INTRODUCTION:

Piperonal also known as helicotropin is an organic compound commonly found in fragrances and flavors. Piperonal is structurally related to aromatic aldehydes such as benzaldehyde and vanillin. Piperonal naturally occurs in various plants like vanilla, black pepper etc¹. Like all aldehydes it can be reduced to its alcohol (piperonyl alcohol) or oxidized to give its acid (piperonylic acid). Piperonal contains a benzodioxole ring system². Due to promising bioactivity of Piperonal derivatives, recently piperonal is subjected for various synthetic conversions. Piperonal nitro derivatives have shown a considerable anti-fungal activity³. Other derivatives such as Pyrazoline and Thiazepine and subsequent derivatives have found to possess anti-cancer, anti-oxidant, anti-bacterial, anti-malarial and anti-mycobacterial activities.

In summary Piperonal derivatives have shown a wide spectrum of pharmacological and therapeutic application. In synthetic chemistry, a series of novel flavone derivatives has been synthesised and reported as antioxidants⁴. Similarly, pyrimidine derivatives were synthesized, screened and reported for antimicrobial and antioxidant activities by Filter disc method and DPPH method, respectively⁵.

Piperonal, a naturally occurring derivative of piperine compound, is an aromatic aldehyde and thus, modifications in piperonal could produce compounds which have the same actions of piperine. Several groups previously reported the synthesis and pharmacological activities of piperonal derivatives. The chemical synthesis of 3,4–methylenedioxyphenyl-2-nitro propene from piperonal in presence of nitroethane and sodium hydroxide has also been reported⁶. Aldol condensation reaction of piperonal and acetophenone in the presence of ethanol and sodium hydroxide was also reported⁷. NMR spectroscopy, Hammett correlations and antimicrobial potency of some of the Schiffs bases derived from piperonal has been proved ⁸. Schiffs base derivative of isatin and its analogs has also been synthesised and found to exhibit DPPH radical scavenging activity⁹.

Ethylenediamine (abbreviated as en when a ligand) is the organic compound with the molecular formula C₂H₄(NH₂)₂. It is a colorless liquid with an ammonia-like odor and also strongly basic in character. It is a widely used building block in chemical synthesis for conjugation and preparation of other derivatives, with approximately 500,000 tonnes produced in 1998. Ethylenediamine readily reacts with moisture in humid air, producing a corrosive, toxic and irritating mist, to which even short term exposures can cause serious damage to health. Ethylenediamine is the first member of series of the so-called polyethylene amines. Ethylenediamine is used in large quantities for production of major synthetic industrial chemicals. It forms derivatives with carboxylic acids (including fatty acids), nitriles, alcohols (at elevated temperatures), alkylating agents, carbon disulfide, and aldehydes and ketones. Ethylene diamine is an organic compound with bifunctional nature, having two amines, due to which it readily forms heterocycles such as imidazolidines^10.

An antioxidant is "any substance that delays or prevents oxidation of any oxidisable species/substrates". Oxidative stress, induced by reactive oxygen species (ROS) produced in the body, is one of the main factors of current slow killer diseases and that population suffering from, like cancer, diabetes, cardiovascular, neurological (Alzheimer's and Parkinson's), inflammatory viral diseases and digestive disorders¹¹. Various chalcones with 3-nitro acetophenone were synthesized, characterized and screened for their antibacterial and antioxidant activities and reported¹². A novel series of flavonoid Derivatives using substituted benzaldehyde were synthesized and screened for biological activities like anticancer and antioxidant activity. Their work has revealed the scope and potential of flavonoids for future exploration to synthesize substituted flavonoids and chalcone derivatives by the use of different substituted benzaldehyde¹³. Hence, in the present work, the schiffs base conjugated piperonal using ethylene diamine was studied for antioxidant and free radical scavenging potential by various invitro methods.

