Anti-inflammatory and antioxidant activity of salicylic acid conjugated dihydropyrazoline analogues

© 2018 Naveen Puttaswamy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Shaukath Ara Khanum, Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysuru. E-mail: shaukathara @ yahoo.co.in Anti-inflammatory and antioxidant activity of salicylic acid conjugated dihydropyrazoline analogues


INTRODUCTION
Design and synthesis of nonsteroidal anti-inflammatory drugs (NSAIDs) is the important field in drug design, hence in recent years the newer strategy of synthesizing molecules for the inhibition of enzyme leading to inflammation is of special interest, PLA2 is a low molecular mass enzyme (Moeira et al., 2011) which is responsible for the release of arachidonic acid and lysophospholipid by catalyzing the hydrolysis of Sn2-ester bond of phospholipids.Arachidonic acid is precursor in the biosynthesis of eicosanoids and the lysophospholipid serves as a precursor for platelet activating factor, these products when produced in excess are responsible for chronic diseases such as cancer and autoimmune disorders (Dennis, 1997).Further, supported by extensive research and clinical evidences, it is found that pathophysiological conditions during inflammation are associated with depleting intrinsic antioxidants and generating free radicals causing oxidative stress.
Inspite, of profoundest development new molecules the urge to screen newer compounds for the development of new antioxidants which also specifically inhibit PLA2 remains constant, proenzyme in the inflammatory pathways, but the protecting cycloxygenase enzyme in gastric mucosa unlike the currently available nonsteroidal anti-inflammatory drugs and also bearing antioxidant property.

Celecoxib (4)
Bearing the above observations in mind, we have made an emphasis on synthesizing a salicylic acid integrated pyrazoline analogues and screening them for their antioxidant and phospholipase A2.

Materials and methods
The Materials and methods are clearly discussed in earlier reference (Naveen et al., 2017).

SYNTHESIS
Synthesis of the compounds 7a-j were clearly discussed earlier (Naveen et al., 2017), to a stirring solution of compound 5a (1.8 mmol) in absolute alcohol, compound 6a (1.9 mmol), was added followed by chloramines-T (2 mmol) the reaction mixture was refluxed on water bath.Further, the completion of the reaction was checked by TLC, the reaction mass was concentrated by evaporating the solvent, the reaction mass was dissolved in dichloromethane and the product was extracted by 10% sodium bicarbonate solution.The sodium bicarbonate extract was neutralizing with 5% hydrochloric acid to achieve compound 7a as solid, which was further purified by column chromatography on silica gel using petroleum ether and methanol as an eluent, compounds 7b-j was synthesized by similar method.

DPPH radical scavenging assay
The antioxidant potential of the synthesized compound was noted through free radical scavenging assay with slightly modified method of Manzocco et al., various concentrations of the synthesized compounds, ranging from 25 to 100 µmole/ml in methanol was added to 4 ml of 0.004% (w/v) of DPPH, prepared in methanol (Scherer and Godoy, 2009).The resulting mixture was incubated for 20 min at room temperature, and the absorbance was measured at 517 nm against a blank.The effective free radical scavenging activity was measured as the decrease in the absorbance of DPPH and calculated using the following equation Percentage of scavenging = 1 − (Absorbance sample (517 nm)/Absorbance control (517 nm)) × 100.

Nitric oxide radical scavenging assay
The title compounds were examined for Nitric oxide radical scavenging assay (Marcocci et al., 1994), the sodium nitroprusside (5 mM) in aqueous solution at physiological pH, spontaneously produces nitric oxide, thus produced nitric oxide reacts with oxygen to generate nitrite ions that can be measured by Griess reagent.Nitric oxide scavengers compete with oxygen leading reduced production of nitric oxide.Sodium nitroprusside (5 mM) in PBS was mixed with the synthesized compounds and kept at 25 o C for 2 hours.The above samples were treated with Griess reagent (1% sulphanilamide, 0.1% napthylethylenediamine dihyrdochlorid and 2% orthophosphoric acid).The diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine results in the formation of chromophore with an absorbance at 540 nm and referred to the absorbance of standard solutions of BHT treated in the same way with Griess reagent.The radical scavenging activity was measured using the equation as described for DPPH assay.

Ferrous ion chelating assay
Ferrous ion chelating activity was recorded according to the method of Suter and Richtes (Gordon et al., 1990).Control was prepared by adding FeCl 3 (200 mM) and K 3 Fe(CN) 6 (400 mM) and the volume was made to 1 ml using water.EDTA (40 mM) was used as positive control in the other set of reactions.Title compounds of different concentrations ranging from 2 to 10 µg/mL, FeCl 3 (200 µM) and K 3 Fe(CN) 6 (400 mM) were added.BHT was used as the reference compound.The tubes were kept for 10 min at room temperature and optical density was measured at 700 nm.The ion chelating activity was found by using formula as described for DPPH radical scavenging assay.

