Journal of Applied Pharmaceutical Science 01 (04); 2011: 89-96

ISSN: 2231-3354
Received: 07-06-2011
Evaluation of antioxidant potential of different parts
Accepted: 10-06-2011
of wild edible plant Passiflora foetida L.

Sasikala. V, Saravana. S. and Parimelazhagan. T

ABSTRACT

The purpose of the present study was to investigate the in vitro anti oxidant activities of
petroleum ether, ethanol and hot water extract of Passiflora foetida root, leaves, flower, fruit peel
and seed. Plant material was extracted in soxhlet extractor successively with petroleum ether and
Sasikala. V, Saravana. S. and ethanol. Extracts of P.foetida root, leaves, flower, fruit peel and seeds were analysed for the
Parimelazhagan. T quantification of total phenolics, tannins and ascorbic acid (vitamin c). The antioxidant property
Bioprospecting Laboratory, Department was estimated using reducing power, metal chelating, hydroxyl radical scavenging, nitric oxide
of Botany, Bharathiar University, radical scavenging, ABTS˙+, DPPH·, antihemolytic and β-carotene assays. P. foetida fruit peel was
Coimbatore, India. found to be much effective when compared to other parts taken for the present study. It is very
clear that this is a plant with tremendous wide spread use and also with extraordinary potential for
the pharmaceuticals.

Key words: Passiflora foetida, DPPH, ABTS˙+, antihemolytic, β-carotene.

INTRODUCTION
Medicinal plants are nature gift to mankind. These plants have been used by man for
centuries in various traditional system of medicine like Ayurveda, Siddha, Unani etc.
(Ranganathan,1999). Nowadays herbal drugs are prescribed widely even when their biologically
active compounds are unknown because of their effectiveness, minimum side effects and relatively
low cost (Valiathan, 1998). Medicinal plants are the important source of life saving drugs for the
majority of the world’s population. Passiflora is the largest genus in the Passifloraceae family and
comprises nearly 500 species (Vanderplank, 1996). Passiflora foetida L. (Stinking passion flower)
is South American origin, which has been spread to many tropical areas. It is found in riverbeds,
dry forest floors, and wayside thickets, covering the top thorny shrubs and also growing near
hamlets. The mostly available wild species are P. edulis, P. leschenaultia, P. mollissima and P.
subpeltata. The traditional medicines are used to cure many diseases like diarrhea, intestinal tract,
throat, ear infections, fever and skin diseases. The ethanobotanical views of P.foetida, suggest that
decoction of leaves and fruits is used to treat asthma and biliousness, leaves and root decoction is
emmenagogue, used in hysteria and leaf paste is applied on the head for giddiness and headache
(Chopra et al., 1956). In Brazil, the herb is used in the form of lotions and skin diseases with
inflammation (Chopra et al., 1944). The major phytoconstituents of this plant are alkaloids,
*For Correspondence: phenols, glycosides, flavonoids and cyanogenic compounds (Dhawan et al., 2004) and
T.Parimelazhagan
Bioprospecting Laboratory passifloricins, polypeptides and alpha-pyrones in P. foetida (Echeverri et al., 2001). Several years
Department of Botany, ago (Echeverri et al., 1991) the resin of the leaves of P. foetida found. Some authors describe the
School of Life sciences, use of Passiflora species in the popular medicine for the inflammatory diseases. About 294
Bharathiar University,
Coimbatore, India. volatile compounds have been identified in several passion fruit extracts (Shibamoto and Tang,
E-mail: [email protected]. 1990). The purpose of the present study was to investigate the in vitro antioxidant activities of
 Journal of Applied Pharmaceutical Science 01 (04); 2011: 89-96

petroleum ether, ethanol and hot water extract of Passiflora foetida supernatant (5mL) was mixed with 5 mL of distilled water and 0.5
root, leaves, flower, fruit peel and seed. mL of 0.1 % ferric chloride. The absorbance of the reaction
mixture was measured spectroscopically at 700 nm.
MATERIALS AND METHODS
(A)
Chemicals
Potassium ferricyanide, ferric chloride, 2,2-diphenyl-1-
picryl-hydrazyl (DPPH), potassium persulfate, 2,2’aninobis(3-
ethylbenzothiozoline-6-sulfonic acid) disodium salt (ABTS), 6-
hydroxy-2, 5,7,8-tetramethylchroman-2-carboxylic acid (Trolox),
linoleic acid, ferrous chloride, ammonium thiocyanate, hydrogen
peroxide, ferrous ammonium sulfate, ethylenediamine tetracetic
acid (EDTA) disodium salt, 2,2’-bipyridyl and hydroxylamine
(B)
hydrochloride were obtained from Himedia, Merck or Sigma. All
other reagents used were of analytical grade.

