Isolation Of Phytoconstituents By Column Chromatography Biology Essay

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Column chromatography is an isolation and purification technique used extensively by phyto-chemists to obtain pure samples of chemicals from natural sources. It is suitable for the physical separation of gram quantities of material.

Table 11: Column Chromatography conditions for all extracts of flowers of Quisqualis indica linn

Parameters

USM of flowers

Ethyl acetate extract of flowers

Ethyl acetate insoluble fraction of methanolic extract of flowers

Column

Glass

Dimension

Diameter: 3 cm, Length: 75 cm

Stationary phase

Silica gel for Column Chromatography 60-120 #

Elution mode

Gradient

Mobile phase

Chloroform

Chloroform

: Methanol

Chloroform Chloroform: Ethyl acetate Chloroform: Ethyl acetate: Methanol

Tolune

Tolune: Chloroform Tolune: Chloroform: Acetone

Volume of each Fraction

7 - 10 ml

Rate of elution

10 - 15 drops / min

Detection of spot

Vanillin- H2SO4

Scanned at 254 nm

Scanned at 254 nm

No. of fractions collected

87

141

25

Table 12: Column chromatography of USM of flowers of Quisqualis indica Linn

Fraction no.

1-10

Mobile phase

Designation

No. of spot

Inference

Chloroform

---

Inseparable

---

Fraction no.

11-29

Chloroform

PEF-A

2

Mixture of 2 compounds

Fraction no.

30-38

Chloroform : Methanol (9:1)

PEF-B

3

Mixture of 3 compounds

Fraction no.

39-46

Chloroform : Methanol (8:2)

PEF-C

2

Mixture of 2 compounds

Fraction no

47-64

Chloroform : Methanol (7:3)

PEF-D

3

Mixture of 3 compounds

Fraction no

65-76

Chloroform : Methanol (6:4)

PEF-E

2

Mixture of 2 compounds

Fraction no

76-87

Chloroform : Methanol (5:5)

--

Inseparable

--

The fractions obtained from column chromatography gives 3 single compounds.

The fractions showing inseparable matter and compounds obtained in trace amount were not taken for further studies. The fraction PEF-A, B, C, and E were combined and concentrated. The two single compounds Fa and Fb were separated by preparative TLC method.

5.1.12 .1 Purification of compound Fa from USM of flowers:

The compound Fa was purified by preparative TLC, after concentration, it was formed white precipitate, remaining solution was decanted. The compound showed positive result for Liberman-Burched test. The homogeneity of the compound was confirmed by using following mobile phase.

1. Hexane: Chloroform: Acetone (5:3:2)

2. Pet ether: Ethyl acetate (9:1)

3. Tolune: Ethyl acetate (9:1)

5.1.12.2 Purification of compound Fb from USM of flowers

The fraction was purified by preparative TLC, after concentration, it was formed white precipitate and purified by adding 90 % ethanol. The precipitate showed positive result of Liberman-Burched test. The homogeneity of the compound was carried out by using the three different mobile phases.

1. Pet ether: Ethyl acetate (9:1)

2. Benzene: Ethyl acetate (9:1)

3. Tolune; Methanol (9:1)

Table 13: Column Chromatography of ethyl acetate extract of flowers

Fraction no.

1-10

Mobile phase

Designation

No. of spot

Inference

Chloroform

---

Inseparable

-

Fraction no.

11-41

Chloroform: Ethyl acetae (9.5:0.5)

EF-1

2

Mixture of 2 compounds

Fraction no.

42-89

Chloroform: Ethyl acetae: Methanol (8.5:1:0.5)

EF-3

5

Mixture of 5 compounds

Fraction no.

90-101

Chloroform: Ethyl acetae: Methanol (7:2:1)

EF-4

2

Mixture of 2 compounds

Fraction no

102-134

Chloroform: Ethyl acetae: Methanol (6:4:2)

EF-5

2

Mixture of 2 compounds

Fraction no

135-138

Chloroform: Methanol (6:4)

EF-6

2

Mixture of 2 compounds

Fraction no

139-141

Chloroform: Ethyl acetae: Methanol (5:2.5:2.5)

---

Inseparable

---

The fractions obtained from column chromatography gives 5 single compounds but three were very less in amount therefore rejected.

The fractions showing inseparable matter and compounds obtained in trace amount were rejected. The fraction EF-1, 2, 4, 5 was combined and concentrated. The two single compounds Fc and Fd were separated by preparative TLC method.

5.1.12 .3 Purification of compound Fc

The fraction was purified by preparative TLC, after concentration, it was formed yellow precipitate, remaining solution was decanted. The precipitate shows positive result for shinoda test and single spot with the mobile phases such as

1. Ethyl acetate: Formic acid: Glacial acetic acid: Water (100:11:11:26)

2. Chloroform: Glacial acetic acid: Water: Methanol (60:32:8:12)

3. Chloroform: Methanol (8:2).

5.1.12 .4 Purification of compound Fd

The fraction was purified by preparative TLC, after concentration, it was formed yellow -orange ppt., remaining solution was decanted. The precipitate shows positive result for shinoda test and single spot with the mobile phases such as

1. Ethyl acetate: Formic acid: Glacial acetic acid: Water (100:11:11:26)

2. Chloroform: Glacial acetic acid: Water: Methanol (60:32:8:12)

3. Ethyl acetate: Methanol: Water (8:1:1).

Table 14: Column chromatography of ethyl acetate insoluble part of methanolic extract of flower

Fraction no.

1-5

Mobile phase

Designation

No. of spot

Inference

Tolune

M-1

Single

Single spot

Fraction no.

6-10

Tolune Chloroform (8:2)

M-2

3

Mixture of 3 compounds

Fraction no.

11-15

Tolune: Chloroform (8:3)

M-3

3

Mixture of 3 compounds

Fraction no.