MATERIALS AND METHODS:

Sample preparation:

Commercial Piperonal was used as reference compound for the investigation and was purchased from Sigma-Aldrich chemicals. The compound Ethylene diamine to be conjugated with piperonal was obtained from Hi-media. Solutions of Ethylene diamine (0.84 g) and Piperonal (1.49 g) were prepared in ethanol. Both the solutions were mixed in a ratio of 1:1 and refluxed for three hours using magnetic stirrer under ice cold condition which resulted in the formation of a white solid substance. This substance was separated out and washed several times with distilled water. Then this crude sample was recrystallized from ethanol¹⁴.

In vitro antioxidant assays of Ethylene Diamine Conjugated Piperonal:

DPPH Radical Scavenging Assay:

The scavenging ability of the synthesised piperonal to remove the Free radicals was measured by DPPH, following the method of Blois ¹⁵. Briefly, 0.1 mM solution of DPPH in methanol was prepared, and 1ml of this solution was added to the test samples: ethylene diamine conjugated and unconjugated piperonal in the concentration range of 100-500 µg/ml. The mixture was shaken well and incubated at room temperature for 30min. Then the absorbance was read at 517 nm in a spectrophometer.

ABTS Radical Scavenging Assay:

ABTS radical elimination activity was measured using the method ¹⁶. First, ABTS solution (2mM) was prepared in 1M phosphate buffer (pH 7.4). ABTS radicals were produced by adding 2.45nM persulfate solution to the mixture. Next, the absorbance of the control solution at 734 nM was adjusted to 700 ± 0.025 nm using phosphate buffer (0.1 M and pH 7.4). ABTS radical solution (0.5 mL) was added to different concentrations (100-500 µg/ml) of the test samples used in this study and incubated for 30min. The absorbance was measured against an ethanol blank at 734 nm.

Hydrogen peroxide scavenging activity:

The hydrogen peroxide scavenging assay was determined by modification of the procedure ¹⁷. The experimental setup consist of 5 test tubes. The solution of hydrogen peroxide (H₂O₂, 10 mM) was prepared in phosphate buffer (0.1 M, pH 7.4). 3.4ml of phosphate buffer was mixed with 0.6 ml of H₂O₂ solution (0.6 ml, 43 mM). and test solution of various concentration of 100-500 µg/ml [1ml] was added to respective test tubes. The absorbance value of the reaction mixture was recorded at 230 nm after 10 minutes incubation at room temperature. Blank solution contains sodium phosphate buffer without H₂O₂. Ascorbic acid with same concentration was used as the standard. The varied concentration in the range of 100 to 500 µg/ ml were taken for both standard and test samples. Control solution containing buffer and H₂O₂were taken.

Nitric Oxide Radical Scavenging Assay:

The method of Garrat was adopted to determine the nitric oxide radical scavenging potential of the synthesized molecules¹⁸. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions estimated by the use of Griess reagents. 2ml of 10 mM sodium nitroprusside was dissolved in 0.5ml phosphate buffer saline (pH 7.4) to which 0.5ml of sample solution at various concentrations (100-500 µg/ml) was added and vortexed. The mixture was incubated at 25^∘C. After 150 min, 0.5 ml of incubation solution was taken in another test tube and 0.5ml of Griess reagent was added. Griess reagent was prepared by mixing equal amounts of 1% sulphanilamide in 2.5% phosphoric acid and 0.1% naphthylethylene diamine dihydrochloride in 2.5% phosphoric acid immediately before use. The mixture was incubated at room temperature for 30 min and the absorbance was spectrophotometrically read at 540 nm.

Deoxyribose Radical Scavenging Assay:

Deoxyribose non-site specific hydroxyl radical scavenging activity of test solution was estimated. Briefly, 2.0 ml aliquots of each of test samples of varied concentration ranging from 100-500 µg/ml were added to the test tube containing reaction mixture of 2.0 ml FeSO₄.7H₂O (10mM), 0.2 ml EDTA (10mM) and 0.2 ml deoxyribose (10mM). The volume was made upto 1.8 ml with 0.1M phosphate buffer (pH-7.4) and finally 0.2 ml H₂O₂ (10mM) was added. The mixture was incubated at 37°C under dark for 4 hours. After incubation, 1 ml of 2.8% TCA and 1%TBA were added and kept in boiling water bath for 10 min. After treatment and subsequent cooling, absorbance was measured at 532nm. If the mixture was turbid, the absorbance was measured after filtration. Ascorbic acid was used as standard¹⁹. Control tube was also measured containing only reagents.