Collection of plant materials

The fresh plant parts of Passiflora foetida L. were
collected from Maruthamalai forest hills, Coimbatore. The
collected plant material was identified and their authenticity was
confirmed by comparing the voucher specimen at the herbarium of Fig: 1. Reducing power of P.foetida root & Reducing power of P.foetida peel. (A)
Botanical survey of India, Southern circle Coimbatore, Tamilnadu. Ethanol, (B) Aqueous extract.
The air-dried, powdered plant material was extracted in soxhlet
Metal chelating activity
extractor successively with petroleum ether and ethanol. Finally,
The chelating of ferrous ions by various extracts of P.
the material was macerated using hot water with occasional stirring
foetida parts were estimated by the method of (Dinis et al., 1994).
for 24 hr and the water extract was filtered. The different solvent
Briefly the extract samples (250 µL) were added to a solution of 2
extracts were concentrated by rotary vacuum evaporator and then
mmol/L FeCl2 (0.05 mL). The reaction was initiated by the
air-dried. The percentage yield was expressed in terms of air-dried
addition of 5 mmol/ L ferrozine (0.2 mL) and the mixture was
weight of plant material. The extracts were freezing dried and
shaken vigorously and left standing at room temperature for 10min.
stored in desiccators until further analysis
Absorbance of the solution was then measured
In vitro antioxidant studies spectrophotometrically at 562 nm. The chelating activity of the
Determination of total phenolic and tannin contents extracts was evaluated using EDTA as standard. The results were
The total phenolic content of P. foetida plant extracts expressed as mg EDTA equivalent/g extract.
was determined by Folin ciocalteu method. Using the same extract
Hydroxyl radical scavenging activity
the tannins were estimated after treatment with polyvinyl
The scavenging activity of petroleum ether, ethanol and
polypyrrolidone (PVPP). The amount of total phenolics and
hot water extracts of P. foetida on hydroxyl radical was measured
tannins were calculated as the tannic acid equivalents (TAE) as according to the method (Klein et al., 1991) various concentrations
described by (Siddhuraju & Becker, 2003).
(50,100,150,and 200µg) of extracts were added with 1.0 mL of
Determination of ascorbic acid (vitamin c) iron-EDTA solution (0.13% ferrous ammonium sulfate and 0.26%
The ascorbic acid was determined using the dichloro EDTA), 0.5 mL of EDTA solution (0.018%), and 1.0 mL of
indophenols (DIP) method with a modification by (Yen and Chen, DMSO (0.85%, v/v in 0.1 M phosphate buffer, pH 7.4). The
1996). The concentrations of the ascorbic acid in the sample were reaction was initiated by adding 0.5 mL of ascorbic acid (0.22%)
determined through comparison with the absorbance of standard and incubated at 80-90oC for 15 min in a water bath. After
ascorbic acid at different concentration. incubation, the reaction was terminated by the addition of 1.0 mL
of icecold TCA (17.5%, w/v). Three mL of Nash reagent (75.0 g of
Reducing power ammonium acetate, 3.0 mL of glacial acetic acid, and 2 mL of
The reducing power of different solvent extracts of P. acetyl acetone were mixed and raised to 1 L with distilled water)
foetida was determined by the method reported by (Siddhuraju et was added and left at room temperature for 15 min. The reaction
al., 2002). 20-100 µg of extract was taken in 1 mL of phosphate mixture without sample was used as control. The intensity of the
buffer and 5 mL of 0.2 M phosphate buffer (pH 6.6) was added. To color formed was measured spectroscopically at 412 nm against
this, 5 mL of 1% Potassium ferricyanide solution was added and reagent blank. The % hydroxyl radical scavenging activity (HRSA)
the mixture was incubated at 50˚ C for 20 min. After the is calculated by the following formula:
incubation, 5mL of 10 % TCA was added. The content was
centrifuged at 1000 rpm for 10 min. The upper layer of the %HRSA= [(Control OD−Sample OD)/Control OD] ×100
 Journal of Applied Pharmaceutical Science 01 (04); 2011: 89-96