16-20

Tolune : Chloroform: Acetone (7:2:1)

M-4

5

Mixture of 5 compounds

Fraction no.

21-25

Tolune: Chloroform :Acetone (5:4:1)

M-5

4

Mixture of 4 compounds

5.1.12 .5 Purification of compound Fe

The isolated compounds Fe was obtained in pure form. The compound showed positive result with lead acetate and ferric chloride solution. It was shown single spot with the mobile phases such as

1. Tolune: Acetone: Ethyl acetate (5:1:4)

2. Tolune: Ethyl acetate: Formic acid (6:6:1)

3. Tolune: Ethyl acetate (3:7)

Table 15: Observation table of isolated compound of Quisqualis indica Linn flowers

Compound Fa

Mobile phase

Rf values

Detecting reagent

Colour

Hexane: Chloroform: Acetone (5:3:2)

0.45

Vanillin - H2SO4

Pink

Pet ether: Ethyl acetate (9:1)

0.48

Tolune : Ethyl acetate (9:1)

0.55

Compound Fb

Pet ether: Ethyl acetate (9:1)

0.67

Vanillin- H2SO4

Violet

Benzene: Ethyl acetate (9.3:0.7)

0.58

Tolune : Methanol (9:1)

0.64

Compound Fc

Ethyl acetate: Formic acid: Acetic acid: Water (100:11:11:26),

0.50

Scanned at 254 nm

Yellowish

Chloroform: Acetic acid: Water: Methanol (60:32:8:12)

0.66

Chloroform: Methanol (8:2)

0.55

Compound Fd

Ethyl acetate: Formic acid: Acetic acid : Water (100:11:11:26)

0.40

Scanned at 254 nm

Yellowish green

Chloroform: Acetic acid: Water: Methanol

(60:32:8:12)

0.36

Ethyl acetate: Methanol: Water (8:1:1)

0.26

Compound Fe

Tolune: Acetone: Ethyl acetate (5:1:4)

0.72

Scanned at 254 nm

Greenish brown

Tolune : Ethyl acetate (3:7)

0.66

Tolune : Ethyl acetate: Formic acid (6:6:1)

0.81

From the above table it was indicated that one steroid, one terpenoid, two flavonoids compounds and one tannin were isolated from Quisqualis indica Linn flowers.

Table 16: Chromatographic conditions for all extracts of leaves of Quisqualis indica Linn

Parameters

USM of leaves

Ethyl acetate extract of leaves

Ethyl acetate insoluble fraction of methanolic extract of leaves

Column

Glass

Dimension

Diameter: 3 cm, Length: 75 cm

Stationary phase

Silica gel for Column Chromatography 60-120 #

Elution mode

Gradient

Mobile phase

Benzene

Benzene: Methanol

Ethyl acetate

Ethyl acetate:

Methanol

Tolune

Tolune: Chloroform

Tolune Chloroform: Acetone

Volume of each Fraction

25 ml

Rate of elution

10-15 drops / min

Detection of spot

Vanillin-sulphuric acid

Scanned at 254 nm

Scanned at 254 nm

No. of fractions collected

35

45

30

Table 17: Column chromatography of USM -2 extract of leaves

Fraction no

1-3

Mobile phase

Designation

No. of spot

Inference

Benzene

---

2

Mixture of 2 compounds

Fraction no

4-9

Benzene

PEL-1

3

Mixture of 3 compounds

Fraction no

10-15

Benzene: Methanol (9.5:0.5)

PEL-2

4

Mixture of 4 compounds

Fraction no

16-21

Benzene: Methanol (9:1)

PEL-3

3

Mixture of 3 compounds

Fraction no

22-27

Benzene: Methanol (8.5:1.5)

PEL-4

2

Mixture of 2 compounds

Fraction no

28-35

Benzene: Methanol (8:2)

PEL-5

inseparable

--

The fractions showing inseparable matter and compounds obtained in trace amount were rejected. The fraction PEL-1, 2, 4 was concentrated. The two single compounds La and Lb were separated by preparative TLC method.

5.1.12 .6 Purification of compound La from USM of leaves:

The fraction was purified by preparative TLC, after concentration, it was formed fine shinny white crystals. The precipitate shows positive result of Liberman-Burched test and single spot with the mobile phases.

1. Hexane: Chloroform: Acetone (5:3:2)

2. Pet ether: Ethyl acetate (9:1)

3. Tolune: Ethyl acetate (9:1)

5.1.12.7 Purification of compound Lb from USM of leaves

The fraction was purified by preparative TLC, after concentration, it was formed white precipitate, remaining solution was decanted. The precipitate shows positive result of Liberman-Burched test and single spot with the mobile phases with Rf value 1. Pet ether: Ethyl acetate (9:1)

2. Benzene: Ethyl acetate (9:1)

3. Tolune: Methanol (9:1)

Table 18: Column chromatography of ethyl acetate extract of leaves

Fraction no.

1-5

Mobile phase

Designation

No. of spot

Inference

Ethyl acetate

---

Inseparable

--

Fraction no.

6-8

Ethyl aceate

EL-1

4

Mixture of 4 compounds

Fraction no.

9-16

Ethyl acetate

EL-2

2

Mixture of 2 compounds

Fraction no.

17-20

Ethyl acetate: Methanol (9.5:0.5)

EL-3

3

Mixture of 3 compounds

Fraction no.

21-28

Ethyl acetate: Methanol (9.5:0.5)

EL-4

2

Mixture of 2 compounds

Fraction no.

29-35

Ethyl acetate: Methanol (9:1)

EL-5

3

Mixture of 3 compounds

Fraction no.

36-40

Ethyl acetate: Methanol (9:1)

EL-6

2

Mixture of 2 compounds

Fraction no.