Superoxide Radical Scavenging Assay:

Scavenging of superoxide radical was studied using the method elaborated²⁰. Assay tubes contained 0.2 ml of the test samples (corresponding to 100-500 µg/ml) with 0.2 ml EDTA (12mM), 0.1 ml Nitro blue tetrazolium, 0.05 ml riboflavin (20µg) and 2.64 ml phosphate buffer (50 mM, 7.6 pH) were taken. The control tube contains DMSO (Dimethyl sulfoxide) solution instead of the test solution. The initial optical densities of the solutions were read spectrophotometrically at 560 nm and the tubes were illuminated uniformly for 30mins with the fluorescent lamp. A560 was measured again and the difference in O.D was taken as the quantum of superoxide production. The percentage of inhibition by the test samples was calculated by comparing with O.D of the control tubes²⁵. The varied concentration ranging from 200 to 1000 µg/ ml were taken for both standard and test samples.

% Inhibition= (Absorbance of control- Absorbance of test) / Absorbance of control × 100

Ferric reducing antioxidant Power (FRAP):

The reduction power as FRAP was determined using the method of Oyaizu with slight modification ²¹. The test samples of varied concentrations ranging from 100-500 µg/ml were prepared from the 1 mg/mL stock solutions of the conjugated and unconjugated piperonal and standard. The sample volume in the tubes was made upto 0.5 mL using acetate buffer (pH 3.6). Subsequently, 2.25 mL of FeCl3 solution and 2.25 mL of FRAP reagent were added to each tube (total volume 5 mL). After incubation at room temperature for 10 min, the absorbance of the mixture was read at 593 nm against the blank. Acetate buffer was used as a blank control sample.

Determination of Radical Scavenging Acticity:

The capability of the test sample to scavenge the free radicals were calculated using the following equation: Radical Scavenging effect (%) = (A0 – A1/A0) x 100 Where, A0 is the absorbance of the control reaction and A1 is the absorbance in the presence of the samples or standards.

Estimation of lipid peroxidation using egg yolk:

Inhibitions of lipid peroxidation in the egg of hen was determined using a modified method thiobarbituric acid- reactive species (TBARS) assay^{26, 27, 28 and 29}. 0.5ml of Egg homogenate (10% in distilled water, v/v) and 0.1 ml of respective test samples were mixed separately in a test tube and the volume in each tube was made up to 1 ml, by adding distilled water. Finally, 0.05ml of 0.07MFeSO₄ was added to the above mixture to induce lipid peroxidation and incubated for 30 min. Subsequently, 1.5 ml of 20% acetic acid and 1.5 ml of 0.8% TBA (w/v) in 1.1% sodium dodecyl sulfate (SDS) and 0.05 ml 20% TCA was added, vortexed and then heated in a boiling water bath for 60 min. After cooling, 5ml of butanol was added to each tube and centrifuged at 3000rpm for 10 min. The absorbance of the separated organic upper layer was measured at 532 nm. The varied concentration in the range of 100-500 µg/ml were taken for both standard and test samples along with the control tube.