Radical scavenging activity using DPPH· method

The DPPH• radical scavenging activity of P. foetida
plants parts extracts was measured according to the method of
(Blios, 1958). IC50 values of the extract i.e., concentration of
extract necessary to decrease the initial concentration of DPPH by
50% was calculated.
Antihemolytic activity
The antihemolytic activity was determined according to
the method described by (Naim et al., 1976). The erythrocytes
from cow blood were separated by centrifugation and washed with
PFPL - P. foetida petroleum ether extract of leaves; PFEL - P. foetida ethanol phosphate buffer (pH 7.4) until the supernatant become colorless.
extract of leaves; PFHL - P. foetida hot water extract of leaves ; PFPR - P. foetida
petroleum ether extract of roots; PFER - P. foetida ethanol extract of roots; PFHR - The erythrocytes were diluted with saline phosphate buffer to give
P. foetida hot water extract of roots; PFPF - P. foetida petroleum ether extract of 4 % suspension. 500 µg of sample and 1 mL saline buffer were
flowers; PFEF - P. foetida ethanol extract of flowers; PFHF - P. foetida hot water
extract of flowers; PFPP - P. foetida petroleum ether extract of peels; PFEP - P. added to 2 mL of the suspension of erythrocytes and the volume
foetida ethanol extract of peels; PFHP - P. foetida hot water extracts of peels; PFPS was made up to 3.5 mL with saline buffer. This mixture was
- P. foetida petroleum ether extract of seeds; PFES - P. foetida ethanol extract of
seeds; PFHS - P. foetida hot water extract of seeds. BHA - Butylated
preincubated for 5 min and then 0.5 mL H2O2 solution of
hydroxyanisole; BHT - Butylated hydroxytoluene; QUE – Quercetin; RUT - Rutin; appropriate concentration in saline buffer was added .The
α-To – α-tocopherol. concentration of H2O2 in the reaction mixture was adjusted so as to
Fig. 2. DPPH˙+ free radical scavenging activity of P.foetida. *Values are means of bring about 90% haemolysis of blood cells after 240 min. After the
three replicate determinations (n=3) ± standard deviation. incubation time the reaction mixture was centrifuged at 1500 rpm
for 10 min and the extent of haemolysis was determined by
Nitric oxide radical scavenging activity measurement of the absorbance at (540 nm) corresponding to
The nitric oxide scavenging activity of different solvent haemoglobin liberation.
extracts of P. foetida on nitric oxide radical was measured
according to the method of (Sreejayan and Rao, 1997). Sodium β-carotene/linoleic acid peroxidation inhibition activity
nitroprusside (10 mM) in phosphate buffered saline, was mixed The antioxidant activity of P. foetida was evaluated by the
with different concentrations (50- 250 µg) of P. foetida extracts β-carotene linoleate model system. One milliliter of a β-carotene
and incubated at room temperature for 150 min. Griess reagent solution in chloroform (1 mg/10 ml) was pipetted into a flask
(0.5mL), containing 1% sulphanilamide, 2% H3PO4 and 0.1% N- containing 20 mg of linoleic acid and 200 mg of Tween 40. The
(1 – naphthyl) ethylene diamine dihydrochloride was added to the chloroform was removed by rotary vacuum evaporator (Yamato
mixture after incubation time. The absorbance of the chromophore RE300, Japan) at 45 ○C for 4 min and, 50 ml of oxygenated
formed was read at 546 nm. Gallic acid and quercetin and the same distilled water was added slowly to the semi-solid residue with
mixture of the reaction without P. foetida extracts were employed vigorous agitation, to form an emulsion. A 5 ml aliquot of the
as positive and negative control. Radical scavenging activity was emulsion was added to a tube containing 200 µl of the antioxidant
expressed as the inhibition percentage of free radical by the (extracts, BHA or α-tocopherol) solution at 1 mg/mL concentration
sample. and the absorbance was measured at 470 nm immediately, against
a blank consisting of the emulsion without β-carotene (Taga,
Total antioxidant activity assay by radical cation (ABTS˙+) Miller, & Pratt , 1984). The tubes were placed in a water bath at 50
○
ABTS was dissolved in water to a 7 mM concentration. C and the absorbance was monitored at 15 min intervals until 180
ABTS radical cation (ABTS•+) was produced by reacting ABTS min. All determinations were carried out in triplicate.
stock solution with 2.45 mM potassium persulfate (final Statistical analysis
concentration) and allowing the mixture to stand in the dark at The values are mean of triplicate analysis of the samples
room temperature for 12-16 hr before use. Prior to assay, the (n=3) and  indicates standard deviation. Analysis of variance and
solution was diluted in ethanol (about 1:89 v/v) and equilibrated to
significant differences (P0.05) among means were tested by one-
30ºC to give an absorbance at 734 nm of 0.70±0.02 in a 1-cm
way ANOVA and Dunnet multiple range test.
cuvette (Re et al., 1999). The concentration of the extracts that
produced between 20-80% inhibitions of the blank absorbance was RESULTS
determined and adapted. After the addition of 1 mL of diluted Total yield percentage
ABTS•+ solution to 10 µL of plant extract or Trolox standards The ethanol extracts of all the parts used for the analysis,
(final concentration 0-15 µM) in ethanol, optical density (OD) was showed higher percentage recovery over other solvent extracts. On
taken at 30ºC exactly 30 min after the initial mixing. The unit of the other hand, petroleum ether extracts of all the parts used,
total antioxidant activity (TAA) is defined as the concentration of showed lower percentage recovery compared to other solvent
Trolox having equivalent antioxidant activity expressed as µmol/g extracts. Thus, the maximum percentage recovery was observed in
sample extracts on dry matter.
 Journal of Applied Pharmaceutical Science 01 (04); 2011: 89-96

