INTRODUCTION:

Free radicals are reactive molecules possessing an unpaired electron produced continuously in cells either by leakage from mitochondrial respiration or as by-products of metabolism (De Zwarttet.al 1999). The free radicals produced in the living organism include the active oxygen species such as super-oxide radical O₂^-, hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl).(V. Loboet.al 2010)

Oxygen consumption during metabolism involves the perpetual generation of free radicals and reactive oxygen species (ROS) (M. Gupta et.al 2007). Active oxygen species H₂O₂ and O₂ interact in the presence of certain transition metal ions yielding highly- reactive oxidizing species like hydroxyl radical (OH). The OH being one of the ROS formed in biological systems rapidly reacts with molecules such as sugars, DNA bases, amino acids and lipids in the living cells. (Duh, et al 2001).

Oxygen free radicals have been shown to cause many pathological conditions such as DNA damage, lipid per-oxidation, and protein damage. Free radicals and ROS are known to be involved in the pathogenesis of a wide variety of clinical disorders as cancer, cardiovascular diseases, inflammatory diseases, asthma and aging (Vani et.al 1997; Slater 1984). Free radicals like the hydroxyl radical, hydrogen peroxide and superoxide anion produce migratory factors, cyclic nucleotides and eicosanoids by mediating components of inflammatory response. These superoxide radicals amplify the intensity of inflammation process by increasing the vascular permeability and by adhesion of polymorphous nuclear leukocytes to the endothelium and also stimulate the platelet aggregation (V. Lobo, et al 2010)

Some enzymatic and non-enzymatic systems exist in the bodies which are involved in the detoxification process like superoxide dismutase, catalase. Antioxidants are required in generous amount to inactivate the production of reactive oxygen species (ROS) which are inevitable resultants of aerobic metabolism. The pro-oxidants and antioxidants may be disrupted during the course of inactivation of ROS leading to oxidative stress and this can have an effect on all types of biological molecules. (Valentão, et.al. 2001)

Antioxidants are a group of substances which often oxidize themselves and significantly inhibit or delay oxidative process when present at low concentrations in relation to oxidizable substrates.

Phytochemicals in plants possess antioxidant properties due to the presence of substituted groups such as carbonyl, phenolic, phenyl side chain, electron withdrawing group, electron donating group etc., Phenolic antioxidants convert the radical to stable non radical product by donating hydrogen to the radical (Tripathi 1998).

PRINCIPLE:

The principle of ABTS assay relies on the scavenging of light by ABTS radicals. An antioxidant with an ability to donate a hydrogen atom will quench the stable free radical. This process is associated with a change in absorption and can be measured using a spectroscope. The relatively stable ABTS radical is quantified in ELISA plate reader at 734nm and has a green color.

MATERIALS:

· PBS (Phosphate Buffered Saline -125mM NaCl in 10mM Sodium phosphate buffer, pH 7.4) : 0.14196g of Di-sodium hydrogen orthophosphate, 0.1560g of Sodium dihydrogen orthophosphate and 0.7305g of Sodium chloride dissolved in 25ml of distilled water. The pH was adjusted to 7.4 using dilute sodium hydroxide solution and the volume was made up to 100ml with de-ionized water.

· ABTS (2, 2’-azinobis-ethyl-benzothiozoline-6-sulphonic acid) (7mM): 38.4mg of ABTS dissolved in PBS and the volume made up to 10ml.

· APS (Ammonium per sulfate) (2.45mM):5.59mg of APS dissolved in PBS and the volume made up to 10ml.

· ABTS (2,2’-azinobis-ethyl-benzothiozoline-6-sulphonic acid) Radical solution:

a) Stock solution: 10ml of ABTS (7mM) and 10ml of APS (2.45mM) solutions were mixed and incubated in dark for 16 hours at room temperature.

b) Working solution: 1.7ml of the stock solution was made up to 50ml with PBS to give an absorbance of 1.000

· Quercetin (standard): 1mg made up to 1ml with PBS.

