Authors: Zian Anzam, Dr. M. Shenbagam Madhavan
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This study reports the antibacterial and antioxidant properties of cinnamaldehyde derivatives. DPPH, Superoxide Anion Radical, Hydroxy Radical, and Nitric Oxide Radical Scavenging Activities were used to assess the test compound\'s antioxidant capacity. The well diffusion method was used to examine the antibacterial efficiency against both Gram positive and Gram-negative microorganisms. Both Gram positive and Gram-negative bacterial strains responded favourably to the antibacterial effects of cinnamaldehyde derivatives. The study revealed the test substance possesses strong antibacterial and radical scavenging abilities.
Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), are created in biological systems as a result of oxygen reduction or the breakdown of xenobiotics. These radicals are unstable and can damage key biomolecules like lipid membranes or membrane proteins, leading to decreased fluidity, loss of enzymes, receptor activity and damage to membrane proteins. Cells have a variety of defence mechanisms to reduce free radical formation or limit its damaging effects. However, these mechanisms are not always sufficient to keep the balance in favour of free radicals’ generation. Oxidative stress is a problem that has been studied extensively for many years, and there are many compounds that can protect against it. Phytochemicals have been shown to scavenge free radicals and inhibit lipid peroxidation, which is a major concern seen in the food industry.
A variety of microorganisms can lead to food spoilage, so it is important to know which ones are present in food products. At first, synthetic chemicals were used to prevent oxidation and microbial contamination of dietary components.
But people are becoming more concerned about the side effects of synthetic compounds and they are in need for a safer material to prevent and control these pathogenic microorganisms that contaminate food and other natural products.
Phytochemicals have the potential to modify human metabolism in a way that is beneficial for the prevention of chronic and degenerative diseases that is commonly seen nowadays. In the present-day spices and aromatic plant materials are used to enhance the flavour and fragrance qualities of food materials in the food industry.
In addition, some natural products are also used as traditional medicine. Spices are a good source of natural antioxidants, and they play a huge role in preventing the growth of bacterial species. Cinnamon's antioxidant activity is due to the components present in them, like cinnamaldehyde, eugenol, cinnamic acid and cineol. Cinnamaldehyde is a member of the phenylpropanoid class of phytochemicals. This study reports the antioxidant potential and antibacterial activities of cinnamaldehyde derivative.
II. MATERIALS AND METHODS
A. DPPH (1,1-diphenyl-2-Picryl Hydraxyl) Radical Scavenging Assay
A dark room was used to measure spectrophotometrically the radical-scavenging capacity of cinnamaldehyde against DPPH. A hydrogen radical or an electron can be accepted by DPPH, to transform it into a stable diamagnetic molecule. DPPH reacts with an antioxidant molecule that can give hydrogen in order to get reduced. Measurements were made of the colour change (from deep violet to blue). According to the quantity and type of radical scavengers present in the sample, a certain degree of blue colour develops.
a. 1,1-diphenyl-2-picryl hydrazine.
In order to make 3ml of water, 1ml of DPPH and 1ml of various amounts of cinnamaldehyde derivative were added. The blue colour developed was measured at 517 nm using vitamin C as the standard.
III. RESULTS AND DISCUSSIONS
A single assay approach is insufficient to study the antioxidant capacity of endogenous substances. The assay concept and testing procedure for various antioxidant tests also varies. For instance, some techniques make use of DPPH or other organic radical producers. They differ from one another in terms of how long it takes for their chemical processes to result in free radicals through an oxidation reaction. Different antioxidants are taken into account as a control for distinct assay procedures according to the time they require for scavenging because the procedure and experimental circumstances differ for different techniques. Additionally, antioxidants can be either non-polar (like vitamin E) or polar (like phenolic, flavonoids, etc.) in nature, and they can scavenge free radicals via either an electron-donating mechanism or a hydrogen-donating mechanism. As a result, various control antioxidants (such AA) were utilised for various antioxidant assays.
A. DPPH Radical Scavenging Activity
According to Baumann et al. (1979), ability of antioxidant to donate hydrogen is assumed to be the reason for their impact on DPPH. Radical scavenging activities are particularly crucial to prevent the detrimental effect of free radicals in many diseases, including CVDs. Scavenging of DPPH free radicals is a common method for determining the antioxidant activity. In the DPPH assay, the addition of the extract reduces the violet colour of the DPPH solution to a yellow-coloured product. Due of the quick turnaround time for analysis, this approach has been widely utilised to predict antioxidant activity.