41-45

Ethyl acetate: Methanol (8.5:1.5)

---

Inseparable

--

The fractions showing inseparable matter and compounds obtained in trace amount were rejected. The fractions EL-3, 4, 5, were concentrated. The two single compounds Lc and Ld were separated by preparative TLC method.

5.1.12 .8 Purification of compound Lc

The fraction was purified by preparative TLC, after concentration, it was formed yellow precipitate. The precipitation shown positive result for shinoda test and single spot with the mobile phases such as

1. Ethyl acetate: Formic acid: Glacial acetic acid: Water (100:11:11:26),

2. Chloroform: Glacial acetic acid: Water: Methanol (60:32:8:12)

3. Chloroform: Methanol (8:2)

5.1.12 .9 Purification of compound Ld

The fraction was purified by preparative TLC, after concentration, it was formed yellow -orange precipitate. The precipitation shows positive result for shinoda test and single spot with the mobile phases such as

1. Ethyl acetate: Formic acid: Glacial acetic acid: Water (100:11:11:26),

2. Chloroform: Glacial acetic acid: Water: Methanol (60:32:8:12)

3. Ethyl acetate: Methanol: Water (8:1:1)

Table 19: Column chromatography of ethyl acetate insoluble part of methanolic extract of leaves

Fraction no.

1-5

Mobile phase

Designation

No.of spot

Inference

Tolune

M-1

Single

Single spot

Fraction no.

6-10

Tolune : Chloroform (8:2)

M-2

3

Mixture of 3 compounds

Fraction no.

11-15

Tolune: Chloroform (7:3)

M-3

3

Mixture of 3 compounds

Fraction no.

16-20

Tolune : Chloroform: Acetone (7:2:1)

M-4

5

Mixture of 5 compounds

Fraction no.

21-30

Tolune: Choloroform: Acetone (6:3:3)

M-5

4

Mixture of 4 compounds

5.1.12.10 Purification of compound Le

The compound Le was obtained in pure form. Compound showed positive result with lead acetate and ferric chloride solution. It was shown single spot with the mobile phases such as

1. Tolune: Acetone: Ethyl acetate (5:1:4)

2. Tolune: Ethyl acetate (3:7)

3. Tolune: Ethyl acetate: Formic acid (6:6:1)

Table 20: Observation table of isolated compound from leaves of Quisqualis indica Linn

Compound La

Mobile phase

RF

Detecting reagent

Colour

Hexane: Chloroform: Acetone (5:3:2)

0.45

Vanillin - H2SO4

Pink

Pet ether: Ethyl acetate (9:1)

0.48

Tolune : Ethyl acetate(9:1)

0.55

Compound Lb

Pet ether: Ethyl acetate (9:1)

0.67

Vanillin - H2SO4

Violet

Benzene: Ethyl acetate (9:1)

0.58

Tolune : Methanol (9:1)

0.64

Compound Lc

EA: Formic acid: Acetic acid: Water (100:11:11:26),

0.50

Scanned at 254 nm

Yellowish

Chloroform: Acetic acid: Water: Chloroform (60:32:8:12)

0.66

Chloroform : Methanol (8:2)

0.55

Compound Ld

EA: Formic acid: Acetic acid: Water (100:11:11:26)

0.40

Scanned at 254 nm

Yellowish green

Chloroform: Acetic acid :Water: Methanol (60:32:8:12)

0.36

Ethyl acetate: Methanol :Water (8:1:1)

0.26

Compound Le

Tolune: Acetone: Ethyl acetate (5:1:4)

0.72

Scanned at 254 nm

Greenish brown

Tolune : Ethyl acetate (3:7)

0.66

Tolune : Ethyl acetate: Formic acid (6:6:1)

0.81

From the above table it was indicated that one steroid, one terpenoid, two flavonoids and one tannin were isolated from Quisqualis indica Linn leaves. Isolated compounds from flowers and leaves of Quisqualis indica Linn subjected for structural elucidation by using spectroscopical method.

5.13 Characterizations of isolated compound by spectroscopical techniques83,84

The pure compounds obtained were subjected to spectral analysis like UV, IR, Mass, NMR and melting point.

5.2 Pharmacological evaluation of Quisqualis indica Linn

5.2.1 Experimental Animals:

Rats: Albino rats of Wistar strain weighing 200- 250 g were obtained from Padmavati College of Pharmacy & Research Institute. Animals of either sex were housed in groups of three under standard laboratory conditions with free access to standard pellet diet and water ad libitum.

Mice: Swiss albino mice weighing 20-30 g were obtained from Padmavati College of Pharmacy & Research Institute. Animals of either sex were housed in groups of five under standard laboratory conditions of 22 ± 30C with relative humidity 30% and 12 h light and dark cycle with free access to standard pellet diet and water ad libitum.

5.2.2 Acute oral toxicity test85:

Three male mice were fasted overnight with free access to water. Each animal received single doses of different extracts of leaves and flowers of QI (2,000 mg/kg, p.o.)

After administration of different extracts of leaves and flowers of QI , animals were observed individually and continuously for 30 min, 2 hr and 24 hr to detect changes in the autonomic or behavioral responses and also for tremors, convulsion, salivation, diarrhoea, lethargy, sleep and coma and then monitored for any mortality for the following 14 days. Different extracts of leaves and flowers of QI which were orally administered in increasing doses found safe up to 2,000 mg/kg dose.

On the basis of acute toxicity the 1/10th of 2000 mg/kg, p.o. dose of different extracts of leaves and flowers of QI were selected at doses as 100 mg/kg, 200 mg/kg, 400 mg/kg, p.o. for evaluation of pharmacological activities.

5.2.3 ANTIDIABETIC ACTIVITY:

5.2.3.1 Hypoglycemic study in nondiabetic rats86

The Wistar rats weighing 200-250 g were used. The overnight fasted animals were divided into fourteen groups (n=5) as follows:

Table 21: Hypoglycemic study in nondiabetic rats

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Plasma glucose

On 0th, 60th, 120th, and 180th min after drug treatment.