% Inhibition of lipid peroxidation = (Absorbance of control- Absorbance of test) / Absorbance of control × 100

RESULTS AND DISCUSSION:

Natural antioxidants are characterized based on their ability to scavenge free radicals. The in vitro scavenging assay was done spectrophotometrically with positive control. Proton-radical scavenging action is a significant attribute of antioxidants, which is measured by the DPPH (2,2- diphenyl-1-picrylhydrazyl) scavenging assay. DPPH, a protonated radical will show maximum absorbance at 517 nm that tends to decrease in the presence of antioxidant because of the scavenging effect of the test sample on the proton radical²⁶. In the present study, DPPH radical scavenging activity was higher even at the lowest concentration and are comparable with the positive drug. The conjugated compound showed good antioxidant activity in DPPH scavenging assay (67.24% - 500 µg/ml) with IC₅₀ value of 386.940 µg/ml when compared to unconjugated piperonal with 432.06 µg/ml and ascorbic acid with 403.815 µg/ml. Test sample exhibits ABTS scavenging activity at lower IC₅₀ values 258.445 µg/ml respectively; as compared to the standard drug 302.609 µg/ml. The percentage of inhibition was higher in test sample i.e. 84.90% than standard that shows 78.75 % at the concentration 500 µg/ml (figure 1). The coupled antioxidant and antidiabetic activity of 2,4-thiazolidone derivatives has been reported²⁷. A series of 5-substituted-arylidene-3-substituted-benzyl-thiazolidine-2, 4-dione derivatives followed by the addition of aromatic aldehydes were synthesized and all the compounds were screened for their in vitro antioxidant activity using DPPH method. The anisaldehyde based thiazolidinedione compounds displayed good activity comparable to those of standard²⁸.

Hydrogen peroxide has strong oxidizing properties. The ability of extract to scavenge hydrogen peroxide was determined and our results demonstrated the presence of higher hydrogen peroxide scavenging activity (figure 2). Hydrogen peroxide scavenging activity of the positive drug at the concentration 100 µg/ml was found to be 23.68% while test sample exhibited 26.94% at the same concentration, respectively. Whereas at concentration 500 µg/ml the test was found to be 88.80% and standard drug shows 84.26%. The overall result of hydrogen peroxide shows that test sample gained higher activity than the standard drug. The hydroxyl radical is an extremely reactive free radical formed in biological systems which has been implicated as a highly damaging species in free radical pathology and is capable of damaging biomolecules found in living cells²⁹.

Nitric oxide is a very unstable species that produce stable nitrate and nitrite when reacting with oxygen molecule. In the presence of a scavenging test compound, the amount of nitrous acid will decrease which can be measured at 546 nm. The ethylene diamine conjugated piperonal has potent nitric oxide activity with IC₅₀ value of 253.457 µg/ml and standard shows 279.100 µg/ml while piperonal showed IC₅₀ value of 253.457 µg/ml. The test sample and standard showed maximum activity of 85.92% and 82.45% respectively at 500 μg /ml, whereas at the concentration 500 µg/ml both the sample exhibited 27.44% and 23.00% inhibition respectively (figure 2).

Figure 1: DPPH and ABTS radical scavenging assay of ethylene diamine conjugated piperonal.

Figure 2: Hydrogen peroxide and NO scavenging activity of ethylene diamine conjugated piperonal.

Figure 3: Deoxy ribose and Superoxide radical scavenging activity of ethylene diamine conjugated piperonal.

Figure 4: Ferric reducing antioxidant Power (FRAP) of ethylene diamine conjugated piperonal.

Figure 5: Estimation of lipid peroxidation of ethylene diamine conjugated piperonal using egg yolks.

The reducing ability of any compound generally depends on the presence of reductants/reducing nature which have been exhibiting antioxidative potential by breaking the free radical chain and donating a hydrogen atom. In present study, the percentage of inhibition of deoxy radical increases as the concentration of the sample increases. The percentage of inhibition was from 29.96% at 100 µg/ml to 78.66% at 500 µg/ml for test sample and for standard they were 34.06% at 100 µg/ml and 85.23% at 500 µg/ml. The IC₅₀ value of both test and standard was 260.203 and 213.581 µg/ml. Similarly, Ethylene diamine conjugated piperonal showed a maximum activity of 84.14% inhibition of SO radical which is comparable to that of positive drug with an activity of 88.70% at the same concentration of 500 µg/ml. The IC₅₀ value of the test was 251.133 µg/ml whereas the standard exerted 230.790 µg/ml (figure 3).

