1-diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging activity ⁽³⁾

Principle

1,1-diphenyl-2-picrylhydrazyl(α,α-diphenyl-β picrylhydrazyl; DPPH [1] is characterised as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, so that the molecules do not dimerise, as would be the case with most other free radicals. The delocalisation also gives rise to the deep violet colour, characterised by an absorption band in ethanol solution at about 520 nm.

When a solution of DPPH is mixed with a substance that can donate a hydrogen atom, then this gives rise to the reduced form [2] with the loss of this violet colour(although there would be expected to be a residual pale yellow colour from the picryl group still present). Representing the DPPH radical by Z• and the donor molecule by AH, the primary reaction is

Z• + AH = ZH + A•

Where ZH is the reduced form and A• is free radical produced in this first step. This latter radical will then undergo further reactions which control the overall stoichiometry, that is, the number of molecules of DPPH reduced (decolorised) by one molecule of the reductant.

The reaction is therefore intended to provide the link with the reactions taking place in an oxidising system, such as the autoxidation of a lipid or other unsaturated substance; the DPPH molecule Z• is thus intended to represent the free radicals formed in the system whose activity is to be suppressed by the substance AH

[1] [2]

1,1-diphenyl-2-picryl-hydrazyl1,1-diphenyl-2-picryl-hydrazine

Reagents required

1, 1- diphenyl-2-picryl hydrazyl (DPPH)

Preparation of test material

Extraction of Trigonella foenum graecum seeds was carried in soxhlet extractor using methanol solvent. The dried seeds were powdered, then extraction followed with methanol. After completion of extraction the solvent was evaporated in evaporating dish on the water bath to obtain dry extract. Extract of Trigonella foenum graecum were reconstituted in methanol to get different concentration like 10, 20, 40, 60, 80 and100 μg/ml and used for assay.

Herbomineral complex of Catechin, Curcumin and Trigonella foenum graecum extract was formed with zinc. Complex was formed in the form of precipitates, solvent was evaporated in evaporating dish and dried precipitates were collected and reconstituted in methanol to get different concentration like 10 ,20 , 40, 60, 80 and100 μg/ml and used for assay.

Methodology⁽⁴⁾

 DPPH, a stable nitrogen centered radical was used to assess the hydrogen donating ability of Catechin, Curcumin, methanolic extract of Trigonella foenum graecum and their complexes, as it offers a convenient and accurate method because of the relatively short time required for analysis.

 The reaction mixture consisted of 1 ml of 0.1mM DPPH in methanol, 0.95 ml of 0.05 M Tris-HCl buffer (pH 7.4), 1 ml of methanol and 0.05 ml of methanolic solution of Catechin complex, Curcumin complex and extract of Trigonella foenum graecum and its complex at various concentrations (10, 20, 40, 60, 80 and 100 µg/ml).Catechin, Curcumin being a phenolic compound was used as positive control, added to a series of other test tubes instead of the complexes

 The absorbance of the mixture was measured at 517 nm exactly after 30 mins.

 The % of scavenging activity was determined by comparing the result of Catechin complex, Curcumin complex, Trigonella foenum graecum extract complex with those of standard antioxidant Catechin, Curcumin and Trigonella foenum graecum extract.The radical scavenging activity was expressed as the inhibition percentage and monitored as per the equation:

Inhibition (%) = [(control − test) ×100] / control

The result was expressed as IC₅₀value that is the concentration of extract required for 50% inhibition of DPPH radicals

The inhibition in free hydroxyl radical scavenging activity of Catechin complex, Curcumin complex and Trigonella foenum graecum extract and its complex were calculated using following graphs and the results are as shown in the table 1.3

Figure 1.2 Plot of % Inhibition Vs Concentration Catechin hydrate

Figure 1.3 Plot of % Inhibition Vs Concentration Catechin hydrate complex

`Figure 1.4 Plot of % Inhibition Vs Concentration Curcumin

Figure 1.5 Plot of % Inhibition Vs Concentration Curcumin complex

Figure 1.6 Plot of % Inhibition Vs Concentration Trigonella foenum graecum seed extract

Figure 1.7 Plot of % Inhibition Vs Concentration Trigonella foenum graecum seed extract complex

Table 1.3Results of free radical scavenging activity by DPPH

Extract	Concentration (µg/ml)	% Inhibition	IC₅₀ (µg/ml)
Catechin hydrate	10	28.56	35.71
	20	46.56
	40	58.12
	60	63.77
	80	73.35
	100	75.98
Catechin hydrate complex	10	30.03	26.31
	20	45.20
	30	69.85
	40	77.09
	60	80.51
	100	80.94
Curcumin	10	4.77	60.29
	20	23.68
	40	32.06
	60	45.26
	80	70.54
	100	77.61
Curcumin complex	10	8.54	53.53
	20	29.41
	40	42.01
	60	55.77
	80	74.86
	100	80.88
Trigonellafoenumgraecum extract complex	10	22.36	62.23
	20	23.69
	40	27.30
	60	48.84
	80	68.70
	100	70.19