II- Glim

Glimepride (0.09 mg/kg, p.o.).

III- MLQ-100

MLQ (100 mg/kg in 0.5% CMC, p.o.).

IV- MLQ-200

MLQ (200 mg/kg in 0.5% CMC, p.o.).

V- MLQ-400

MLQ (400 mg/kg in 0.5% CMC, p.o.).

VI- PLQ-100

PLQ (100 mg/kg in 0.5% CMC, p.o.).

VII- PLQ-200

PLQ (200 mg/kg in 0.5% CMC, p.o.).

VIII- PLQ-400

PLQ (400 mg/kg in 0.5% CMC, p.o.).

IX- MFQ-100

MFQ (100 mg/kg in 0.5% CMC, p.o.).

X- MFQ-200

MFQ (200 mg/kg in 0.5% CMC, p.o.).

XI- MFQ-400

MFQ (400 mg/kg in 0.5% CMC, p.o.).

XII- PFQ-100

PFQ (100 mg/kg in 0.5% CMC, p.o.).

XIII- PFQ-200

PFQ (200 mg/kg in 0.5% CMC, p.o.).

XIV- PFQ-400

PFQ (400 mg/kg in 0.5% CMC, p.o.).

CMC = Carboxy methyl cellulose, Glim = Glimepride, MLQ = Methanolic extract of leaves of Quisqualis indica Linn, PLQ = Pet. ether extract of leaves of Quisqualis indica Linn, MFQ = Methanolic extract of flowers of Quisqualis indica Linn, PFQ = Pet. ether extract of flowers of Quisqualis indica Linn.

Parameters evaluated

Biochemical Parameters: At the 0th, 60th, 120th and 180th min after drug administration blood was collected by puncturing retro-orbital plexus under mild ether anesthesia by using fine glass capillary in epindroff tubes containing anticoagulant. Plasma was separated by centrifugation. Plasma glucose level was estimated by GOD/POD method.

5.2.3.2 Anti-hyperglycemic study in glucose induced rats86

The Wistar rats weighing 200-250 g were used. The overnight fasted animals were divided into fourteen groups (n=5) as follows:

Table 22: Anti-hyperglycemic study in glucose induced rats

Groups (n=5)

Treatment

GlucoseLoad

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

10% Glucose solution

(2 g/kg, p.o).

30 min after vehicle/drug administration

Plasma glucose

on 0th, 30th, 90th, 150th min.

after glucose loaded

II- Glim

Glimepride (0.09 mg/kg, p.o.).

III- MLQ-100

MLQ (100 mg/kg in 0.5% CMC, p.o.).

IV- MLQ-200

MLQ (200 mg/kg in 0.5% CMC, p.o.).

V- MLQ-400

MLQ (400 mg/kg in 0.5% CMC, p.o.).

VI- PLQ-100

PLQ (100 mg/kg in 0.5% CMC, p.o.).

VII- PLQ-200

PLQ (200 mg/kg in 0.5% CMC, p.o.).

VIII PLQ-400

PLQ (400 mg/kg in 0.5% CMC, p.o.).

IX MFQ-100

MFQ (100 mg/kg in 0.5% CMC, p.o.).

X MFQ-200

MFQ (200 mg/kg in 0.5% CMC, p.o.).

XI MFQ-400

MFQ (400 mg/kg in 0.5% CMC, p.o.).

XII PFQ-100

PFQ (100 mg/kg in 0.5% CMC, p.o.).

XIII PFQ-200

PFQ (200 mg/kg in 0.5% CMC, p.o.).

XIV PFQ-400

PFQ (400 mg/kg in 0.5% CMC, p.o.).

CMC = Carboxy methyl cellulose, Glim = Glimepride, MLQ = Methanolic extract of leaves of Quisqualis indica Linn, PLQ = Pet. ether extract of leaves of Quisqualis indica Linn.

Parameters evaluated

Biochemical Parameters: At the 0th, 30th, 90th and 150th min after glucose load, the blood samples were collected by puncturing retro-orbital plexus under mild ether anesthesia by using fine glass capillary, in epindroff tubes containing anticoagulant. Plasma was separated by centrifugation. The plasma glucose level was determined by GOD/POD Method.

5.2.3.3 Study in dexamethasone induced insulin resistance in mice87.

The male swiss mice weighing 25-30 g were used. The overnight fasted animals were divided into nine groups (n=5) as follows:

Table 23: Study in dexamethasone induced insulin resistance in mice

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o., b.i.d.)

Biochemical Parameters

Plasma glucose

and triglyceride

Morphological

Parameter

Body weight

Antioxidants

GSH, SOD, Catalase, LPO levels in liver homogenate.

Glucose uptake

In isolated hemi-diaphragms.

II- Dexa + Control

(1mg/kg/day, i.m.) From day 1 to day 22.

III- Dexa + Pio

Dexamethasone (1 mg/kg/day, i.m.) daily for 22 days and pioglitazone (2 mg/kg/day, p.o., b.i.d.) from day 8 to day 22.

IV- Dexa + MLQ-100

Dexamethasone (1 mg/kg/day, i.m.) daily for 22 days and MLQ (100 mg/kg/day, p.o., b.i.d.) from day 8 to day 22.

V- Dexa + MLQ-200

Dexamethasone (1 mg/kg/day, i.m.) daily for 22 days and MLQ (200 mg/kg/day, p.o., b.i.d.) from day 8 to day 22.

VI- Dexa + MLQ-400

Dexamethasone (1 mg/kg/day, i.m.) daily for 22 days and MLQ (400 mg/kg/day, p.o.) from day 8 to day 22.

VII Dexa + MFQ-100

Dexamethasone (1 mg/kg/day, i.m.) daily for 22 days and MFQ (100 mg/kg/day, p.o.) from day 8 to day 22.