P. foetida ethanolic extract of root while the minimum one was lipid peroxidation, minimizing Fe2+ concentration in Fenton
shown by P. foetida petroleum ether extract of peel. reactions affords protection against oxidative damage. In the metal
chelating assay, ferrozine can quantitatively form complexes with
Total phenolics and Tannins Fe 2+. The chelating effects on the ferrous ions by the various
The total phenolic content determination showed that P. solvent extracts of different parts of the P. foetida were shown in
foetida ethanolic peel extract possess higher (10.09 %) phenolic (Table 2). All the sample extracts exhibited the ability to chelate
content over the other extracts such as ethanol extract of root metal irons. Among the different samples extracts, the ethanol
(9.3%), petroleum ether leaf extract (7.80 %), (6.95 %), hot water extract of flower showed higher metal chelating activity
extract of seed (5.42%), and petroleum ether extract of flower (6748.1mg/g) than other sample extracts.
(5.26 %). Total phenolics content of different parts of the solvent
extract of P. foetida were shown in the table.1. Among the Hydroxyl radical scavenging activity
different samples analysed, the tannin content was found to be Hydroxyl radical scavenging activity was estimated by
maximum (4.73%) in ethanol root extract, whereas the other generating the hydroxyl radicals using ascorbic acid-iron EDTA.
sample extracts showed minimum tannin content. The results are The hydroxyl radical formed by the oxidation will react with
showed in the (Table 1). dimethyl sulfoxide (DMSO) to yield formaldehyde, which provides
a convenient method to detect hydroxyl radicals, by treatment, with
Table 1.Total phenolics, tannins and vitamin C of P.foetida. Nash reagent (Singh, Murthy & Jayaprakasha, 2002). The
Extraction Sample Total Tannins Vitamin C
medium Phenolics scavenging activities of different plant parts were shown in (Table
Petroleum ether Leaf 7.8 ± 0.1 3.1 ± 0.2 2.3 ± 0.0
2). The OH˙ scavenging activities of all the samples were
Root 4.7 ± 0.0 0.1 ± 0.3 1.7 ± 0.0 investigated at the concentration of 250µg in the reaction mixture.
Flower 5.2 ± 0.2 0.5 ± 0.0 1.3 ± 0.0 The ethanol extracts of peel showed the highest level of scavenging
Peel 6.9 ± 0.3 0.6 ± 0.3 2.4 ± 0.0
Seed 5.0 ± 0.0 0.6 ± 0.1 1.3 ± 0.0 activity (92.5%). The flower samples exhibited moderate
Ethanol Leaf 4.8 ± 0.2 0.004 ± 0.3 1.6 ± 0.0 scavenging activity (62.5%, 64.2%, and 65.5%) compared to other
Root 9.3 ± 0.1 4.7 ± 0.2 1.5 ± 0.0
Flower 4.3 ± 0.0 0.8 ± 0.2 1.3 ± 0.0
sample extracts.
Peel 10.0 ± 0.0 2.7 ± 0.3 1.5 ± 0.0
Seed 4.5 ± 0.1 0.7 ± 0.1 1.4 ± 0.0 Nitric oxide radical scavenging activity
Hot water Leaf 4.3 ± 0.0 0.1 ± 0.2 1.3 ± 0.0
Root 4.9 ± 0.1 0.3 ± 0.2 1.2 ± 0.0 Nitric oxide is a very unstable species, so under aerobic
Flower 4.8 ± 0.0 1.1 ± 0.2 1.3 ± 0.0 condition it can react with O2 to produce its stable products such as
Peel 4.8 ± 0.1 0 ± 0.0 1.2 ± 0.0
Seed 5.4 ± 0.2 0.5 ± 0.0 1.4 ± 0.0
nitrate and nitrite through intermediates NO2, N2O4. The nitric
oxide radical scavenging activity was estimated by using Griess
Values are means of three replicate determinations (n=3) ± standard deviation.
reagent. In the presence of a scavenging test compound, the
Vitamin C amount of nitrous acid will decrease and can be measured at
The ascorbic acid content of P foetida was found to be 546nm. The nitric oxide radical scavenging activity of different
higher in petroleum ether extract of peel (2.4%) and petroleum solvent extracts of all the samples were shown in (Table 2). The
ether extract of leaf (2.3%), while the other extracts showed higher free radical scavenging activity was exerted by ethanol
Vitamin C content ranging from 1.2% - 1.7%. The results are extract of peel (120.48µg) followed by ethanol extract of flower >
showed in the (Table 1). hot water extract of leaf > hot water extract of peel > ethanol
extract of root > ethanol extract of leaf > hot water extract of leaf >
Reducing power hot water extract of flower > petroleum ether extract of seed >
In the reducing power assay, the presence of reductants ethanol extract of seed > hot water extract of seed > petroleum
(antioxidants) in tested samples would result in the reduction of ether extract of leaf > petroleum ether extract of flower > ethanol
Fe3+/ ferricyanide complex to the ferrous form. The Fe2+ can
therefore be monitored by measuring the formation of Perl’s extract of leaf > petroleum ether extract of peel.
Prussion blue at 700 nm (Chung et al., 2002). The reducing power
of different solvent extracts of the samples was shown in (Figure ABTS radical scavenging activity
2). A strong reducing power was noted for the samples ethanol In ABTS˙+ cation radical scavenging method, the activity
extract of root and hot water extract of peel (0.57 OD700). Much of tested extracts were expressed as micromolar equivalent of
lower reducing power was found for the sample petroleum ether Trolox solution having an antioxidant equivalent to one gram dry
extract of root (0.27 OD700). matter of the sample under the experimental investigation. The
effect of various solvent extracts of different plant parts of P.
Metal chelating activity foetida is shown in (Table 2). Eventhough the samples exhibited
The transition metal iron is capable of generating free good ABTS radical scavenging activity, the petroleum ether extract
radicals from peroxides by Fenton reactions and may be implicated of flower showed the highest activity (3991.9µmolg-1). Whereas
in human cardiovascular diseases (Chung et al.,2002). Since Fe2+ the ethanol extract of peel showed lower level of activity (1649.
also has been shown to cause the production of oxyradicals and 9µmolg-1), when compare to other samples extracts.
 Journal of Applied Pharmaceutical Science 01 (04); 2011: 89-96

