INTRODUCTION:

The Schiff bases play a vital role find use in analytical chemistry, agriculture, dyes and polymer industries besides their utility as model systems in the field of bio-inorganic chemistry. Schiff bases serve as useful gravimetric, colorimetric agents and anti-coagulant agent. The oxygen carrying property and electron transfer reactions of various schiff bases have been reported. Schiff bases are capable of forming coordinate bonds with many of metal ions through both azomethine group and phenolic group or via its azomethine or phenolic groups [1-13]. A large number of Schiff bases and their complexes have shown significant interest and attention because of their biological activity including anti-tumor, antibacterial, fungicidal and anti-carcinogenic properties [3-8] and catalytic activity [8-13]. Schiff base of salicylaldehyde-4-aminophenol have shown cytotoxic effect towards brine shrimo naupli[14].

Knowing the importance of 2-((4-hydroxyphenylimino) methyl)phenol Schiff bases, we plan to synthesis Schiff base by standard procedure. The present work focus on the synthesis of the above Schiff base and their pharmacological activities like Anti-inflammatory and CNS activities determination. In addition to these activities, we plan to predict the bioactivity by using online bioactivity predicting software PASS.

MATERIALS AND METHODS:

Materials used

The chemicals such as salicylaldehyde, 4-Aminophenol of E. Merck grade and distilled ethanol were used. The melting point was determined using melting point apparatus and IR spectra was recorded in FT-IR Affinity-1 Shimadzu.

Drugs

Diclofenac sodium(standard for anti-inflammatory) and Chlorpromazine (standard for CNS) were chosen for the work.

Animals used

For the anti-inflammatory and CNS depressant activity studies twelve albino rats of both sexs of weight 100-165g for each studies were used. . The animals were kept in poly propylene cages in a dark/light cycle, 12hrs/12hrs and animals were fed with pelleted diet and drinking water ad libitum. All the experimental protocols were approved by the committee for the purpose of control and supervision on experiments on animals (CPCSEA), animal ethics committee vide number SBCP/ 2011-2012/ IAEC/ CPCSEA/6.

Methods used Preparation of Schiff base

The salicylaldehyde and 4-aminophenolwere taken in a equimolar ratio of 1mmol and refluxed with ethanol for 2hours. After refluxing, the product obtained was filtered, dried and recrystallized using ethanol.

The melting point was recorded using melting point apparatus the melting point of 2-((4-hydroxyphenylimino) methyl)phenol Schiff base observed as 189.5^oC and the IR spectral studies reveals the presence of C=N & O-H functionalities and showed the following stretching frequencies. uC=N stretching at 1624cm-¹ and uOH stretching was 3417cm-¹.

ANTI-INFLAMMATORY ACTIVITY DETERMINATION [15, 16]

Carrageenan induced paw edema model

Carrageenan-induced hind paw edema is the standard experimental model of acute inflammation. Carrageenan is the phlogiston agent of choice for testing anti-inflammatory drugs as it is not known to be antigenic and is devoid of apparent systemic effects. Moreover, the experimental model exhibits a high degree of reproducibility.

Carrageenan-induced edema is a biphasic response. The first phase is mediated through the release of histamine, serotonin and kinins. Whereas, the second phase is related to the release of prostaglandin. The animals were divided into three groups each consisting of four animals. Group 1 served as control, Group 2 received diclofenac sodium (10 mg/kg) as standard and Group 3 received 250mg/kg of 2-((4-hydroxyphenylimino)methyl)phenol Schiff base respectively. Acute inflammation was produced by sub plantar injection of 0.1 ml of 1% suspension of carrageenan in normal saline, in the right hind paw of the rats, one hour after oral administration of the drugs. The paw diameter was measured with the aid of a verniar caliper at 0, 1, 2, 3 and 4 hours after the injection of carrageenan. The difference between the readings at time zero hour and the different time intervals were taken as the thickness of edema. The values were recorded in Table-1. Percentage inhibition of paw edema was calculated by comparing the control. The percentage inhibition of inflammation was calculated for each dose at different hours as given below.