Ferric reducing activity of the test sample ethylene diamine conjugated piperonal and standard was represented in figure 4. The FRAP reducing ability of the test samples showed an increase in reduction of ferric ions in a dose dependent manner. As represented in figure 5, test sample significantly showed higher lipid peroxidation inhibition compared with the standard drug. Test sample and standard have IC50 values of 272.866 and 313.030 µg/ml, respectively. These results implied that test samples have the highest scavenging activity of 84.32% when compared with the standard 78.01%.

The present investigation demonstrates the antioxidant effect of ethylene diamine conjugated piperonal compound by various methods. The bioactivity of piperonal has been reported by several studies demonstrating their antioxidant, antimicrobial, antifungal activity against different organisms^30,31. The synthesis of various substituted chalcones and their 1,3-dipolar cycloaddition with appropriate aldoximes to give spiro compounds and heterocyclization using amines to yield isoxazolines and pyrazolines has been studied and reported³². All the compounds were also screened for their antimicrobial and antitubercular activity. Their results have shown that among the spiro compounds, the one having benzo[d] [1,3]dioxole as a substituent showed excellent activity against both bacterial and fungal strains, however none of the compounds were found to exert antitubercular activity. The active compounds of these series would be promising structural templates for the development of novel and more efficient antimicrobial agents.

A series of 5-substituted-arylidene-3-substituted-benzyl-thiazolidine, 2,4-dione derivatives were synthesized and all the compounds were screened for their in vitro antioxidant activity using DPPH method. The anisaldehyde based thiazolidinedione compounds were reported to exhibit good activity comparable to those of standard³³. A series of 2-((2-3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-(4hydroxyphenyl) propanoic acid (3a) and its analogs were synthesized from N,N′-Dicyclohexylcarbodiimide (DCC) by acetylating and finally condensing with amino acids to yield good antioxidant molecules which are showing the potency nearer to ascorbic acid and exploring their anti inflammatory activity³⁴. Likewise, series of substituted pyrazolo [4, 5-e]-4H-pyrimido [2, 3-b] [1, 3] benzimidazoles were synthesised, screened and proved for their antioxidant activity³⁵.

The efficacy of piperonal as a potent anti-obesity agent, provide scientific evidence for its traditional use and the possible mechanism of action has also been demonstrated³⁶. Vidhya K R and Syed Shafi S, 2014 have successfully synthesised a new series of 2-pyrazoline derivatives and moreover, some of compounds contains bioactive heterocyclic moiety^37. The antimicrobial screening suggests that all the newly synthesised compounds showed moderate to good antimicrobial activity against the tested organisms. Mathew A et al., 2011 research work focused on design and development of pyrazole analogues of piperine as novel anticancer agents and anti-inflammatory drugs³⁸. The analogues also showed good binding affinity with Cycloxygenase and farnesyl transferase receptors, which was proved from their insilico docking studies. Thus, the derivatives of piperonal have been confirmed for their different therapeutic role in combating clinical symptoms of disease involving cellular oxidative damage.

CONCLUSION:

The data of the present study clearly demonstrated the antioxidant and free radical scavenging potential of conjugated and unconjugated piperonal. In all the assays, the activity of EDP is comparable to that of standard. The potent ability of the conjugated piperonal is due to the schiffs base conjugation. The antioxidant and cytotoxic role of many piperidine and chalcone derivatives has been reported. Hence, the novel conjugated piperonal has the effect of reducing oxidative stress and can be explored for various pharmacological activities that requires further validation.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors would like to thank the management of Mohamed Sathak College of Arts and Science for providing all the lab facilities to carry out the research work.

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Received on 25.06.2021 Modified on 27.11.2021

Research J. Pharm. and Tech 2022; 15(11):4987-4993.

DOI: 10.52711/0974-360X.2022.00838