Table 2. ABTS*, metal chelating and hydroxyl radical scavenging activities of

P.foetida
Extracti Sample TAA (µmol/g Metal Hydroxyl Nitric oxide
on DM) chelating radical radical
medium Property scavengin scavenging
(mg EDTA/g g activity
extract) activity
Petroleu Leaf 3570.7±217.2 5903.7±325.1 49.9±0.4 204.0 ± 0.5.
m ether Root 3914.9±57.6 6270.3±89.8 55.8±0.5 259.0 ± 0.3
Flower 3991.4±54.9 5777.7±86.7 64.2±0.7 206.0 ± 1.5
Peel 3739.4±35.7 5985.1± 587.9 62.3±2.1 228.3 ± 1.9
Seed 3140.9±237.0 6348.1± 193.1 58.6±0.6 141.2 ± 0.4
Ethanol Leaf 3845.2±57.4 5540.7±511.8 25.0±1.2 227.0 ± 0.7
Root 3725.9±128.6 5129.6±588.9 50.9±1.6 143.2 ± 1.0
Flower 3710.2±89.1 6748.1±281.3 62.5±0.1 127.2 ± 0.3
Peel 1649.9±157.0 6325.9± 83.3 92.5±0.6 120.4 ± 0.6
Seed 3667.4±67.6 6451.8± 145.0 55.9±0.7 180.5 ± 1.3
Hot Leaf 3631.4±210.5 5574.0±409.2 44.0±10.0 131.5 ± 1.4
water Root 2094.7±295.7 5562.9±272.2 55.4±0.2 144.5 ± 0.4
Flower 3680.2±218.2 6562.9±212.0 65.5±2.1 162.8 ± 0.9 PFPL - P. foetida petroleum ether extract of leaves; PFEL - P. foetida ethanol
Peel 3543.7±201.2 6077.7± 539.8 78.2±0.5 132.6 ± 0.2 extract of leaves; PFHL - P. foetida hot water extract of leaves; PFPR - P. foetida
Seed 2857.4±116.1 6340.7± 73.9 63.0±0.3 200.0 ± 0.6 petroleum ether extract of roots; PFER - P. foetida ethanol extract of roots; PFHR -
Values are means of three replicate determinations (n=3) ± standard deviation. P. foetida hot water extract of roots; PFPF - P. foetida petroleum ether extract of
flowers; PFEF - P. foetida ethanol extract of flowers; PFHF - P. foetida hot water
extract of flowers; PFPP - P. foetida petroleum ether extract of peels; PFEP - P.
DPPH radical scavenging activity foetida ethanol extract of peels; PFHP - P. foetida hot water extracts of peels; PFPS
The free radical-scavenging activities of different parts of - P. foetida petroleum ether extract of seeds; PFES - P. foetida ethanol extract of
seeds; PFHS - P. foetida hot water extract of seeds. BHA - Butylated
the P. foetida samples along with standards such as BHA, BHT, hydroxyanisole; BHT - Butylated hydroxytoluene; QUE – Quercetin; RUT - Rutin;
Quercitin, Tannic acid and α-Tocopherol were determined by the TAN - Tannin acid; α-To – α-tocopherol; ASC - Ascarbic acid.
DPPH radical scavenging activity and the results were shown in
Fig. 3. Antihemolytic activity of different solvent of P. foetida. Values are means
(Figure 2). The decrease in absorbance of the DPPH radical caused of three replicate determinations (n=3) ± standard deviation.
by antioxidant was due to the scavenging of the radical by
hydrogen donation. It is visually noticeable as a colour change inhibition of 18.52 when compared to other extracts. The results
from purple to yellow. A lower value of IC50 indicates a higher were shown in the (Figure 4).
antioxidant activity. The highest free radical scavenging activity
was exerted by ethanol extract of leaf (595.23µg). The free radical