VIII Dexa + MFQ-200

Dexamethasone (1 mg/kg/day, i.m.) daily for 22 days and MFQ (200 mg/kg/day, p.o., b.i.d.) from day 8 to day 22.

IX Dexa + MFQ-400

Dexamethasone (1 mg/kg/day, i.m.) daily for 22 days and MFQ (400 mg/kg, p.o., b.i.d.) from day 8 to day 22.

CMC = Carboxy methyl cellulose, Dexa = Dexamethasone, Pio = Pioglitazone, MLQ = Methanolic extract of leaves of Quisqualis indica Linn, MFQ = Methanolic extract of flowers of Quisqualis indica Linn.

Parameters evaluated

Biochemical Parameters: The plasma glucose and triglyceride levels were estimated on day 1 and day 22.

2) Morphological: The body weights were taken on day 1 and day 22.

3) Antioxidants: The antioxidant parametres (GSH, SOD, Catalase, LPO levels) were estimated from liver homogenate.

4) Glucose uptake in isolated hemi-diaphragms.

5.2.3.4 Study in nondiabetic and alloxan induced diabetic rats with reference to diabetic complications88,89.

Diabetes was induced by a single intraperitonial injection of alloxan monohydrate in citrate buffer (pH 4.5) at a dose of 140 mg/kg of body weight of the rat. The diabetic state was confirmed 48 hr after alloxan injection by hyperglycemia. Surviving rats with fasting blood glucose level higher than 250 mg/dl were included in the study.

Procedure

Total of 90 Wistar rats were used (50 diabetic surviving and 40 non-diabetics). The rats were divided in to 18 groups (n=5) as follows:

Table 24: Alloxan induced diabetic rats with reference to diabetic complications

Groups (n=5)

Treatment and Dose/ Day

Parameters evaluated

I- Control

(1% gum acacia 1 ml/kg, p.o., b.i.d)

Biochemical

Parameters

Plasma glucose,

Plasma triglyceride,

Plasma total cholesterol,

Plasma LDL- cholesterol,

Plasma HDL- cholesterol,

Plasma creatinine,

Plasma albumin,

Plasma total protein

Hemodynamic parameters

Blood pressure,

Heart rate

Morphological parameter

Body weight

II-D Control

Alloxan (140 mg/kg, i. p.) on 0th day

III-D + Glim

Alloxan (140 mg/kg, i. p.) on 0th day + Glimepride (0.09 mg/kg, p.o., b.i.d) for 42 days

IV-D + Losar

Alloxan (140 mg/kg, i. p.) on 0th day + Losartan (2 mg/kg, p.o., b.i.d.) for 42 days

V- D + MLQ -100

Alloxan (140 mg/kg, i.p.) on 0th day + MLQ (100 mg/kg, p.o., b.i.d) for 42 days

VI- D + MLQ-200

Alloxan (140 mg/kg i.p.) on 0th day + MLQ (200 mg/kg, p.o., b.i.d) for 42 days

VII- D + MLQ- 400

Alloxan (140mg/kg i.p.) on 0th day + MLQ (400 mg/kg, p.o., b.i.d) for 42 days

VIII- D + MFQ -100

Alloxan (140 mg/kg, i.p.) on 0th day + M

FQ (100 mg/kg, p.o., b.i.d) for 42 days

IX- D + MFQ-200

Alloxan (140 mg/kg i.p.) on 0th day + MFQ (200 mg/kg, p.o., b.i.d) for 42 days

X- D + MFQ- 400

Alloxan (140mg/kg i.p.) on 0th day + MFQ (400 mg/kg, p.o., b.i.d) for 42 days

XI- Glim

Glimepride (0.09 mg/kg, p.o., b.i.d.) for 42 days

XII- Losar

Losartan (2 mg/kg, p.o., b.i.d) for 42 days

XIII- MLQ -100

MLQ (100 mg/kg, p.o., b.i.d) for 42 days

XIV- MLQ-200

MLQ (200 mg/kg, p.o. b.i.d) for 42 days

XV- MLQ- 400

MLQ (400 mg/kg, p.o., b.i.d) for 42 days

XVI - MFQ -100

MFQ (100 mg/kg, p.o., b.i.d) for 42 days

XVII - MFQ-200

MFQ (200 mg/kg, p.o., b.i.d) for 42 days

XVIII - MFQ- 400

MFQ (400 mg/kg, p.o., b.i.d) for 42 days

CMC = Carboxy methyl cellulose, Glim = Glimepride, Losar = Losartan, MLQ = Methanolic extract of leaves of Quisqualis indica Linn, MFQ = Methanolic extract of flowers of Quisqualis indica Linn.

Parameters evaluated

Biochemical Parameters: The plasma glucose, triglyceride, total cholesterol,

LDL-cholesterol, HDL-cholesterol, creatinine, albumin and total protein levels were estimated on day 1 and day 43.

Morphological: In this study, body weight of the animals were mesured on day 1 and day 43.

Hemodynamic parameters: In this study the blood pressure measurment was done by using BIOPAC (Invasive method).

5.2.4 ANTI-INFLAMMATORY ACTIVITY:

Carrageenan induced paw edema in rats (Quisqualis indica Linn flowers extract)

The anti-inflammatory activity using carrageenan induced hind paw edema was carried out as described by Winter et al90.

Male wistar rats of 200-250 g were used. Rats were divided into eight groups (n=5) as follows:

Table 25: Carrageenan induced paw edema in rats (Quisqualis indica Linn flowers)

Groups (n=5)

Treatment

Parameters

I- Control

% sodium CMC (1 ml/kg, p.o.)

Edema volume

And Percent inhibition

II- Indo

Indomethacin (10 mg/kg, p.o.)

III- MFQ-100

MFQ (100 mg/kg in 0.5% CMC, p.o.).

