Extraction:

Dried root powder (200 g) was exhaustively extracted using methanol (3 X 500 ml). The marc and filter paper were washed with methanol each time. Methanol extract of P. integrifolia (PIM) obtained was concentrated, dried and used for the evaluation.

Chemicals:

All the chemicals for antioxidant evaluation were purchased from Sigma Chemicals Co. Other reagents and chemicals were of analytical grade and were either from Sigma or Merck chemicals.

Antioxidant Activity:

(i) Antiradical activity (DPPH):

Antiradical activity was measured by observing decrease in absorbance at 516 nm of a methanolic solution of colored DPPH (1,1-diphenyl-2-picryl hydrazyl, a stable free radical) brought out by the sample. A stock solution of DPPH (4.3 mg/3.3 ml methanol) was prepared such that, 75 µl of it in 3ml methanol gave an initial absorbance of 0.9. Decrease in absorbance in the presence of PIM at different concentration (20-70 µg) was measured at 516 nm up to 2 min at an interval of 30 sec. Ascorbic acid was used as a reference standard and 75 µl of stock solution of DPPH in 3 ml methanol as a control. The activity was expressed as an effective concentration at 50 % (EC₅₀) i.e. the concentration of the test solution required to give a 50 % decrease in the initial absorbance, which was calculated as percentage inhibition using formula¹⁰.

% Inhibition = OD (control) - OD (extract) X 100

OD (control)

(ii) Superoxide radical scavenging activity:

Superoxide anion radical scavenging assay was based on the reduction of nitroblue-tetrazolium (NBT) to a blue colored formazan, which is directly proportional to the concentration of superoxide anion in the system in presence of riboflavin-light-NBT system¹¹. The mixture contained 200 μl EDTA (12 mM), 300 μl NBT (1 %), 200 μl riboflavin and different concentration of PIM in 3 ml of phosphate buffer (50 Mm, 7.4 pH). The reaction mixture was vortexed. The reaction was started by illuminating the sample cuvette with light and measured the increased absorbance at 590 nm on regular interval of 30 seconds up to 2.5 min and last reading was taken at 4 min. The superoxide radical scavenging activity was calculated as percentage inhibition.

(iii) Lipid peroxidation:

Hydroxyl radical is known to attack lipid-constituent of cell membrane and fragments it to malonyldialdehyde (MDA). This binds with thiobarbituric acid (TBA) to form pink MDA-TBA chromogen and absorb at 532 nm. For preparing liver homogenate, wistar rats were anesthetized and dissected from the ventral side; liver was exposed and perfused with phosphate buffer saline (pH-7.4). Blood free liver was taken out and prepared liver homogenate in phosphate buffer (1 %) with the help of glass Teflon homogenizer. The reaction mixture contained 0.5 ml liver homogenate, 0.1 ml tris-HCl buffer (pH- 7.2), 0.05 ml of 4 mM FeSO₄ and various concentration of drug extract (10 μl, 100 μl, 1000 μl) was incubated at 37 ºC for 1 hr. To this 0.5 ml of incubated mixture, 9 ml distilled water, 2 ml thio-barbituric acid TBA (0.6 %) was added and shaken vigorously. The assay mixture was kept in water bath for heating for 30 min. After cooling the assay mixture was shaken with butanol and measured the absorbance of separated butanol layer at 532 nm. α-tocopherol was taken, as standard and control solution is test minus tested sample¹⁰. Malonaldehyde formed was calculated by following formula:

Nm of MDA production =

OD (Test/Control) X 0.025 X 10⁹(3)

1.56 X 10⁵

(iv) Hydroxyl radical (OH^-) scavenging activity

The formaldehyde formed during the oxidation of dimethylsulphoxide (DMSO) by Fe⁺³- ascorbic acid was used to detect hydroxyl radicals. The reaction mixture contain 1.55 ml phosphate buffer (50 Mm, pH- 7.4), 20 μl FeCl₃(0.1mM), 60 μl EDTA (0.3 mM), 167 μl (DMSO), different concentration of PIM (10 μl, 100 μl, 1000 μl) dissolved in phosphate buffer and 100 μl ascorbic acid (2 mM). The mixture was incubated for 30 min at 37 ºC and the reaction was stopped by adding 125 μl (17.5 %w/v). The formaldehyde formed was determined spectrophotometrically at 412 nm¹².