Procedure:

Mixture of ABTS and APS (Ammonium per sulfate) are incubated in dark for 16 hours at room temperature to produce ABTS radical cations. The solution obtained was diluted with PBS to give an absorbance of 1.000. Different concentrations of reference standard (highest volume taken was 50ml) and of the test sample are added to 225 µl of ABTS working solution and made up by adding PBS to give a final volume of 250µl. The absorbance was recorded at 734nm immediately and percent inhibition is calculated at different concentrations. The IC₅₀values were calculated by using Graph pad prism. (Auddy (2003) and L. Lavanya, et al., 2020)

RESULTS AND DISCUSSION:

Quercetin, an antioxidant invitro contains poly phenolic chemical substructure that stops or inhibits the oxidation (loss of electrons) by acting as scavenger of free radicals. In this study, the plant products are assessed against the control as Quercetin for its antioxidant properties and the results are tabulated below.

Table 1: ABTS radical scavenging activity of Quercetin as standard.

Standard
sample	con	OD 734	% inhibition	IC50
Quercetin	control	0.66	0	5.86
	0.312	0.61	7.44
	0.625	0.57	13.85
	1.25	0.48	27.12
	2.5	0.43	34.2
	5	0.34	48.05
	10	0.22	66.4
	20	0.11	82.11

Fig 1: ABTS radical scavenging activity of Quercetin as standard.

Table 2: ABTS radical scavenging activity of MEOH. MEOH

Methanol
sample	con	OD 734	% inhibition	IC50
Cinnamon	control	0.66	0	38.96
	3.125	0.61	6.95
	6.25	0.59	10.4
	12.5	0.51	22.67
	25	0.39	40.99
	50	0.27	59.17
	100	0.16	75.48
Cumin	3.125	0.62	5.93	53.12
	6.25	0.59	9.8
	12.5	0.52	20.41
	25	0.46	30.51
	50	0.34	47.64
	100	0.24	63.77
Pepper	3.125	0.61	6.93	48.53
	6.25	0.57	13.64
	12.5	0.53	19.91
	25	0.43	34.07
	50	0.32	50.52
	100	0.22	65.62
Clove	6.25	0.60	8.14	107.7
	12.5	0.54	17.06
	25	0.52	21.15
	50	0.44	32.23
	100	0.34	48.56
	200	0.24	62.73
Star anise	3.125	0.57	13.43	18.09
	6.25	0.49	25.62
	12.5	0.39	40.32
	25	0.29	54.8
	50	0.14	78.66
	100	0.11	83.03

Fig 2: ABTS radical scavenging activity of MEOH .

Fig 3: ABTS radical scavenging activity of clove MEOH.

Table 3: ABTS radical scavenging activity of Chloroform.

Chloroform
sample	con	OD 734	% inhibition	IC50
Cinnamon	control	0.66	0.00	142
	6.25	0.62	5.99
	12.5	0.59	10.19
	25	0.54	17.22
	50	0.46	29.59
	100	0.39	40.03
	200	0.26	59.73
Cumin	3.125	0.56	14.80	51.25
	6.25	0.54	17.53
	12.5	0.48	26.61
	25	0.42	35.41
	50	0.30	53.47
	100	0.23	65.11
Pepper	3.125	0.57	13.55	34.97
	6.25	0.53	19.85
	12.5	0.45	30.95
	25	0.36	44.78
	50	0.25	61.80
	100	0.16	75.77
clove	3.125	0.53	19.82	33.23
	6.25	0.49	25.80
	12.5	0.40	38.80
	25	0.35	46.85
	50	0.24	63.52
	100	0.15	76.14
Star anise	3.125	0.58	11.56	42.46
	6.25	0.55	15.69
	12.5	0.48	26.46
	25	0.38	41.23
	50	0.27	58.73
	100	0.17	74.18

Fig 4: ABTS radical scavenging activity of chloroform.

Fig :5: ABTS radical scavenging activity of cinnamon chloroform.

CONCLUSION:

Spices Cumin, Pepper, Clove, Star anise and Cinnamon in chloroform exhibited convincing antioxidants when compared to Quercetin in ABTS free radical assay. The IC50 values are compared for the above plant products and tabulated. Among all the samples Cinnamon has exhibited the highest IC50 value being 142 in chloroform extracts and the lowest being 18.09 is of star anise in methanol extracts. Although all the spices contain the antioxidant properties, Cinnamon seems to be having convincingly high concentration of antioxidants.

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Received on 16.07.2022 Modified on 14.08.2022

Research J. Science and Tech. 2022; 14(4):213-218.

DOI: 10.52711/2349-2988.2022.00035