Chapter VI

Evaluation of In Vitro Free Radical

Scavenging Activity

67

6. EVALUATION OF IN VITRO FREE RADICAL SCAVENGING ACTIVITY

OF PHYLLANTHUS AMARUS Schum & Thonn.

6.1 Introduction

The origin of disease is multifactorial in nature and the majority of the present

day diseases are due to the shift in the balance of the pro-oxidant and the antioxidant

homeostatic phenomenon in the body. Pro-oxidant conditions dominate either due to

the increased generation of the free radicals caused by excessive oxidative stress of

the current life or due to the poor scavenging/quenching in the body caused by

depletion of the dietary antioxidants (Rakesh and Rajesh, 2006).

From the few decades, there has been a considerable growth in the field of

herbal medicine. It is getting popularized in developing and developed countries due

to its natural origin and lesser side effects (Khopde et al., 2001; Naik et al., 2003).

6.1.1 Free radicals

Free radicals are continuosly produced in the body as accidental by products

of metabolism or deliberately during phagocytosis, they are very active and they

possess an unpaired electron.

It is the excessive generation of the free radicals, reactive oxygen (ROS) and

nitrogen species, such as superoxide anions, peroxyl, alkoxyl, hydroperoxyl, nitric

oxide and nitrogen dioxide radicals and hydroxyl radicals are constantly generated in

aerobic organisms in response to both exogenous chemicals and endogenous

metabolic processes.

These freeradicals are involved in the development of various diseases such

as cancer, rheumatoid arthritis, hepatic damage, certain neurodegenerative diseases,

tissue damage and also ageing (Larkins, 1999; Mates et al.,1999; Sarikurkcu et

al.,2008).

68

Table.6.1. Free radicals implicated in human diseases

S.No. Disease/Diseased State Free Radical

Implicated Damage inflicted

1 Cancer (Dreher and Junod,

1996) OH

- Oxidative DNA damage

2 Myocardial damage injury

(Chan, 1996) OFR Myocardial reperfusion

3 Atherosclerosis

(Terasawa et al., 2000) OFR

Oxidative modification of

LDL

4 Parkinson’s disease

(Evans, 1992) OFR Mediates neuronal loss

5 Alzheimer’s disease

(Pedersen et al., 2001) OFR

Promotes neuro-

degenerative damage

6 Ischemic hepatitis

(Poli, 1992) OFR

Acute lethal damage of

hepatocytes

7 Lung disease

(Ryrfeldt et al., 1992) ROS

Lipid peroxidation leading

to lung fibrosis

8 Diabetes mellitus

(Wolff, 1992) H2O2, OH

-

Oxidative stress leading to

diabetic complications.

OFR = Oxygen Free Radicals ROS = Reactive Oxygen Species

6.1.2 Oxidative stress damage in human diseases

The oxidative stress damage was implicated in many diseases like

atherosclerosis, diabetic complications, hepatitis, arthritis, ulcer formation and cancer.

Though the higher organisms have developed effective antioxidant systems, the

oxidative stress in biological systems can be induced by the depletion of antioxidants

and/or by an overload of oxidant species, i.e., reactive oxygen and nitrogen species

(ROS, RNS) and other radicals (R•), so that antioxidant levels become insufficient

(Droge, 2002).

69

Fig.6.1. ROS, oxidative damage and human diseases.

The above figure (Fig.6.1) shows different pathways, the pathways toward the

upper part showed the interrelationship between the effect of imbalance in the ROS

and their consequences on the cellular growth and the cellular function while lower

part showed interrelationship between ROS imbalance and the mechanism and

pathways from oxidative damage to mutation.

The imbalanced production of free radicals play an important role in the

pathogenesis of several human diseases such as ischemia, reperfusion injury (Chan,

1996), atherosclerosis (Terasawa et al., 2000), neurodegenerative diseases (Buscigllo

and Yankner, 1995) and cancer (Dreher and Junod, 1996). In addition, oxidative stress

due to inadequate antioxidant enzymes has been related with much other specific

pathologies as chronic granulomatous diseases, diabetic compilations, hepatitis,

arthritis, influenza virus, ulcer, pneumonia, HIV infection, cataract and glaucoma

(Ames et al., 1993; Halliwell et al., 1994; Van Dam et al., 1995; Araujo et al; 1998).