IV- MFQ-200

MFQ (200 mg/kg in 0.5% CMC, p.o.).

V- MFQ-400

MFQ (400 mg/kg in 0.5% CMC, p.o.).

VI- PFQ-100

PFQ (100 mg/kg in 0.5% CMC, p.o.).

VII- PFQ-200

PFQ (200 mg/kg in 0.5% CMC, p.o.).

VIII PFQ-400

PFQ (400 mg/kg in 0.5% CMC, p.o.).

CMC = Carboxy methyl cellulose, Indo = Indomethacin, MFQ = Methanolic extract of flowers of Quisqualis indica Linn, PFQ = Pet. ether extract of flowers of Quisqualis indica Linn.

5.2.4.1 Carrageenan induced paw edema in rats (Quisqualis indica Linn leaves extract)

Table 26: Carrageenan induced paw edema in rats (Quisqualis indica Linn leaves extract)

Male wistar rats of 200-250 g were used. Rats were divided into eight groups (n=5) as follows:

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Edema volume and

Percent inhibition

II- Indo

Indomethacin (10 mg/kg, p.o.)

III- MLQ-100

MLQ (100 mg/kg in 0.5% CMC, p.o.)

IV- MLQ-200

MLQ (200 mg/kg in 0.5% CMC, p.o.)

V- MLQ-400

MLQ (400 mg/kg in 0.5% CMC, p.o.)

VI- PLQ-100

PLQ (100 mg/kg in 0.5% CMC, p.o.)

VII- PLQ-200

PLQ (200 mg/kg in 0.5% CMC, p.o.)

VIII PLQ-400

PLQ (400 mg/kg in 0.5% CMC, p.o.)

CMC = Carboxy methyl cellulose, Indo = Indomethacin, MLQ = Methanolic extract of leaves of Quisqualis indica Linn, PLQ = Pet. ether extract of leaves of Quisqualis indica Linn.

Procedure:

After 60 minutes of administration of test drugs, 0.1 ml of 1% carrageenan was injected into right hind paw. Paw volume was measured by using Plethysmometer at a time interval of 30 min, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr and 6 hr after administration of carrageenan.

Edema volume and % inhibition were calculated by using following formulae

Edema Volume = Vt - Vc

Vt = Paw volume in ml, at time t, after carrageenan administration

Vc = Paw volume in ml, before carrageenan administration

Percent Inhibition = Ec-Et / Ec Ã- 100

Ec = Edema volume of control group

Et = Edema volume of treated group

5.2.4.2 Acetic acid-induced vascular permeability in mice91 (Quisqualis indica Linn flowers extract)

The swiss albino mice weighing 25-30 g were used. The overnight fasted animals were divided into eight groups (n=5) as follows:

Table 27: Acetic acid-induced vascular permeability in mice (Quisqualis indica Linn leaves extract)

Groups (n=5)

Treatment

Parameters

I- Control

% sodium CMC (1 ml/kg, p.o.)

Amount of dye leakage

Percent inhibition of dye leakage

II- Indo

Indomethacin (10 mg/kg, p.o.)

III- MFQ-100

MFQ (100 mg/kg in 0.5% CMC, p.o.).

IV- MFQ-200

MFQ (200 mg/kg in 0.5% CMC, p.o.).

V- MFQ-400

MFQ (400 mg/kg in 0.5% CMC, p.o.).

VI- PFQ-100

PFQ (100 mg/kg in 0.5% CMC, p.o.).

VII- PFQ-200

PFQ (200 mg/kg in 0.5% CMC, p.o.).

VIII PFQ-400

PFQ (400 mg/kg in 0.5% CMC, p.o.)

CMC = Carboxy methyl cellulose, Indo = Indomethacin, MFQ = Methanolic extract of flowers of Quisqualis indica Linn, PFQ = Pet. ether extract of flowers of Quisqualis indica Linn.

5.2.4.3 Acetic acid-induced vascular permeability in mice (Quisqualis indica Linn flowers extract)

The swiss albino mice weighing 25-30 g were used. The overnight fasted animals were divided into eight groups (n=5) as follows:

Table 28: Acetic acid-induced vascular permeability in mice (Quisqualis indica Linn flowers extract)

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Amount of dye leakage

Percent inhibition of dye leakage

II- Indo

Indomethacin (10 mg/kg, p.o.)

III- MLQ-100

MLQ (100 mg/kg in 0.5% CMC, p.o.).

IV- MLQ-200

MLQ (200 mg/kg in 0.5% CMC, p.o.).

V- MLQ-400

MLQ (400 mg/kg in 0.5% CMC, p.o.).

VI- PLQ-100

PLQ (100 mg/kg in 0.5% CMC, p.o.).

VII- PLQ-200

PLQ (200 mg/kg in 0.5% CMC, p.o.).

VIII PLQ-400

PLQ (400 mg/kg in 0.5% CMC, p.o.).

CMC = Carboxy methyl cellulose, Indo = Indomethacin, MLQ = Methanolic extract of leaves of Quisqualis indica Linn, PLQ = Pet. ether extract of leaves of Quisqualis indica Linn

Procedure

After 60 minutes of test drug administration, 0.6 % solution of acetic acid was injected (10 ml/kg, i.p.) in mice. Immediately, 10 % (w/v) Evan's blue was injected (10 ml/kg, i.v.) through the tail vain. Thirty minutes after Evan's blue injection, the animals were anaesthetized with ether anaesthesia and sacrificed. Peritoneal fluid was removed from each animal, filtered and volume to make up to 10 ml with distilled water. Absorbance of each sample was measured spectrophotometrically at 610 nm.

Results were expressed as,

Percent inhibition = Wc-Wt/Wc

Wc = Amount of dye leakage in control group

Wt = Amount of dye leakage in test group

5.2.4.4 Cotton pellet granuloma formation in rats92 (Quisqualis indica Linn leaves extract)

Male rats of 200-250 g of rats were divided into eight groups containing six animals in each group.