Hydroxyl radical scavenging assay
Hydroxyl radical scavenging assay of the synthesized compounds (Halliwell et al., 1987) was done by incubating the pyrazoline analogues with H 2 O 2 (1 mM), deoxyribose (2.8 mM), EDTA, FeCl 3 and ascorbic acid in phosphate buffer 0.02 M, pH 7.4 for 1 hour at 37 o C. 1% TBA was added to quench the reaction.The tubes were boiled in water bath for 20 min.The optical density was measured at 535 nm using a suitable reagent blank.The % radical scavenging activity was done using the formula as described for DPPH scavenging assay.

Anti-inflammatory activity by inhibition of PLA2
Protein concentration in the Russel viper venom was calculated (Lowry et al., 1957), using bovine serum albumin fraction (0-75 µg).PLA2 inhibition activity was calculated by (Boman et al., 1957).Indirect hemolytic assay, a semi quantitative method was employed.Briefly, egg yolk, packed human erythrocytes and phosphate buffer saline was mixed (1:1:8 V/V). 1 ml of this as substrate was incubated with 60 µg of enzyme which was pretreated with various concentration title compounds for 30 min at room temperature.9 ml of cold phosphate buffer saline was added to stop the reaction and centrifuged at 4 o C for 10 min at 1500 rpm.The released hemoglobin in the supernatant was measured at 540 nm.The assay was also performed in the presence of various concentrations 3, 6, 9, 12 and 15 µg/ml of test compound, and the percent enzyme inhibition was calculated.

Anti-inflammatory activity (PLA2 inhabition)
The synthesized compound 7d, with para hydroxy and methoxy group (Alam et al., 2016) substitution to the phenyl ring at 3 rd and 4 th position of the heyerocyclic ring, compound 7h, with chloro and methoxy groups (Geronikaki and Gavalas, 2006) at para position of the phenyl ring at 3 rd and 4 th position, 7i with chloro at meta and methoxy at para position at the phenyl ring at 3 rd and 4 th position respectively and 7a compound with no substitution showed maximum inhibition with IC 50 value of 0.020, 0.019, 0.018 and 0.015 µmole/mL respectively which is followed by compound 7j, having chloro group at para position of the phenyl ring attached to 3 rd and 4 th position, compound 7b bearing chloro and bromo group at para to the phenyl ring attached to 3 rd and 4 th position and compound 7f with chloro at meta and hydroxy to para position of phenyl ring attached to 3 rd and 4 th position of heterocyclic moiety showed moderate inhibition with an IC 50 values of 0.022, 0.025 and 0.024 µmole/mL respectively.Compounds 7c, 7e and 7g showed least potency with IC 50 values of 0.026, 0.027 and 0.027 µmole/mL respectively.

Antioxidant assays
The title compounds 7a-j are screened for their in vitro antioxidant activity by DPPH (Di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium) radical scavenging, nitric oxide radical scavenging, ferrous ion chelating and hydroxyl radical scavenging assays, which are represented in Table 1.Compound 7d with para hydroxy and methoxy groups substitution (Burguete et al., 2007) at phenyl ring attached to 3 rd and 4 th position of heterocyclic ring, 7f bearing chloro group at meta position and hydroxyl substitutions at the para position of phenyl ring attached to 3 rd and 4 th position of heterocyclic ring respectively and compound 7h with chloro and methoxy groups both para to the phenyl ring at 3 rd and 4 th position of heterocyclic ring has shown significant antioxidant activity Compound 7a with no substitution, 7b with chloro and bromo group at para position of phenyl ring attached to 3 rd and 4 th position of heterocyclic ring respectively, 7c bearing chloro at meta position of phenyl ring attached to 3 rd position and bromo group substituted to para position of phenyl ring of 4 th position, 7e bearing chloro group at meta and para position of phenyl ring at 3 rd and 4 th position respectively, 7g with chloro substitution at para and meta position of phenyl ring at 3 rd and 4 th position of the heterocyclic ring, 7i with chloro substitution at meta position and methoxy at para position of the phenyl ring attached at 3 rd and 4 th position of the heterocyclic ring and 7j bearing chloro substitutions at the para position of phenyl ring attached to 3 rd and 4 th position of heterocyclic ring showed moderate activity in comparison with the standard BHT (2,6-di-tert-butyl-4-methyl phenol).

CONCLUSION
In this present study we have the synthesized the series of salicylic acid integrated dihydropyrazoline analogous (7a-j), which were then evaluated for PLA2 inhibition as well as anti-oxidant activity.All the molecules have exhibited good anti-oxidant activity and in addition the study reveals that the compounds with donating substitution have shown excellent anti-oxidant activity, whereas the compounds with hydroxy and chloro groups have shown good PLA2 enzyme inhibition, which opens up for the further study of the synergetic effect of the two biologically potent molecules for the discovery of new bioactive molecules.

Table 1 :
Antioxidant and Anti-inflammatory activity of synthesized compounds.