The role of oxidative stress is also implicated in inflammation, hypersensitivity and

autoimmune conditions (Maurice et al., 1997).

6.1.3 Antioxidants

Defence provided by antioxidant systems is crucial to survive and they can act

at different stages within the cells through the prevention of radical formation. The

antioxidants act by removal of or reduction in the local oxygen concentration,

removal of catalytic metal ions, scavenging the radicals such •OH, RO

• and RO2

• and

70

removal of ROS such as O2- and H2O2

-, scavenging the singlet oxygen (Gutteridge,

1994).

To minimize the stress induced by ROS, complex combination of enzymatic

and nonenzymatic functions will act as antioxidants. Non-enzymatic anti-oxidants are

classified into water-soluble and lipid-soluble, depending on whether they act

primarily in the aqueous phase or in the lipophilic region of the cell membranes.

Vitamin C (ascorbic acid) and certain polyphenol flavonoid groups act as hydrophlilic

antioxidants and ubiquinone, retinoids, carotenoids, apocynin, procyanidins and

tocopherols act as lipophilic anti-oxidants (Mates, 1999; Sarikurkcu et al., 2008;

Middleton et al., 2000). Other non-enzymatic anti-oxidants are antioxidant enzyme

cofactors, oxidative enzyme inhibitors and transition metal chelators such as ethylene

diamine tetra-acetic acid (EDTA).

According to Mittal (1999), these antioxidants may be classified as

(i) Endogenous antioxidants, those which are from physiological origin.

(ii) Exogenous antioxidants are those which cannot be produced by .the

human body but may protect against pro-oxidant forces when administered

as supplements.

More number of compounds destroys single oxygen molecules (free radicals)

in the body, thereby protecting against oxidative damage of cells. They are essential

for good health and are found naturally in a wide variety of foods and plants including

many vegetables and fruits (Orhan et al., 2003).

6.1.4 Phytoconstituents with antioxidant activity

The vast majority of plant based aromatic natural products are phenols.

Numerous categories of those compounds exists viz., simple phenols, phenyl

propanoids, flavonoids, tannins, lignans and quinines. A few phytochemical

antioxidants are terpenoids (lycopene, β- carotene, α-carotene, lutein), flavonoid

polyphenolics (rutin, hesperidin and naringin), anthocyanins, Isoflavone (genistin,

glycitein), silymarin, curcumin, resveratrol and ellagic acid (Rakesh and Rajesh,

2006).

Synthetic anti-oxidants, such as butylated hydroxyanisole and butylated

hydroxytoluene, have been developed, but their uses are limited due to toxicity (liver

damage and carcinogenesis) (Gulcin et al., 2002). In a search for sources of novel

anti-oxidants with low toxicity, medicinal plants have been studied extensively for

71

their radical scavenging activity over the past few years (Molyneux et al., 2004). The

antioxidant potential of the phenolic compounds in plants has been recognized (Wang

et al., 2008). As plants produce a large number of anti-oxidants to control the

oxidative stress caused by sunbeams and oxygen, it is clear that plants may represent

a source of new compounds with antioxidant activity (Scartezzini and Speroni, 2000).

Epidemiological studies also strongly suggested that consumption of certain plant

materials may reduce the risk of chronic diseases related to oxidative stress, on

account of their antioxidant activity (Fang Tian et al., 2009). Hence, development

and utilization of effective antioxidants of plant origins are highly desirable.

Anti-oxidant properties elicited by plant species have a full range of

applications in human healthcare, as they protect against these radicals. The anti-

oxidant activity of the plant materials was measured by the generation of radicals (and

their related compounds) and the addition of anti-oxidants, the latter resulting in the

reduction of the radical and its consequent disappearance (Arnao et al., 1999).