Percentage inhibition = 1- Vt / Vc *100

Where Vc = volume of paw edema in control animals

Vt = volume of paw edema in treated animals.

DETERMINATION OF CNS ACTIVITY [17].

Preparation of the drug for the experimental study

Schiff base and the standard drugs were administered in the form of suspension in water with 1% Sodium Carboxy Methyl Cellulose (SCMC) as suspending agent.

Locomotor activity

Locomotor activity was recorded with using Actophotometer (digital activity cage). The animals were divided into three groups (n = 4). Each rat was individually placed in the actophotometer for 10 min. Animals of group 1 were intraperitoneally treated with Caffeine (30 mg/kg) (i.p). Group 2 was treated orally with Chlorpromazine (3 mg/kg, i.p.) and group 3 was treated orally with 250 mg/kg dose levels of 2-((4-hydroxyphenylimino)methyl)phenol drugs. Basal reaction time was noted before and 30 min after the administration of treatment. Account is recorded when the beam of light falling on the photocell of actophotometer is cut off by rat. Sample 1 received reference standard Chlorpramazine at a dose of 3 mg/kg (i.p.) 30 min before the test. Mean changes in the locomotor activity was recorded for each group and were listed in Table -2.

Chemdraw ultra11.0 software

The structure of 1,3-bis(2-hydroxybenzylidene)thiourea schiff base was drawn in chemultra11.0 appear as given in Fig.1. and their structure was saved as molfiles (*.mol).

Fig 1: Structure of 2-((4-hydroxyphenylimino)methyl)phenol Schiff base

Docking

The possible bioactivities were predicted with PASS software (V. Poroikov et al, version 1.917) as given in Fig.2 and the result was given as Table-3.

Fig.2. PASS Prediction window

RESULT AND DISCUSSION:

Anti-inflammatory activity

The results showed that dose dependent decrease in the size of the edema range from 0.235±0.01106 to 0.74±0.0094 for 5mg/kg of Standard Diclofenac Sodium and from 0.25±0.01491 to 0.75±0.01490 for 250mg/kg of 2-((4-hydroxyphenylimino)methyl)phenol. The Schiff base was found to show higher activity at third hour than the standard with a probability<0.5 and the graph was illustrated as in Fig.3.

Table-1.

Anti-inflammatory activity of 2-((4-hydroxyphenylimino)methyl) phenol

Drug treatment

Dose

(mg/kg)

Mean time (in seconds) ±SEM

2hr

3hr

4hr

control

0.3325±0.01092

0.64±0.009434

0.84±0.0163

1.045±

0.01105

Diclofenac

sodium

0.235±

0.01106

(29.32)

0.43±0.01154

(32.81)

0.615±0.0057

(26.98)

0.74±0.0094

(29.18)

HPIMP

250

0.25±

0.01491

(24.81)

0.435±0.01106

(32.03)

0.625±0.0110

(48.21)

0.75±0.01490

(28.22)

*HPIMP- 2-((4-hydroxyphenylimino)methyl)phenol

Data are expressed as Mean±SEM, n=3 in each group, statistical analysis done by paired t test. P<0.5, compared to diclofenac sodium standard.

One way ANOVA
F	23.76647	166.7419	153.0789	277.1538
df	(2,9)	(2,9)	(2,9)	(2,9)
P	<0.5	-	-	-

Parameter	2-((4-hydroxyphenylimino)methyl)phenol
Parameter	1 Hour	2 Hour	3 Hour	4 Hour
t values	0.8532	0.3302	0.5963	0.5963
P-values	<0.5	-	-	-

Fig.3 . *STD-Standard

CNS depressant activity

Table-2.

Locomotor activity of 2-((4-hydroxyphenylimino)methyl)phenol .