scavenging activity was found to be least in petroleum ether extract
of peels (980.39 µg). The scavenging effects of various solvent
extracts with the DPPH radical in the ascending order is given by
ethanol extract of leaf > petroleum ether extract of root > ethanol
extract of peel > hot water extract of peel > ethanol extract of seed
> petroleum ether extract of seed > petroleum ether extract of
flower > hot water extract of seed > ethanol extract of root > hot
water extract of root > ethanol extract of flower > hot water extract
of flower > hot water extract of leaf > petroleum ether extract of
leaf > petroleum ether extract of peel.

Antihemolytic activity PFPL - P. foetida petroleum ether extract of leaves; PFEL - P. foetida ethanol
extract of leaves; PFHL - P. foetida hot water extract of leaves; PFPR - P. foetida
Lipid oxidation of cow blood erythrocyte membrane, petroleum ether extract of roots; PFER - P. foetida ethanol extract of roots; PFHR -
mediated by H2O2 induces membrane damage and subsequently P. foetida hot water extract of roots; PFPF - P. foetida petroleum ether extract of
flowers; PFEF - P. foetida ethanol extract of flowers; PFHF - P. foetida hot water
haemolysis. Moreover, the RBC haemolysis is a more sensitive extract of flowers; PFPP - P. foetida petroleum ether extract of peels; PFEP - P.
system for evaluating the antioxidant properties of the foetida ethanol extract of peels; PFHP - P. foetida hot water extracts of peels; PFPS
- P. foetida petroleum ether extract of seeds; PFES - P. foetida ethanol extract of
phytoceuticals. The antihaemolytic activity of different solvent
seeds; PFHS - P. foetida hot water extract of seeds. ASC - Ascarbic acid; GAL -
extracts was given in (Figure 3). At the concentration of 500µg in Gallic acid; TAN - Tannic acid.
the final reaction mixture, all the sample extracts exhibited the
Fig. 4. Antioxidant activity of β- carotene bleaching method of P. foetida.
antihaemolytic activity. Inhibition of haemolysis was found to be Values are means of three replicate determinations (n=3) ± standard deviation.
the highest in hot water extract of roots (89.09%).
DISCUSSION
β- Carotene/linoleic acid peroxidation inhibition activity
Phenolics compounds are commonly found in both edible
The β-carotene bleaching method estimates the relative
and non-edible plants, and they have multiple biological effects.
ability of antioxidants compounds to scavenge the radical of
Among the different sample extract, total phenol content present in
linoleic acid peroxide that oxidizes β-carotene in the emulsion
the peel ethanol extract was found to be maximum. Phenolic
phase. In the β-carotene bleaching system, the highest antioxidant
compounds are widely distributed in plants, which have gained
activity was shown by ethanol extract of leaf with percentage
much attention, due to their antimutegenic, antitumor, antioxidant
 Journal of Applied Pharmaceutical Science 01 (04); 2011: 89-96