6.2. Materials and methods

6.2.1 Chemicals

1, 1- diphenyl-2-picrylhydrazyl (Sigma Chemical Company, St. Louis, USA).

Riboflavin (Loba Chemie Pvt Ltd., Bombay).

Deoxyribose (Sisco Research Laboratories Pvt Ltd., Mumbai).

Nitroblue tetrozolium (Sisco Research Laboratories Pvt Ltd., Mumbai).

All other chemicals and reagents used were of analytical grade.

6.2.2. Plant material

The aerial parts of Phyllanthus amarus were successively extracted with hexane,

ethyl acetate and methanol in Soxhlet apparatus. Hexane extract (PAHE), ethyl acetate

(PAEA), methanolic extracts (PAME) and the compounds namely phyllanthin (PAPH)

and hypophyllanthin (PAHP) isolated from hexane extract were assessed for free radicals

scavenging activity against superoxide, hydroxyl and DPPH radicals. The selected

extracts and isolated lignan compounds were dissolved in dimethyl sulphoxide (DMSO)

respectively.

72

6.3. Determination of Superoxide Radical Scavenging Activity

6.3.1 Reagents

i) Phosphate buffer (58 mM) solution

Solution A: 1.068 g of NaH2PO4 was weighed, transferred to a volumetric flask and

the volume made up to 100 mL with distilled water.

Solution B: 0.936 g of DiSodium hydrogen Phhosphate (Na2HPO4) was weighed,

transferred to a volumetric flask and the volume made up to 100 mL with distilled

water.

From the above solutions, 91.5 mL of solution A and 8.5 mL of solution B

were mixed and the pH was adjusted to 7.8.

ii) Ethylenediamine tetraacetic acid (EDTA-6 M) containing Sodium cyanide

(NaCN-3g) solution

3.72 g of EDTA and 1.5 mg of NaCN were weighed, transferred to a

volumetric flask and the volume made upto 100 mL with distilled water.

iii) Nitroblue tetrazolium (50 M) solution

12.3 mg of Nitroblue tetrazolium was weighed transferred to a volumetric

flask and the volume made upto 10 mL with distilled water.

iv) Riboflavin (2.0 M) solution:

4.5 mg of Riboflavin was weighed, transferred to a volumetric flask and the

volume made up to 100 mL with distilled water.

6.3.2 Procedure

6.3.2.1 Riboflavin photoreduction method

Superoxide scavenging activity of the plant extract was determined by

McCord and Fridovich method, 1969, which depends on light induced superoxide

generation by riboflavin and the corresponding reduction of nitroblue tetrazolium.

0.1mL of different concentrations of plant extract and 0.1 mL of 6 µM

ethylenediamine tetraacetic acid containing NaCN, 0.1 mL of 50 µM nitroblue

tetrazolium, 0.05 mL of 2 µM riboflavin were transferred to a test tube, and final

73

volume was made up to 3 mL using phosphate buffer. Then the assay tubes were

uniformly illuminated with an incandescent light (40 Watts) for 15 minutes and

thereafter the optical densities were measured at 560 nm. A control was prepared

using 0.1 mL of respective vehicle in the place of plant extract/compound/ascorbic

acid. The percentage inhibition of superoxide production was evaluated by comparing

the absorbance values of control and experimental tubes.

6.3.2.2 Calculation of percentage inhibition

The percentage inhibition of superoxide production by the extract was calculated

using the formula:

Inhibitory ratio = (A0 - A1) ×100

A0

Where, A0 is the absorbance of control; A1 is the absorbance with addition of

plant extract/ ascorbic acid.

6.3.2.3 Calculation of 50% inhibition concentration

The optical density obtained with each concentration of the

extract/compound/ascorbic acid was plotted taking concentration on X-axis and

percentage inhibition on Y-axis. The graph was extrapolated to find the 50%

inhibition concentration of extract/compound/ascorbic acid.

6.4 Determination of Hydroxyl Radical Scavenging Activity

Hydroxyl radical scavenging activity is commonly used to evaluate the free

radical scavenging effectiveness of various antioxidant substances (Elizabeth and

Rao, 1990).