Table 29: Cotton pellet granuloma formation in rats (Quisqualis indica Linn leaves extract)

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Granuloma weight

Percent inhibition

Ulcerogenic Index

Antioxidant in liver

LPO

SOD

Catalase

GSH

II- Ibup

Ibuprofen (10 mg/kg, p.o.)

III- MLQ-100

MLQ (100 mg/kg in 0.5% CMC, p.o.).

IV- MLQ-200

MLQ (200 mg/kg in 0.5% CMC, p.o.).

V- MLQ-400

MLQ (400 mg/kg in 0.5% CMC, p.o.).

VI- PLQ-100

PLQ (100 mg/kg in 0.5% CMC, p.o.).

VII- PLQ-200

PLQ (200 mg/kg in 0.5% CMC, p.o.).

VIII PLQ-400

PLQ (400 mg/kg in 0.5% CMC, p.o.).

CMC = Carboxy methyl cellulose, Ibup = Ibuprofen, MLQ = Methanolic extract of leaves of Quisqualis indica Linn, PLQ = Pet. ether extract of leaves of Quisqualis indica Linn.

5.2.4.5 Cotton pellet granuloma formation in rats (Quisqualis indica Linn flower extract)

Male rats of 200-250 g of rats were divided into eight groups containing six animals in each group.

Table 30: Cotton pellet granuloma formation in rats (Quisqualis indica Linn flowers extract)

Groups (n=5)

Treatment

Parameters

Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Granuloma weight

Percent inhibition

Ulcerogenic Index

Antioxidant in liver

LPO

SOD

Catalase

GSH

Ibup

Ibuprofen (10 mg/kg, p.o.)

MFQ-100

MFQ (100 mg/kg in 0.5% CMC, p.o.).

MFQ-200

MFQ (200 mg/kg in 0.5% CMC, p.o.).

MFQ400

MFQ (200 mg/kg in 0.5% CMC, p.o.)

PFQ-100

PFQ (100 mg/kg in 0.5% CMC, p.o.).

PFQ-200

PFQ (200 mg/kg in 0.5% CMC, p.o.).

PFQ-400

PFQ (200 mg/kg in 0.5% CMC, p.o.).

CMC = Carboxy methyl cellulose, Ibup = Ibuprofen, MFQ = Methanolic extract of flowers of Quisqualis indica Linn, PFQ = Pet. ether extract of flowers of Quisqualis indica Linn.

Procedure

The cotton pellet weighing 50 ± 2 mg was sterilized in an autoclave (Lab hosp, Mumbai, India) handled with sterile instrument. The pellet was inserted in each animal on the back. On 8th day, animals were anaesthetized by ether and blood was withdrawn by retro orbital plexus by using fine glass capillary and collected into epindroff tubes for separation of plasma.

After blood withdrawal animals were sacrificed by decapitation, cotton pellets were carefully removed and made free from extraneous tissues.

Each Pellet was then dried at 60â-¦C in an oven until constant weight was obtained. Granuloma formation was evaluated by the dry weight of the pellet. Liver of each animal was removed rapidly and washed with ice cold TRIS buffer.

Liver of each animal was cut into a small pieces and homogenated with homogenizer, so that clear homogenate is formed. These lever homogenate were used for estimation of LPO, GSH, SOD and Catalase levels.

5.2.5 ANALGESIC ACTIVITY

5.2.5.1 Acetic acid induced writhing in mice93 (Quisqualis indica Linn leaves extract)

The swiss albino mice weighing 25-30 g were used. The overnight fasted animals were divided into eight groups (n=5) as follows:

Table 31: Acetic acid induced writhing in mice (Quisqualis indica Linn leaves extract)

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Number of writhes during 60 min

Percent inhibition

II- Penta

Pentazocine (10 mg/kg, i.p.)

III- MLQ-100

MLQ (100 mg/kg in 0.5% CMC, p.o.).

IV- MLQ-200

MLQ (200 mg/kg in 0.5% CMC, p.o.).

V- MLQ-400

MLQ (400 mg/kg in 0.5% CMC, p.o.).

VI- PLQ-100

PLQ (100 mg/kg in 0.5% CMC, p.o.).

VII- PLQ-200

PLQ (200 mg/kg in 0.5% CMC, p.o.).

VIII PLQ-400

PLQ (400 mg/kg in 0.5% CMC, p.o.).

CMC = Carboxy methyl cellulose, Penta = Pentazocin, MLQ = Methanolic extract of leaves of Quisqualis indica Linn, PLQ = Pet. ether extract of leaves of Quisqualis indica Linn.

5.2.5.2 Acetic acid induced writhing in mice (Quisqualis indica Linn flowers extract)

The swiss albino mice weighing 25-30 g were used. The overnight fasted animals were divided into eight groups (n=5) as follows:

Table 32: Acetic acid induced writhing in mice (Quisqualis indica Linn flowers extract)

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Number of writhes during 60 min

Percent inhibition

II-Penta

Pentazocine (10 mg/kg, i.p.)

III- MFQ-100

MFQ (100 mg/kg in 0.5% CMC, p.o.).

IV- MFQ-200

MFQ (200 mg/kg in 0.5% CMC, p.o.).

V- MFQ-400

MFQ (400 mg/kg in 0.5% CMC, p.o.).

VI- PFQ-100

PFQ (100 mg/kg in 0.5% CMC, p.o.).

VII- PFQ-200

PFQ (200 mg/kg in 0.5% CMC, p.o.).

VIII- PFQ-400

PFQ (400 mg/kg in 0.5% CMC, p.o.).

CMC = Carboxy methyl cellulose, Penta = Pentazocin, MFQ = Methanolic extract of flowers of Quisqualis indica Linn, PFQ = Pet. ether extract of flowers of Quisqualis indica Linn.