activities and free radical-scavenging abilities, which potentially estimated using Griess reagent. In the present study, all the extract
have beneficial implication for human health (Li et al., 2006). of P.foetida exhibited dose dependent nitric oxide radical
Among the different samples, the tannin content was found to be scavenging activity. The ABTS·+ radical assay can be used to
maximum in the ethanol extract. Tannins were identified as another measure the substances, i.e., both aqueous phase radicals and lipid
large class of phenolics in the tested medicinal herbs. Vitamins are peroxyl radicals (Robert et al., 1999). The scavenging activity of
the small molecule dietary antioxidant, quickly moving into an the extract on the radical ABTS, generated by potassium persulfate
important position in medicinal and nutritional circles (Milton, was compared with a standard amount of trolox. In general,
1999). Vitamin C is highly bio-available and is consequently one medicinal plants may play an important role in chemical protection
of the most important water-soluble antioxidants in cells. In the from oxidative damage by possessing endogenous antioxidants
present study, vitamin C content was strongly correlated with such as phenolic compounds. Hagerman et al., (1998) have
antioxidant capacity. This supports the observation that vitamin C reported that the high molecular weight phenolics (tannins) have
may be one of the major antioxidants in P.foetida fruits. Reducing more ability to quench free radicals (ABTS·+). In the present
power Fe (III) reduction is often used as an indicator of electron investigation, all the plant extract possessed the free radical
donating activity, which is an important mechanism of phenolic property. The ethanol extract of peel exhibited the highest radical
antioxidant action, can be strongly correlated with other scavenging activity. Li et al., (2006) had recently reported that the
antioxidant properties (Dorman et al., 2003). P.foetida root and peels of pomegranate have higher antioxidant activity than its pulp
peel extracts showed the highest reducing power and the values and seed. Regarding these result, it could be considered that the
were comparable to that of ascorbic acid standard. Iron is essential peel extracts of P.foetida contain strong antioxidative agents, and
for life because it is required for oxygen transport, respiration and had the highest potential. DPPH scavenging is widely used to test
activity of many enzymes. However, iron was an extremely the free radical scavenging activity of several natural products
reactive metal and will catalyze oxidative change in lipid, protein (Ahn et al. 2007). DPPH is a stable free radical and any molecule
and other cellular components (Decker & Hultin, 1992). An that can donate an electron or hydrogen to DPPH* can react with it
important mechanism of antioxidant activity is the ability to and bleach the DPPH* absorption at 517 nm (Huang, Ou, & Prior,
chelate/deactivate transition metals, which possess the ability to 2005). There is a reverse correlation between IC50 values and
catalyze hydroperoxide decomposition and Fenton-type reaction. DPPH scavenging activity. The degree of discoloration indicates
Therefore, it was considered of importance to screen the iron (II) the scavenging potentials of the antioxidant extract. In the present
chelating ability to the extracts. In the present study, all the extracts study, the highest free radical scavenging activity was extracted by
demonstrated the ability to chelate iron. From the iron (II) ethanol extract of leaf. The result indicates that medicinal plants
chelating data, the extracts may be able to play a protective role have significant effects on scavenging free radicals. Hemolysis has
against oxidative damage by sequestering iron (II) ions that may a long history of use in measuring free radical damage and its
otherwise catalyze Fenton-type reaction or participate in metal- inhibition by antioxidant but only few studies have been performed
catalyzed hydroperoxide decomposition reactions (Dorman, et al., with erythrocytes in whole blood. Zhu et al., (2002). Demonstrated
2003) Among the different sample extract of P. foetida, the ethanol a dose dependent activity for three fraction of Oolong tea effective
extract of flower showed higher activity. Hydroxyl radical against lipid oxidation in the erythrocytes membrane. In this study,
scavenging activity was esrimated by generating the hydroxyl we used a biological test based on the free radical-induced
radical using ascorbic acid iron EDTA. The hydroxyl radical erythrocytes lysis in cow blood. This assay is useful either for
formed by the oxidation will react with dimethyl sulfoxide screening studies on various molecules and their metabolites,
(DMSO) to yield formaldehyde, which provides a convenient especially on the one hand molecules have an oxidizing or
method to detect hydroxyl radicals, by treatment, with Nash antioxidizing activity and on the other hand molecule having a
reagent (Singh, Murthy & Jayaprakasha, 2002). The hydroxyl long-term action (Djeridane et al., 2006). More over the RBC
radical is an extremely reactive free radical formed in biological hemolysis is a more sensitive system for evaluating the antioxidant
system and has been implicate as a highly damaging species in free properties of the phytoceuticals. The higher antihemolytic potential
radical pathology, capable of damaging almost every molecule was shown by P.foetida hot water extract of root, which might
found in living cells (Hochestein and Atallah, 1988). In P.foetida attribute to the presence of Vitamin C. β-carotene bleaching
peel samples a significant correlation between hydroxyl radical method estimates the relative ability of antioxidants compounds to
scavenging activity and total phenolic content was observed. Nitric scavenging the radical of linoleic acid peroxide that oxidizes β-
oxide is a free radical with a single unpaired electron. Nitric oxide carotene in the emulsion phase. The antioxidant assay using the
is formed from L-argenine by NO synthase (Fang, Yang, & Wu, discoloration of β-carotene is widely used because β-carotene is
2002). Nitric oxide, exposed in human blood plasma, can deplete extremely susceptible to free radical mediated oxidation. Due to its
the concentration of ascorbic acid and initiate lipid peroxidation 11 pairs of double bonds, which are extremely sensitive to
(Halliwell, 1996). The nitric oxide scavenging assay procedure is oxidation, β-carotene decolorized easily with the oxidation of
based on the principle that the sodium nitroprusside in aqueous linoleic acid (Unten, Koketsum & Kim, 1997). The protective
solution at physiological pH spontaneously generates nitric oxide, effect of β-carotene can be explained by scavenging of free radicals
which interacts with oxygen to produce nitrite ions that can be before they cause damage to cellular macromolecules because
 Journal of Applied Pharmaceutical Science 01 (04); 2011: 89-96