6.4.1 Reagents

i) 2- deoxyribose (10 mM) solution

37.54 mg of 2- deoxyribose was weighed, transferred to a volumetric flask and

the volume made up to 100 mL with distilled water.

ii) EDTA (10 mM) solution

372.2 mg of EDTA was weighed, transferred to a volumetric flask and the

volume made up to 100 mL with distilled water.

74

iii) Ferrous sulphate (10 mM) solution

278 mg of ferrous sulphate was weighed, transferred to a volumetric flask and

the volume made up to 100 mL with distilled water.

iv) Phosphate buffer (0.1 M, pH 7.4) solution

Solution A: 276 mg of NaH2PO4 was weighed, transferred to a volumetric

flask and the volume made up to 100 mL with distilled water.

Solution B: 568 mg of Na2HPO4 was weighed, transferred to a volumetric

flask and the volume made up to 100 mL with distilled water.

From the above solutions, 12 mL of solution A and 88 mL of solution B were

mixed and pH was adjusted to 7.4.

v) Hydrogen peroxide (1.0 mM) solution

11L of Hydrogen peroxide was transferred to a volumetric flask and the

volume made up to 10 mL with distilled water.

6.4.2 Procedure

6.4.2.1 Deoxyribose degradation method

Hydroxyl radical scavenging activity was measured by studying the

competition between deoxyribose and the extracts for hydroxyl radicals

generated from the Fe2+

/EDTA/H2O2 system (Fenton reaction).

The hydroxyl radical attacks deoxyribose, which eventually results in the

formation of thiobarbituric acid reacting substances (TBARS) (Elizabeth and

Rao, 1990).

Fenton reaction mixture consisting of 200 L of 10 mM ferrous sulphate

(FeSO4. 7H2O), 200L of 10mM EDTA and 200 L of 10mM 2-deoxyribose

and was mixed with 1.2mL of 0.1 M phosphate buffer (pH 7.4) and 200L of

plant extract.

Thereafter, 200L of 10 mM H2O2 was added before the incubation at 37oC

for 4 h. Then, 1mL of this Fenton reaction mixture was treated with 0.2mL of

8.1% sodium dodecyl sulphate, 1.5mL of 0.8% thiobarbituric acid and 1.5mL

of 20 % acetic acid.

75

The total volume was then made to 5 mL by adding distilled water and kept in

an oil bath at 1000

C for 1 hour. After the mixture had been cooled, 5 mL of

15:1, v/v butanol-pyridine mixture was added.

Following vigorous shaking, the tubes were centrifuged at 4000 rpm for

10 mins and the absorbance of the organic layer containing the thiobarbituric

acid reactive substances was measured at 532 nm.

A control was prepared using 0.1mL of vehicle in the place of plant

extract/ascorbic acid.

The percentage inhibition of hydroxyl radicals by the extract/compound was

determined by comparing the absorbance values of the control and the

experimental tubes as calculated for Hydroxyl radical assay.

6.5 Determination of 1, 1- Diphenyl-2-Picrylhydrazyl (DPPH) Radical

Scavenging Activity

6.5.1 Principle

In DPPH assay method is based on the reduction of alcoholic DPPH solution

(dark blue in colour) in the presence of a hydrogen donating antioxidant converted to

the non radical form of yellow colored diphenyl–picrylhydrazine.

6.5.2. Reagents

1, 1- diphenyl-2-picrylhydrazyl (DPPH, 0.004%) solution

4 mg of DPPH was dissolved in 100 mL of ethanol and kept it overnight in

dark place for the generation of DPPH radical.

6.5.3. Procedure

The scavenging activity for DPPH free radicals was measured according to the

procedure described by Braca et al., 2003. An aliquot of 3mL of 0.004% DPPH

solution in ethanol and 0.1 mL of plant extract at various concentrations were mixed.

The mixture was shaken vigorously and allowed to reach a steady state at room

temperature for 30 min. Decolorization of DPPH was determined by measuring the

absorbance at 517 nm. A control was prepared using 0.1 mL of respective vehicle in

the place of plant extract/ascorbic acid. The percentage inhibition activity was

76

calculated as [(A0-A1)/A0] ×100, where A0 was the absorbance of the control, and A1

was the absorbance of the plant extract/ ascorbic acid.