Drug treatment	Dose (mg/kg)	Before treatment	After treatment	% Change in activity
caffeine	3 mg/ kg(i.p)	66.5± 5.4672	108.75± 3.9265	64.15
Chlorpromazine	30 mg/Kg (p.o.)	85.25± 10.5451	34.5±8.0693	60.08
HBIMP	(250 mg/kg)p.o	88± 0.8165	30.5± 10.8579	65.45

One way ANOVA
F	3.863022	39.38807
df	(2,9)	(2,9)
P	-	-

Parameter	2-((4-hydroxyphenylimino)methyl)phenol
Parameter	Before	After
t- values	0.2828	0.2608
P-values	-	-

The dose dependent depression in the locomotor activity was 60.08% for 3mg/kg of chlorpromazine and 65.45% for 250mg/kg of 2-((4- hydroxy phenylimino) methyl)phenol. Higher depressant activity was observed for 250mg/kg of 2-((4-hydroxy phenyl imino)methyl) phenol Schiff base than that of chlorpromazine.

PASS prediction activity

Table-3

PASS Prediction activity of 2-((4-hydroxyphenylimino)methyl) phenol

S.NO	Activity	Pa	Pi
1.	Laccase inhibitor	0.892	0.005
2.	3-Hydroxybenzoate 4-monooxygenase inhibitor	0.859	0.003
3.	Monophenol monooxygenase inhibitor	0.848	0.005
4.	Glutathione thiolesterase inhibitor	0.844	0.009
5.	Glucan endo-1,6-beta-glucosidase inhibitor	0.835	0.011
6.	Antiseborrheic	0.828	0.039
7.	Glucose oxidase inhibitor	0.811	0.030
8.	Alkane 1-monooxygenase inhibitor	0.804	0.014
9.	Corticosteroid side-chain-isomerase inhibitor	0.804	0.003
10.	Cysteamine dioxygenase inhibitor	0.802	0.009
11.	Arylacetonitrilase inhibitor	0.794	0.034
12.	Taurine dehydrogenase inhibitor	0.788	0.034
13.	Hydroxylamine reductase (NADH) inhibitor	0.785	0.007
14.	Anthranilate 3-monooxygenase (deaminating) inhibitor	0.779	0.003
15.	Cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase inhibitor	0.758	0.010

The PASS prediction of 2-((4-hydroxyphenylimino) methyl)phenol Schiff base showed the Laccase inhibitor (Pa=0.892) and 3-Hydroxybenzoate 4-monooxygenase inhibitor (Pa=0.859) activities. Monophenol monooxygenase inhibitor was found to be 0.848Pa. They can also exhibit Glutathione thiolesterase inhibitor (Pa=0.844), Glucan endo-1,6-beta-glucosidase inhibitor (Pa=0.835), Antiseborrheic (Pa=0.828), Glucose oxidase inhibitor (Pa=0.811), Alkane 1-monooxygenase inhibitor (Pa=0.804), Corticosteroid side-chain-isomerase inhibitor (Pa=0.804), Cysteamine dioxygenase inhibitor (Pa=0.802), Arylacetonitrilase inhibitor (Pa=0.794), Taurine dehydrogenase inhibitor (Pa=0.788), Hydroxylamine reductase (NADH) inhibitor (Pa= 0.785), Anthranilate 3-monooxygenase (deaminating) inhibitor (Pa=0.779) and Cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase inhibitor activities (Pa=0.758).

CONCLUSION:

The Schiff bases derived from salicylaldehyde and 4-Aminophenol exhibit anti-inflammatory and CNS drug activities. The anti-inflammatory activity of the Schiff base compared to the standard drug Diclofenac sodium(29.32%) was found to be higher in the third hour(48.21%) 2-((4-hydroxyphenylimino)methyl)phenol of 250mg/kg possessed highest CNS depressant activity (65.45%) than the standard chlorpromazine(60.08%). The PASS prediction of bioactivity have shown that the above schiff base was found to possess Laccase inhibitor,3-Hydroxybenzoate 4-monooxygenase inhibitor, Monophenol monooxygenase inhibitor activities greater than 80% and can also exhibit Arylacetonitrilase inhibitor, Taurine dehydrogenase inhibitor activity , Hydroxylamine reductase (NADH) inhibitor activities greater than 70%.

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Research J. Pharm. and Tech. 5(11):November, 2012; Page 1457-1460