Trigonellafoenumgraecum extract complex	10	25.81	55.89
	20	29.45
	40	30.05
	60	53.78
	80	70.87
	100	74.63

METHOD:

Lipid Peroxidation

1 Principle

The inhibition in lipid peroxidation of liver homogenate of mice is used as method to determine the antioxidant activity of plant extracts. In this method in vitro free radicals were generated using ferrous ascorbate system. These free hydroxyl radicals during aerobic incubation with liver homogenate leads to formation of malonadialdehye (MDA) which reacts with thiobarbituric acid (TBA). The formation of this thiobarbuturic acid reactive substance (TBARs) was monitored calorimetrically at 532 nm using UV spectrophotometry. The reaction between lipid peroxides and TBA is used as sensitive method for lipid peroxidation in animal tissues ⁽⁵⁾

Reagents Required

 0.15 M Potassium chloride solution.

 Tris buffer.

 100 µM ferrous sulphate

 100 µM Ascorbic acid

 5 % Thiobarbituric acid

 Ice cold saline (0.95 NaCl)

Preparation of test material

The extract of Trigonella foenum graecum and its complex, Catechin complex and Curcumin complex were reconstituted in methanol. Catechin, Curcumin was dissolved in methanol which was used as a positive standard.

Methodology ⁽⁶⁾

 Mice liver homogenate (10%) was prepared by homogenizing the fresh liver in 0.15 M KCl solution.

 This fresh liver homogenate was mixed with 0.15 M KCL and tris hydrochloride buffer.

 The test reagents then added in various concentrations. Catechin and Curcumin was used as standard.

 In vitro lipid peroxidation was initiated by addition of 100 µM ferrous sulphate and 100 µM Ascorbic acid.

 After incubation for 1 hour at 37⁰C, the reaction was terminated by addition of thiobarbituric acid and then boiled at 95⁰C for 15 minutes for development of colored complex.

 On cooling test tubes were centrifuged at 4000 RPM for 10 minutes.

 Absorbance of supernatant was determined by colorimetrically at 532 nm and percent inhibition of TBARs formation was calculated with respect to control in which no test sample was added.

Evaluation

The inhibition of lipid peroxidation was determined by calculating percent inhibition in formation of TBARs using the following formula.

% Inhibition = A_control– A _test

------------------

A_control

The IC₅₀ values were calculated for all test material by subjecting the results to linear regression.

The inhibition in lipid peroxidation of Trigonella foenum graecum extract and its complex as well as Catechin complex and Curcumin complex were calculated using following graphs and the results are as shown in the table 1.4

Figure. 1.9 Plot of % Inhibition Vs Concentration Catechin hydrate

Figure 1.10 Plot of % Inhibition Vs Concentration Catechin hydrate complex

Figure1.11 Plot of % Inhibition Vs Concentration Curcumin

Figure 1.12 Plot of % Inhibition Vs Concentration Curcumin complex

Figure 1.13 Plot of % Inhibition Vs Concentration Trigonella foenum graecum seed extract

Figure. 1.14 Plot of % Inhibition Vs Concentration Trigonella foenum graecum seed extract complex

RESULT AND DISCUSSION

The evaluation and comparison of antioxidant activity of Catechin, Curcumin and Trigonella foenum graecum extract and their herbomineral complex by DPPH and by lipidperoxidation method showed increase in free radical scavenging activity by forming complex with zinc.

Catechins are widely known to be very potent antioxidants. They are able to scavenge reactive oxygen species (ROS) before ROS can cause damage to the cell. Catechin is able to scavenge free radicals directly by hydrogen atom donation, antioxidant activity depends on the arrangement of functional groups on its core structure. Both the configuration and total number of hydroxyl groups substantially influence the mechanism of antioxidant activity The B ring hydroxyl configuration is the most significant determinant of ROS scavenging. Catechins are excellent in vitro antioxidants due to the high number of hydroxyl groups in their molecules. Catechins were effective in scavenging peroxyl radicals in the liposomal system. criteria for effective radical scavenging are (a) the o-dihydroxyl structure in the B ring, which confers higher stability to the radical form and participate in electron delocalization, (b) the 2,3-double bond in conjugation with a 4-oxo function in the C ring, which is responsible for electron delocalization from the B ring, and (c) the 3- and 5-OH groups with 4-oxo function in the A and C rings for maximum radical scavenging potential. ⁽⁷⁾

Table 1.4 Results of free radical scavenging activity by Lipid Peroxidation method

Extracts	Concentration (µg/ml)	% inhibition in TBARS Formation	IC₅₀ (µg/ml)
Catechin hydrate.	10	32.06	43.19
	20	35.34
	40	48.76
	60	59.43
	80	71.60
	100	75.14
Catechinhyrate complex	10	34.09	37.00
	20	41.98
	40	51.95
	60	63.47
	80	73.74
	100	76.19
Curcumin	10	13.5	70.98
	20	28.65
	40	34.84
	60	44.76
	80	52.08
	100	66.16
Curcumin complex	10	23.95	55.69
	20	31.43
	40	42.53
	60	53.72
	80	63.82
	100	71.82
Trigonellafoenumgraecumextract	10	26.12	42.90
	20	38.31
	40	52.39
	60	65.39
	80	68.43
	100	78.84
Trigonellafoenumgraecumextract complex	10	39.14	22.63
	20	50.68
	40	61.79
	60	68.32
	80	74.33
	100	80.52