6.6. Results

Superoxide anion plays an important role in the formation of more reactive

species such as hydrogen peroxide, hydroxyl radical, and singlet oxygen which induce

oxidative damage in lipids, proteins, and DNA (Pietta, 2000). Therefore, studying the

scavenging activity of plant extracts on superoxide radical is one of the most

important ways of clarifying the mechanism of antioxidant activity.

In the present study, the hexane (PAHE), ethyl acetate (PAEA), methanolic

(PAME) extracts and isolated compounds like phyllanthin (PAPH) and

hypophyllanthin (PAHP) of P.amarus aerial parts showed concentration dependent

scavenging activity on the three tested radicals i.e., superoxide, hydroxyl and DPPH

radicals. The lower the IC50 values the higher the antioxidant activity. The results of

scavenging activity on superoxide radicals were given Table.6.2 and Fig.6.2. The

mean IC50 values for superoxide radical of PAHE, PAEA, PAME, PAPH and PAHP

of P.amarus were found to be 1147.2µg, 550.8µg, 395.6µg, 36.9µg and 46.25µg

respectively. The mean IC50 value of standard ascorbic acid was found to be 30.42µg.

The results were summarized in Table.6.5 and depicted in Fig.6.5

The PAHE, PAEA, PAME, PAPH and PAHP of P.amarus were found to

possess concentration dependent scavenging activity on hydroxyl radicals and the

results were given Table.6.3 and Fig.6.3. The mean IC50 values for hydroxyl radical of

PAHE, PAEA, PAME, PAPH and PAHP of P.amarus were found to be 1124.5µg,

514.27µg, 241.6µg, 31.59µg and 39.85µg respectively. The mean IC50 value of

ascorbic acid was found to be 27.61µg.

The PAHE, PAEA, PAME, PAPH and PAHP of P.amarus showed

concentration dependent scavenging activity on DPPH radicals and the results were

given Table.6.4 and Fig.6.4. The mean IC50 values for hydroxyl radical of PAHE,

PAEA, PAME, PAPH and PAHP of P.amarus were found to be 729.26µg, 388.56µg,

214.4µg, 26.56µg and 28.14µg respectively. The mean IC50 value of ascorbic acid

was found to be 20.88µg.

The order of free radical scavenging activity of the three extracts (PAHE,

PAEA & PAME) and two isolated compounds (PAPH & PAHP) of P.amarus aerial

77

parts against the tested radicals (superoxide, hydroxyl and DPPH radicals) was in the

following manner: Ascorbic acid>PAPH>PAHP>PAME>PAEA>PAHE.

Table 6.2. Concentration dependent percent inhibition of Superoxide radical by

hexane (PAHE), ethylacetate (PAEA), methanol extracts (PAME), phyllanthin

(PAPH), hypophyllanthin (PAHP) of P.amarus and Ascorbic acid in in vitro studies.

Results are expressed as Mean±SEM (n=3).

0 150 300 450 600 750 900 1050120013500

20

40

60

80

100PAME

PAEA

PAHE

Ascorbic acid

PAPH

PAHP

Mean±SEM (n=3)

Conc((µg/mL)

% I

nhib

itio

n

Fig 6.2.Concentration dependent percent inhibition of Superoxide radical by hexane

(PAHE), ethylacetate (PAEA), methanol extracts (PAME), phyllanthin (PAPH),

hypophyllanthin (PAHP) of P.amarus and ascorbic acid in in vitro studies.

