.

ns: non significant compared to normal control.

Table no 8.7 Effect of methanolic extract of Scutia myrtina on immobility time, swimming and climbing in modified forced swimming

Group

Treatment

Dose

Immobility time (sec)

Swimming count in 6 min

Climbing count in 6 min

I

Control

Distilled water

203.16±6.14

37±0.85

6.5±0.7

II

Caffeine (standard)

10 mg/kg

85±3.65***

114±3.06***

33.16±2.18***

III

SCME

400 mg/kg

121.6±5.57***

59.16±1.13***

23±2.30***

Data represents mean ± S.D. (n=6).

*p< 0.05 Significant as compared to control.

**p< 0.01 Significant as compared to control.

***p< 0.001 Significant as compared to control.

ns: non significant compared to normal control.

8.8 RESULT

Table no 8.1 showed the levels of glucose in rats in different groups. The glucose level was significant (p<0.01) high in alloxan control rats compared with normal control. But the level of glucose was significant (p<0.05) decreased in diabetic rats treated with extract as compared with diabetic control rats. On repeated administration of the extract for 21 days, a significant decrease in the glucose level was observed in the diabetic rats as compared to diabetic control. There was no significant difference normal control and rats treated with only with extract.

It was evident from the table that untreated diabetic rats has elevated blood glucose levels and that the methanolic extract of whole plant were able to correct this metabolic deviation from the Diabetic Control significantly since there was no significant difference between Normal Control and Diabetic Control. So the whole plant extract has anti-diabetic activity.

Biochemical Parameters in experimental rats was depicted in Table no 8.2. In alloxan induced diabetic rats, there was significant (p<0.001) decrease in total protein when compared to Normal Control and the level was restored to nearly normal after the treatment. There was no significant difference between Normal Control and rats only treated with extract.

In alloxan induced diabetic rats, there was a significant (p<0.001) increase in Cholesterol, Triglycerides, SGOT and SGPT in serum compared to Normal Control. The plant extract used in the experimental study significantly (p<0.001) decreased the levels of Cholesterol, Triglycerides, SGOT and SGPT. This shows that the plant extract had favorable effect on lipid metabolism of diabetic rats.

Diabetes Mellitus is a multifactorial disease, which is characterized by hyperglycemia and lipoprotein abnormalities. Diabetes Mellitus leads to various metabolic aberrations in the animals viz. increased blood glucose, decreased protein content, increased cholesterol, Triglycerides, SGOT and SGPT.

The increased levels of transaminases (SGOT, SGPT) which are active in the absence of insulin because of the availability of amino acids in the blood of diabetics are responsible for the increased gluconeogenesis and ketogenesis observed in diabetes. In the present study, the whole plant extract significantly decreased the enzymatic activities. Hence restoration of the SGOT, SGPT to the normal levels may also indicate revival of insulin secretion to normal levels.

Caffeine, a mild stimulant is the most widely used psychoactive drug in the world. It increases norepinephrine secretion and enhances neural activity in numerous brain areas. Many of its effects are believed to occur by means of competitive antagonism at adenosine receptors. Tolerance occurs rapidly to the stimulating effects of caffeine, thus a mild withdrawal syndrome has been produced.

The methanolic extract produced significant (p<0.001) increase in locomotor activity when compared to the Normal Control. The methanolic extract showed 20.11% increase in activity where as the standard drug Caffeine showed 34.69% increase in activity (Table no 8.3).

The extract and the caffeine (standard) had no significant effect on the mean time spent on the Rota rod (Table no 8.4). The extract's CNS stimulant effect was further confirmed by its ability to maintain the animals on the Rota rod, thus indicating muscle co-ordination.

In the Elevated plus maze (Table no 8.5), the rats treated with caffeine and extract showed significant (p<0.01 & p<0.001) decrease in the no. of entries in open arm and the time spent in the open arm compared with the control. Caffeine and the plant extract showed stimulant and anxiogenic effect.

In Y-maze test, the animals treated with methanol extract of Scutia myrtina at the dose of 400 mg/kg and caffeine showed a Significant (p< 0.001) marked increase in the no. of entries compared with control (Table no 8.6).

In modified forced swim test, the animals treated with caffeine and plant extract showed significant (p<0.001) reduction in immobility time and increased the frequency of climbing and swimming (Table no 8.7).

The results obtained in this study indicate that the extract possesses CNS stimulant properties which probably act via competitive antagonism at adenosine receptors leading to increase in nor-epinephrine secretion and enhanced neural activity in numerous brain areas.

Fig 8.1 Effect of methanolic extract of Scutia myrtina on blood glucose level in Alloxan induced Diabetic rats

NC: Normal control DC: Diabetic control

STD: Glibenclamide SCME: methanolic extract of Scutia myrtina

Fig 8.2 Effect of the methanolic extract of Scutia myrtina on biochemical parameters

NC: Normal control DC: Diabetic control

STD: Glibenclamide SCME: methanolic extract of Scutia myrtina

Fig 8.3 Effect of methanolic extract of Scutia myrtina on locomotor activity

NC: Normal control

STD: Caffeine

SCME: methanolic extract of Scutia myrtina

Fig 8.4 Effect of methanolic extract of Scutia myrtina on muscle coordination

NC: Normal control

STD: Caffeine SCME: methanolic extract of Scutia myrtina

Fig 8.5 Effect of methanolic extract of Scutia myrtina on Elevated plus maze (time spent in arms)

NC: Normal control

STD: Caffeine

SCME: methanolic extract of Scutia myrtina

Fig 8.6 Effect of methanolic extract of Scutia myrtina on Elevated plus maze (no. of entries)

NC: Normal control

STD: Caffeine SCME: methanolic extract of Scutia myrtina

Fig 8.7 Effect of methanolic extract of Scutia myrtina on Y maze

D:\pjpreethi\thesis\y maze.bmp

NC: Normal control

STD: Caffeine

SCME: methanolic extract of Scutia myrtina

Fig 8.8 Effect of methanolic extract of Scutia myrtina on immobility time, swimming and climbing in modified forced swimming

NC: Normal control

STD: Caffeine SCME: methanolic extract of Scutia myrtina

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