The dose-response curve of cinnamaldehyde's DPPH radical scavenging activity is depicted in this picture. Our findings showed that cinnamaldehyde has the same free radical-scavenging properties as standard vitamin C. It was clear that cinnamaldehyde had proton-donating ability and this might serve as free radical inhibitors or scavengers, possibly acting as major antioxidants, even if its DPPH radical scavenging skills were slightly lower than those of vitamin c. The amount of the cinnamaldehyde extract that inhibited the DPPH radical was 9.5, 17.4, 18.9, and 20.5% at 100, 200, 400, and 800 g/ml. The findings of this study indicate that the ability to donate hydrogen or to transfer electrons can enhance the ability to scavenge cinnamaldehyde radicals.
B. Nitric Oxide Scavenging Assay
Nitric oxide is a strong pleiotropic modulator of many physiological processes. It is a diffusible free radical that functions in a variety of biological systems as an effector molecule. Due to its extreme instability, it interacts with oxygen molecules to form stable nitrate and nitrite that may be measured using the NEDD reagent. The amount of nitrous acid, which is detectable at 546 nm, will decrease in the presence of a scavenging test agent. The below graph illustrates a large clearance of NO radical caused by the scavenging activity of cinnamaldehyde in a dose-dependent manner.
above graph plots percentage-free radical scavenging against the various cinnamaldehyde concentrations. Cinnamaldehyde had the highest percentage of inhibition at 22.5%, and its ability to scavenge NO rose as its concentration did as well. When cinnamaldehyde and vitamin c are compared in terms of the percentage of inhibition, cinnamaldehyde's inhibition rate is highly dependent on its concentration, and its percent of inhibition is comparable to vitamin c's.
C. Superoxide Anion
Although superoxide is seen as a relatively inactive oxidant with little chemical reactivity, it can nonetheless produce more hazardous species such singlet oxygen and hydroxyl radicals, which can lead to damaging of lipids. Numerous enzyme systems create these species. Thus, active free radicals that have the ability to react with biological macromolecules and cause tissue damage are considered as the precursors of superoxide anion. NBT will be decreased in this system by superoxide anions produced by the riboflavin/methionine/illuminate system from dissolved oxygen. In this procedure, the yellow dye (NBT2+) is reduced by the superoxide anion to create the blue formazan, which is detected spectrophotometrically at 560 nm. Antioxidants have the ability to prevent the production of blue NBT. Antioxidants cause the absorbance at 560 nm to drop, which shows that superoxide anion consumption has occurred.
The graph above demonstrates how effectively and efficiently cinnamaldehyde inhibits the synthesis of blue formazan in a dose-dependent way. The proportion of inhibitory concentration rises with dose, from 9.9% to 38.5%, at low concentrations of 100 g/ml to high concentrations of 800 g/ml.
D. Hydroxy Radical Scavenging Activity
OH radicals are short lived and can seriously harm other biomolecules and cell membranes. Therefore, OH radical scavenging is required to shield cells from oxidative harm. The Fenton reaction, which includes the transition metal-catalysed breakdown of hydrogen peroxide to form hydroxyl radicals, is the most significant of the numerous ways that OH radicals can be produced.
E. Effect Of Cinnamaldehyde On Antimicrobial Activity
The antibacterial activity of CAHD Sample against bacterial pathogens were tested by a well diffusion method.
The CAHD Sample revealed that, at the specific concentration of 100 µl, the zone of inhibition against Klebsiella pneumoniae was the largest (26 mm), followed by that against Streptococcus pyogenes (24 mm), Staphylococcus aureus (16 mm), and Escherichia coli (18 mm).
As per the findings of the present study, cinnamaldehyde was revealed to be an effective antioxidant by conducting a variety of in vitro assays, such as DPPH, hydroxy radical scavenging, nitric oxide, and superoxide anion, when compared to conventional antioxidants like vitamin C. At a concentration of 100 µl, the antibacterial activity of cinnamaldehyde was highly effective against Klebsiella pneumonia (26 mm), Streptococcus pyogenes (24 mm), Staphylococcus aureus (16 mm), and Escherichia coli (18 mm).
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