Extract/isolated

compound

Percentage inhibition of Superoxide radical

Quantity of extracts/isolated compounds/ ascorbic acid in micrograms (µg/mL)

20 40 80 160 320 640 1280

PAHE 9.14±0.16 14.24±0.22 24.89±0.18 29.26±2.17 36.79±0.14 42.74±2.81 50.26±0.36

PAEA 14.5±0.16 22.5±0.29 30.25±0.2 38.92 ±0.14 42.19±0.11 51.94±0.25 60.19±0.22

PAME 18.2±0.35 25.5±2.11 34.27±0.14 42.22±0.11 47.4±1.07 59.61±2.7 71.16±0.17

PAPH 28.15±0.5 54.19±1.1 76.17±0.2 84.46±0.7 85.72±0.4 86.11±1.5 86.91±1.1

PAHP 25.56±0.28 47.82±1.1 69.58±0.27 78.15±0.14 81.89±0.52 84.8±0.17 85.87±0.24

Ascorbic acid 35.22±0.41 68.28±2.2 76.64±2.2 85.12±0.64 85.87±0.22 87.45±0.15 88.95±0.36

78

Table 6.3. Concentration dependent percent inhibition of Hydroxyl radical by hexane

(PAHE), ethylacetate (PAEA), methanol extracts (PAME), phyllanthin (PAPH),

hypophyllanthin (PAHP) of P.amarus and ascorbic acid in in vitro studies

Extract/isolated

compound

Percentage inhibition of Hydroxyl radical

Quantity of extracts/isolated compounds/ ascorbic acid in micrograms (µg/mL)

20 40 80 160 320 640 1280

PAHE 8.25±0.23 11.37±0.18 18.72±0.26 24.15±0.11 35.25±0.27 42.81±1.9 51.12±0.6

PAEA 10.44±0.29 19.5±0.16 26.52±0.41 35.12±0.18 46.45±2.42 54.13±0.26 62.19±0.17

PAME 15.14±0.14 22.79±0.85 35.25±0.19 44.65±0.22 55.75±1.13 62.89±2.16 74.15±2.6

PAPH 32.15±0.22 58.19±0.95 81.87±0.41 85.46±0.27 88.72±0.14 89.16±0.16 89.91±0.64

PAHP 27.56±0.14 50.82±0.36 74.58±0.29 81.15±0.18 86.19±0.31 86.8±0.27 87.17±0.44

Ascorbic acid 38.32±0.4 74.12±0.6 82.61±1.0 86.31±0.6 88.25±0.4 90.11±1.0 90.92±1.3

Results are expressed as Mean±SEM (n=3).

0 150 300 450 600 750 900 1050120013500

20

40

60

80

100PAME

PAEA

PAHE

Ascorbic acid

PAPH

PAHP

Conc((µg/mL)

% I

nh

ibit

ion

Mean±SEM (n=3)

Fig.6.3. Concentration dependent percent inhibition of Hydroxyl radical by hexane

(PAHE), ethylacetate (PAEA), methanol extracts (PAME), phyllanthin (PAPH),

hypophyllanthin (PAHP) of P.amarus and ascorbic acid in in vitro studies.

79

Table 6.4. Concentration dependent percent inhibition of DPPH radical by hexane

(PAHE), ethylacetate (PAEA), methanol extracts (PAME), phyllanthin (PAPH),

hypophyllanthin (PAHP) of P.amarus and ascorbic acid in in-vitro studies.

Extract/isolated

compound

Percentage inhibition of DPPH radical

Quantity of extracts/isolated compounds/ ascorbic acid in micrograms (µg/mL)

20 40 80 160 320 640 1280

PAHE 11.29±0.54 20.35±0.24 28.71±0.57 35.57±0.33 39.21±2.67 49.34±1.13 58.71±0.35

PAEA 13.28±0.24 22.36±1.2 31.26±0.41 37.2±0.2 47.15±0.7 58.11±0.25 64.19±0.22

PAME 17.54±0.28 27.34±0.52 39.6±0.22 46.99±0.31 58.32±0.55 67.54±0.45 74.22±2.57

PAPH 38.25±0.58 65.14±0.14 84.25±0.27 87.87±0.54 88.2±0.26 88.55±0.25 89.29±0.27

PAHP 35.2±0.22 61.57±0.31 79.15±0.51 84.52±0.24 87.14±0.15 87.85±0.22 88.56±0.25

Ascorbic acid 49.32±0.4 78.12±0.6 82.61±1.0 86.31±0.6 88.25±0.4 90.11±1.0 90.92±1.3

Results are expressed as Mean±SEM (n=3).