Procedure

The writhing syndrome was elicited by intraperitonial injection of acetic acid (0.1 ml of 0.6% solution) and numbers of writhes displayed from 5 to 20 min were recorded118

After 60 minutes of drug administration, 0.6 % acetic acid solution (10 ml/ kg, i.p.) was injected to each animal and resulting writhes and stretching were observed and counted over a period of 60 min after acetic acid injection. Results were expressed as,

Percent inhibition = Wc-Wt/Wc

Wc= Number of wriths in control group

Wt = Number of wriths in test group

5.2.5.3 Formalin induced nociception94 (Quisqualis indica Linn leaves extract)

The Swiss albino mice weighing 25-30 g were used. The overnight fasted animals were divided into eight groups (n=5) as follows:

Table 33: Formalin induced nociception (Quisqualis indica Linn leaves extract)

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Number of writhes during 60 min

Percent inhibition

II- Asp

Aspirin (100 mg/kg, p.o.)

III- MLQ-100

MLQ (100 mg/kg in 0.5% CMC, p.o.).

IV- MLQ-200

MLQ (200 mg/kg in 0.5% CMC, p.o.).

V- MLQ-400

MLQ (200 mg/kg in 0.5% CMC, p.o.).

VI- PLQ-100

PLQ (100 mg/kg in 0.5% CMC, p.o.).

VII- PLQ-200

PLQ (200 mg/kg in 0.5% CMC, p.o.).

VIII PLQ-400

PLQ (200 mg/kg in 0.5% CMC, p.o.).

CMC = Carboxy methyl cellulose, Asp = Aspirin, MLQ = Methanolic extract of leaves of Quisqualis indica Linn, PLQ = Pet. Ether extract of leaves of Quisqualis indica Linn

5.2.5.4 Formalin induced nociception (Quisqualis indica Linn flowers extract)

The Swiss albino mice weighing 25-30 g were used. The overnight fasted animals were divided into eight groups (n=5) as follows:

Table 34: Formalin induced nociception Quisqualis indica Linn flowers extract

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Number of writhes during 60 min

Percent inhibition

II- Asp

Aspirin (100 mg/kg, p.o.)

III- MFQ-100

MFQ (100 mg/kg in 0.5% CMC, p.o.)

IV- MFQ-200

MFQ (200 mg/kg in 0.5% CMC, p.o.)

V- MFQ-400

MFQ (400 mg/kg in 0.5% CMC, p.o.)

VI- PFQ-100

PFQ (100 mg/kg in 0.5% CMC, p.o.)

VII- PFQ-200

PFQ (200 mg/kg in 0.5% CMC, p.o.)

VIII PFQ-400

PFQ (400 mg/kg in 0.5% CMC, p.o.)

CMC = Carboxy methyl cellulose, Asp = Aspirin, MFQ = Methanolic extract of flowers of Quisqualis indica Linn, PFQ = Pet. Ether extract of flowers of Quisqualis indica Linn.

5.2.5.5 Hot plate method95 (Quisqualis indica Linn leaves extract)

The Swiss albino mice weighing 25-30 g were used. The overnight fasted animals were divided into eight groups (n=5) as follows:

Table 35: Hot plate method (Quisqualis indica Linn leaves extract)

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Time of reaction

(sec)

II- Penta

Pentazocine (10 mg/kg, i.p.)

III- MFQ-100

MFQ (100 mg/kg in 0.5% CMC, p.o.)

IV- MFQ-200

MFQ (200 mg/kg in 0.5% CMC, p.o.)

V- MFQ-400

MFQ (400 mg/kg in 0.5% CMC, p.o.)

VI- PFQ-100

PFQ (100 mg/kg in 0.5% CMC, p.o.)

VII- PFQ-200

PFQ (200 mg/kg in 0.5% CMC, p.o.)

VIII- PFQ-400

PFQ (400 mg/kg in 0.5% CMC, p.o.)

Penta= Pentazocine, CMC = Carboxy methyl cellulose, MLQ = Methanolic extract of leaves of Quisqualis indica Linn, PLQ = Pet. ether extract of leaves of Quisqualis indica Linn.

5.2.5.6 Hot plate method95 (Quisqualis indica Linn flowers extract)

The Swiss albino mice weighing 25-30 g were used. The overnight fasted animals were divided into eight groups (n=5) as follows:

Table 36: Hot plate method (Quisqualis indica Linn flower extract)

Groups (n=5)

Treatment

Parameters

I- Control

0.5 % sodium CMC (1 ml/kg, p.o.)

Time of reaction (sec)

II- Penta

Pentazocine (10 mg/kg, i.p.)

III- MLQ-100

MLQ (100 mg/kg in 0.5% CMC, p.o.).

IV- MLQ-200

MLQ (200 mg/kg in 0.5% CMC, p.o.).

V- MLQ-400

MLQ (400 mg/kg in 0.5% CMC, p.o.).

VI- PLQ-100

PLQ (100 mg/kg in 0.5% CMC, p.o.).

VII- PLQ-200

PLQ (200 mg/kg in 0.5% CMC, p.o.).

VIII- PLQ-400

PLQ (400 mg/kg in 0.5% CMC, p.o.).

Penta= Pentazocine, CMC = Carboxy methyl cellulose, MFQ = Methanolic extract of flowers of Quisqualis indica Linn, PFQ = Pet. ether extract of flowers of Quisqualis indica Linn.

Procedure

After 60 minutes of test drug administration, animals were individually placed on hot plate with temperature adjusted to 55 ± 10o C. The latency to the first sign of paw licking or jump response to avoid the heat was taken as index of pain threshold; the cut off time was 20 secs in order to avoid damage to the paw. Procedure was performed after every 30th, 60th, 120th, 180th and 240th minutes of interval.

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