(v) Nitric oxide scavenging activity:

Sodium nitroprusside in phosphate buffer solution (PBS) at physiological pH generates NO, which interacts with oxygen to produce nitrite ions that was estimated by Griess reagent¹³. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite ions. The reaction mixture contained sodium nitroprusside (0.3 ml, 10 mM) in phosphate buffer saline and PIM extract in different concentrations incubated at room temperature for 150 min. The same reaction mixture without the test served as control. After the incubation, 1 ml Griess reagent (1 % sulphanilamide, 2 % H₃PO₄ and 0.1 % Naphthylene diamine dihydrochloride) was added. The absorbance of reaction mixture was measured at 546 nm.

Estimation of total phenolic content:

The air-dried powder of P. integrifolia root (0.1 g) was extracted with methanol (100 ml) and the final volume of the extract was adjusted to 100 ml with methanol. 5 ml of this was diluted with an equal volume of methanol and was used for the estimation of phenols. To 1 ml of the methanolic extract was added 10 ml of distilled water and 1.5 ml of diluted (1:2) folin ciocalteu reagent and the mixture was kept aside for 5 min. After adding 4 ml of 20 % Na₂CO₃ solution the final volume was adjusted to 25 ml using distilled water. Absorbance was measured at 765 nm at an interval of 30 min up to 2 h, against distilled water as a blank¹⁴. The total phenol content was measured using following formula:

C = A * 282.6 - 8.451, where A = Absorbance

RESULTS AND DISCUSSION:

As a consequence of metabolism, production of Reactive Oxygen Species (ROS) is thought to results in random damage to protein, lipids and DNA, which may even shorten the life span of an individual. Incompletely reduced oxygen species-superoxide (O₂^-), peroxynitrite (OONO-) and hydroxyl radical (OH^-) are particularly reactive and dangerous. Thus agents such as anti oxidants that can control states of oxidative stress represent a major line of defense, regulating general health status. There are several proteins and bio-molecules in the living organisms, which act as free radical scavengers. Besides these bio-molecules, several dietary components containing vitamins, polyphenols, glycosides, terpenoids, lignans and flavones act as natural free radical scavengers¹⁵. To screen antioxidant potential, the methanolic extracts of PIM was tested for their interaction with ROS in various ROS-generating in vitro chemical reactions.

(i) Antiradical activity (DPPH):

The DPPH radical is considered to be a model of lipophilic radical. A chain reaction in lipophilic radicals was initiated by lipid autoxidation¹⁶. PIM and standard antioxidant ascorbic acid showed significant (p<0.001) dose dependent anti-radical (DPPH) activity with an EC₅₀ value of 69.89 and 3.67 μg/ml respectively (Table 1). The effective scavenging of the DPPH radical (an iron-free system)suggests that the antioxidant activity is most likely caused by a chain-breaking action with the lipid peroxidation cascade.

Table 1: Antiradical activity (DPPH) of PIM

Sample

Tested

Concentration

µg/ml

% Inhibition

EC50 µg/ml

(r2)

PIM

33.33

66.66

100

133.33

22.51 ± 0.41

50.29 ± 0.13

74.79 ± 0.22

88.02 ± 0.08

69.89

(0.97)

Ascorbic acid

1.66

2.5

3.33

4.166

23.59 ± 0.53

32.66 ± 0.28

47.06 ± 0.20

56.90 ± 0.19

3.67

(0.98)

All values are Mean ± S.E.M, n=3, r²= Correlation coefficient

(ii) Superoxide radical scavenging activity:

Superoxide(O2˙ˉ) anion scavenging assay performed by monitoring the reduction of yellow dye, nitro-blue tetrazolium (NBT) to produce a blue colored formazan, which is directly proportional to the concentration of superoxide(O2˙ˉ) anion in the system¹⁷. NBT in presences of O2˙ˉ is reduced to tetrazolium, two of which combine together to give a blue dye (monoformazan). The extracts scavenged superoxide radical in a concentration dependent manner. EC₅₀ values of PIM and ascorbic acid observed were 303.16 and 94.28 μg/ml respectively (Table 2). The action may depend on hydrogen atom donation by their components leading to the formation of secondary radical species that are resonance stabilized, like many phenolic antioxidants¹⁸.

Table 2: Super oxide radical scavenging activity of PIM

Sample

Tested

Concentration

µg/ml

% Inhibition

EC₅₀ µg/ml

(r²)

PIM

16.66

41.66

83.33

125

18.26 ± 0.94

19.25 ± 0.35

21.42 ± 0.17

31.31 ± 0.60

303.16

(0.85)

Ascorbic acid

8.33

16.66

33.33

9.74 ± 0.40

13.65 ± 0.61

17.93 ± 0.56

21.30 ± 0.51

94.28

(0.99)

All values are Mean ± S.E.M, n=3, r²= Correlation coefficient

(iii) Lipid peroxidation:

PIM inhibited lipid peroxidation in liver homogenate in a dose dependent manner (Table 3). Lipid peroxidation is a chain reaction initiated by the attack on the membrane-lipids by free radicals that has sufficient reactivity to abstract a hydrogen atom from the methylene group¹⁹. This leaves behind an unpaired electron on the carbon atom. The carbon radical is stabilized by molecular rearrangement to produce conjugated diene, which then reacts with an oxygen molecule to form a peroxy radical. These peroxy radicals form cyclic peroxide, which is fragmented in to aldehyde like, 4-OH 2,3 transnoneal and malonaldehyde (MDA). These 4-OH 2,3 transnoneal attacks essential sulfhydryl group of many protein and MDA attack amino groups of the protein molecule to form intra and intermolecular cross-link. These aldehydes react with thiobarbituric acid forming thio-barbituric acid reactive species (TBARS). Continued fragmentation of fatty acid side chains to produce aldehydes and hydrocarbons will eventually lead to loss of membrane integrity²⁰. FeSO₄ catalyzed oxidation and lead to formation of MDA. In short, lipid peroxidation is a complex process which can be influenced by different compounds through different mechanism, such as metal chelation and free radical scavenging²¹. The inhibitory effect of the extracts could be partly due to their action as superoxide and hydroxyl radical scavengers. The action may be attributed to the presence of flavanoids (scutellarein, pectolinaringenin, hispidulin, apigenin and luteolin).

Table 3: Protective effect of PIM against MDA formed by lipid peroxidation

Sample

Tested

Concentration

µg/ml

Nm of MDA Formed

Inhibition

EC₅₀

µg/ml

(r²)

Control

22.19 ± 0.264

PIM

100

1000

15.25 ± 0.098*

10.43 ± 0.049*

6.31± 0.039*

31.26 ± 1.22

52.99 ± 0.78

71.55 ± 0.19

310.4

(0.78)

α-tocopherol

0.066

0.66

6.6

21.93 ±0.019*

16.31 ±0.015*

11.84 ±0.019*

1.11 ± 1.10

13.31 ± 1.10

46.61 ± 0.72

7.08

(0.76)

All values are Mean ± S.E.M, n=3, r²= Correlation coefficient, *P<0.001

(iv) Hydroxyl radical (OH^-) scavenging activity:

In the hydroxyl scavenging assay, DMSO reacts with OH˙, formed from ascorbate-Fe⁺³ systems and the result is production of formaldehyde. The competition between the test extract and DMSO for hydroxyl radicals, generated from the ascorbate-Fe⁺³ systems; expressed as percent inhibition of formaldehyde production, was used for the estimation of the hydroxyl radical scavenging activity²². PIM scavenged hydroxyl radicals in a dose dependent manner. EC₅₀ value of PIM was found to be 3.32 mg/ml (Table 4). This result is conjugation with the inhibitory activity of the PIM in lipid peroxidation assay.

Table 4: Hydroxyl radical scavenging activity of PIM

Sample

Tested

Concentration

mg/ml

%Inhibition

(n=3)

EC₅₀ mg/ml

(r²)

PIM

0.025

0.25

2.5

5.74 ± 0.24

40.03 ± 0.19

63.82 ± 0.32

1.66

(0.73)

All values are Mean ± S.E.M, n=3, r²= Correlation coefficient

(v) Nitric oxide scavenging activity:

Physiological production of NO is beneficial for the host’s defense against microorganism, parasites and tumor cells. Over production of Nitric oxide is implicated in inflammation, cancer, and other pathological conditions²³. PIM significantly scavenged (P<0.001) nitric oxide radical, in a concentration dependent manner (Table 5).

Table 5: Nitric oxide radical scavenging activity of PIM

Sample

Tested

Concentration

mg/ml

%Inhibition

EC₅₀ mg/ml

(r²)

PIM

0.025

0.25

2.5

7.44 ± 0.041

14.57 ± 0.057

27.66 ± 0.074

5.55

(0.92)

All values are Mean ± S.E.M, n=3, r²= Correlation coefficient

Estimation of total phenolic content:

The PIM was found to have 0.028 % of phenolics. The phenolics compounds may contribute directly to the antioxidant action.

CONCLUSION:

It may be concluded that the methanolic extract of P. integrifolia have significant antioxidant activity. The anti-oxidant potential may be attributed to the presence of phenolic compounds. Thus, these findings also seem to; in part justify the folkloric uses of this plant.

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Received on 24.12.2010

Accepted on 24.03.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(3): May-June 2010, 196-199

MATERIAL AND METHODS:

RESULTS AND DISCUSSION:

PIM

Ascorbic acid

PIM

Ascorbic acid

PIM

α-tocopherol

PIM

PIM