0 150 300 450 600 750 900 1050120013500

20

40

60

80

100PAME

PAEA

PAHE

Ascorbic acid

PAPH

PAHP

Mean±SEM (n=3).

Conc((µg/mL)

% I

nhib

itio

n

Fig.6.4. Concentration dependent percent inhibition of DPPH radical by hexane (PAHE),

ethylacetate (PAEA), methanol extracts (PAME), phyllanthin (PAPH), hypophyllanthin

(PAHP) of P.amarus and ascorbic acid in in vitro studies.

80

Table 6.5. In vitro 50% inhibition concentration (IC50) of hexane (PAHE),

ethylacetate (PAEA), methanol extracts (PAME), phyllanthin (PAPH), hypophyllanthin

(PAHP) of P.amarus on Superoxide, Hydroxyl and DPPH free radicals.

Extract/isolated

compound

IC50 value (µg)

Superoxide radical Hydroxyl radical DPPH radical

PAHE 1147.2 1124.5 729.26

PAEA 550.8 514.27 388.56

PAME 395.6 241.6 214.4

PAPH 36.9 31.59 26.56

PAHP 46.25 39.85 28.14

Ascorbic acid 30.42 27.61 20.88

Superoxid

e radic

al

Hydroxyl r

adical

DPPH radic

al0

150

300

450

600

750

900

1050

1200

1350

PAEA

PAHE

Ascorbic acid

PAPH

PAHP

PAME

IC50

(µg

)

Fig.6.5. In vitro 50% inhibition concentration (IC50) of hexane (PAHE), ethylacetate

(PAEA), methanol extracts (PAME), phyllanthin (PAPH), hypophyllanthin (PAHP)

of P.amarus on Superoxide, Hydroxyl and DPPH free radicals.

81

6.7. Discussion

Oxidation is one of the body’s natural chemical processes that produce “free

radicals,” which are highly unstable molecules that can damage cells. Free radicals

can cause damage, known as “oxidative stress,” which is thought to play a role in the

development of many diseases, including Alzheimer’s disease, cancer, eye disease,

heart disease, Parkinson’s disease, and rheumatoid arthritis. In laboratory

experiments, antioxidant molecules counter oxidative stress and its associated

damage.

The body produces its own antioxidants and some of the antioxidants were

obtained from food. Antioxidants are abundant in vegetables, fruits and are also found

in grain cereals, teas, legumes, and nuts. Examples of antioxidants include

anthocyanins, beta-carotene, catechins, coenzyme Q10, flavonoids, lipoic acid, lutein,

lycopene, selenium, and vitamins C & E.

Numerous natural products are effective antioxidants, and many medicinal

plants with a long history of use in folk medicine in different countries against a

variety of diseases have turned out to be rich sources of antioxidants. (Madhukiran

and Ganga Rao, 2011; Mantle et al., 2000; Mathisen et al., 2002; Lee et al., 2003a).

It was reported that some medicinal plants contain a wide variety of natural

antioxidants, such as lignans, phenolic acids, flavonoids and tannins, which possess

more potent antioxidant activity (Wang et al., 2008). Many investigations indicate

that these compounds are of great value in preventing the onset and or progression of

many human diseases (Halliwell and Gutteridge, 1989; Halliwell et al., 1992). The

health-promoting effect of antioxidants from plants is thought to arise from their

protective effects by counteracting reactive oxygen species (ROS) (Wong et al.,

2006).

In the present study, the tested plant extracts showed the presence of the

compounds such as lignans, phenols, alkaloids, steroids, glycosides, flavanoids,

tannins and saponins in the qualitative phytochemical screening (Table 3.02). The

different extracts of the P.amarus aerial parts, isolated compounds like phyllanthin

and hypophyllanthin produced concentration dependent inhibition against the tested

three free radicals. Among all the test substances, phyllanthin showed better free

radical scavenging of the three tested radicals.