these are known antioxidant with the capacity for free radical phenolics compounds and their antioxidant activity. Eur Food Res
trapping. This indicates that the P.foetida possess antioxidant Technol 2006; 224: 801-809.
activity. Hence, the result justifies the employment of P. foetida in Dorman HJD, Kosar M, Kahlos K, Holm Y, Hilturien R
Antioxidant properties and composition of aqueous extract from
treating various ailments and their related disorders..
Mentha species, hybrid varieties and cultivars. J Agric Food Chem
2003; 51: 4563-4569.
CONCLUSION Echeverri F, Arango V, Quinones W, Terres F, Escobar
G, Rosero Y, et al. Passiflorocins, Polyketides and -pyrones
This study supports the idea that the medicinal plants of From Passiflora foetida resin. Phytochemistry 2001; 56: 881-885.
P.foetida have a good source of natural antioxidants. A good Echeverri F, Torres F, Cardona G, Lopez J, Quinones W,
correlation between total phenols content and the FRAP values Gallego L, et al. Isolation of an ingestion deterrent from Passiflora
foetida L. Revist Boliviana de Quimica 1991;10: 25-29.
also support the idea that phenols may be the principal contributor
Fang YZ, Yang S, Wu G Free radicals, antioxidants and
of the antioxidant power of botanical materials. It is very clear that nutrition. Nutrition 2002; 18: 872-879.
this is a plant with tremendous wide spread use and also with Hagerman AE, Riedl KM, Jones GA, Sovik KN, Ritchard
extraordinary potential for the pharmaceuticals. Hence further NT, Hartzfeld PWHigher molecular weight oplant
studies should be considered in isolating bioactive compounds polyphenolics(Tannins) as biological antioxidant. J Agric Food
from P.foetida and it will pave a way for promoting natural drugs Chem 1998; 46: 1887-1892.
for various health benefits. Halliwel B. Uric acid; an examble of antioxidant
evalution. In: Handbook of antioxidants. Cadenas, E. and Packer,
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ACKNOWLEDGMENTS Hochestein P, Atallah S The nature of oxidant and
antioxidant systems in inhibition of mutation and cancer. Mutation
The authors are very grateful to the University Grants Research 1988; 202: 363-3745.
Commission New Delhi (UGC letter No: F.No.37-95/2009 (SR)) Huang D, Ou B, Prior RL. The Chemistry behind
for financial support of this major research project work. antioxidant capacity assays. J Agric Food Chem 2005; 53: 1841-
1856.
Klenin SM, Cohen G, Cederbaum AI Production of
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