The antioxidant mechanism of Curcumin was a H-atom abstraction from the phenolic group. The antioxidant activity of Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) was determined by inhibition of controlled initiation of styrene oxidation. Synthetic Curcumin is a classical phenolic chain-breaking antioxidant, donating H atoms from the phenolic groups not the CH₂ group as has been suggested ⁽⁸⁾

Flavonoids belong to a large family of compound with a common diphenylpropane structure (C6C3C6) with different degree of hydroxylation, oxidation and substitution. Flavonoids have been reported to be able to interfere with the activities of enzyme involved in the reactive oxygen species generation, quenching free radicals, chelating transition metals and rendering them redox inactive. Thus the flavonoids present in Trigonella foenum graecum extract may be responsible for its antioxidant activity. ⁽⁹⁾

These are the possible different mechanism by which Catechin, Curcumin and Trigonella foenum graecum extract gives free radical scavenging activity.

Hrebomineral complex of Catechin, Curcumin and Trigonella foenum graecum extract was prepared by the process of chelation. Chelation form a special kind of bond, called a coordinate covalent bond, with the ions of certain metals. If all the requirements are met, then bingo—the metal ion is grabbed and “locked up” in the chelator’s clawlike embrace. The resulting molecular entity, called a coordination complex, is typically very stable and will not easily yield back the metal ion. Thus, if this complex is excreted, metal ions go with drug.

The protonated phenolic group is not a particularly good ligand for metal cations, but once deprotonated, an oxygen center is generated that possesses a high charge density, a so-called "Hard" ligand. Although the pKa value of most phenols is in the region of 9.0-10.0, in the presence of suitable cations for instance iron(III) or copper(H), the proton is displaced at much lower pH values, e.g., 5.0-8.0. Thus metal chelation by phenols can occur at physiological pH values

For good scavenging activity, a catechol moiety on ring B is required. The 3-OH moiety can function as a chelation site and can also be oxidized. The 3-OH group in combination with a C2 C3 double bond increases the scavenging activity. Chelation can then raise the activity to the level of the most active scavengers; possibly by site-specific scavenging .Thus chelation will increase antioxidant activity by site- specific scavenging ⁽¹⁰⁾

From the literature search it was revealed that excess Zn will suppress absorption of iron and copper, which are the trace metals responsible for producing oxidative stress. Hence oxidative stress produced by these trace metals is suppressed. Thus increase antioxidant activity.

Zinc itself is a good antioxidant. Mechanism of antioxidation for zinc can be divided into acute and chronic effects. Chronic effects involve exposure of an organism to zinc on a long-term basis, resulting in induction of some other substance that is the ultimate antioxidant, such as the metallothioneins. Chronic zinc deprivation generally results in increased sensitivity to some oxidative stress. The acute effects involve two mechanisms: protection of protein sulfhydryls or reduction of ^·OH formation from H₂O₂ through the antagonism of redox-active transition metals, such as iron and copper. Protection of protein sulfhydryl groups is thought to involve reduction of sulfhydryl reactivity through one of three mechanisms: (1) direct binding of zinc to the sulfhydryl, (2) steric hindrance as a result of binding to some other protein site in close proximity to the sulfhydryl group or (3) a conformational change from binding to some other site on the protein. Antagonism of redox-active, transition metal-catalyzed, site-specific reactions has led to the theory that zinc may be capable of reducing cellular injury that might have a component of site-specific oxidative damage, such as postischemic tissue damage ⁽¹¹⁾

Catechin ,Curcumin and flavonoids present in Trigonella foenum graecum extract showed potent antioxidant activity invitro, but due to their poor bioavailability these are not that much effective in vivo. From the literature search if the pharmacokinetic of Zn and these compounds were compared, chelation with Zn will increase the bioavailability of poorly bioavailable drug. Catechin and flavonoids are excreted through urine, where as90% zinc is excreted in feaces, and small traces in urine due to reabsorption of zinc occurs in kidney. Thus the complex will reabsorb and gives free radical scavenging activity for longer period of time. But in case of Curcumin, both Curcumin and zinc are excreted through feaces. Hence bioavailability of Curcumin is not increased.

Catechin, curcumin and Trigonella foenum graecum extract showed good antioxidant activity by their own mechanism. Formation of complex, will increase activity in vitro due to site specific scavenging, suppression of absorption of trace metals responsible for producing oxidative stress and antioxidant activity of Zinc. Also, invivo by increasing bioavailability. Thus hrebomineral complex will added more way and gives synergistic and multifunctional activity.

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Received on 12.03.2012

Modified on 15.03.2012

Accepted on 18.03.2012

Research Journal of Pharmacognosy and Phytochemistry. 4(3): May-June 2012, 171-177