European Journal of Scientific Research

ISSN 1450-216X Vol.31 No.4 (2009), pp.592-598

EuroJournals Publishing, Inc. 2009
http://www.eurojournals.com/ejsr.htm

Antibacterial, Cytotoxic and Antioxidant Activity of

Passiflora Edulis Sims

Farhana Alam Ripa

LecturerDepartment of Pharmacy, Southeast University
Banani, Dhaka 1213, Bangladesh
E-mail: ripa_rubd@yahoo.com
Tel: +8801726216153

Mahmuda Haque
Corresponding AuthorSenior LecturerDepartment of Pharmacy
Southeast University, Banani, Dhaka 1213, Bangladesh
E-mail: mh_sumi@yahoo.com
Tel: +8801716586770

Laizuman Nahar
LecturerDepartment of Pharmacy, Southeast University
Banani, Dhaka 1213, Bangladesh
E-mail: laboni4@yahoo.com
Tel: +8801712676754

Md. Monirul Islam

Department of Agricultural Extension, Khamarbari
Farmgate, Dhaka 1215, Bangladesh
E-mail: mmi75@live.com
Tel: +8801712101035

Abstract

The petroleum ether and chloroform fractions of ethanol extract of leaf and stem
from the plant Passiflora edulis (Fam: Passifloraceae) was subjected to antioxidant,
antibacterial and cytotoxic activity. All the fractions showed potent antioxidant activity, of
which the chloroform and petroleum ether fraction of stem demonstrated the strongest
antioxidant activity with the IC50 value of 51.28 g/ml and 54.01 g/ml, respectively. In
case of antibacterial screening, crude chloroform extract of leaf showed moderate
antibacterial activity ranged from 7-10 against twelve microorganisms at a concentration of
500 g/disc but no activity is observed by the petroleum ether extract. The crude
chloroform and petroleum ether extracts of stem showed notable antibacterial activity at a
concentration 500 g/disc ranged from 8-17 and 7-12 respectively against twelve
microorganisms However, in the brine shrimp lethality bioassay, all the crude extracts of
leaf and stem possessed considerable cytotoxic activity. It was evident that, the chloroform
and petroleum ether extracts of stem and leaf have significant cytotoxic potentials with the
LC50 value of 6.63 g/ml, 6.89 g/ml and 7.91 g/ml 11.17g/ml respectively.
Antibacterial, Cytotoxic and Antioxidant Activity of Passiflora Edulis Sims 593

Keywords: Passiflora edulis, Passifloraceae, antibacterial, antioxidant, brine shrimp.

1. Introduction
Undoubtedly medicinal plants are relevant in both developing and developed nations of the world as
sources of drugs or herbal extracts for various chemotherapeutic purposes. Also the use of plant-
derived natural compounds as part of herbal preparations used as alternative sources of medicaments
continues to play major roles in the general wellness of people all over the world. Higher plants, as
sources of medicinal compounds continue to play a dominant role in maintenance of human health
since antiquities. Over 50% of all modern clinical drugs are of natural product origin (Stuffness and
Douros, 1982) and natural products play an important role in drug development programs of the
pharmaceutical industry (Baker et al., 1995; Cordell, 1995). In developing countries, especially in rural
contexts people usually turn to traditional healers when in diseased conditions and plants of ethno
botanical origin are often presented for use. In the continuation of this strategy of new drug discovery
we have studied only the aerial parts of the plant P. edulis for their antibacterial, cytotoxic and
antioxidant properties.
P. edulis Sims (Passion fruit) belongs to the genus Passiflora, comprising about 500 species that
are distributed in warm temperatures and tropical regions. The passion fruit, in general, prefer
subtropical and frost free environments. P. edulis Sims (Family- Passifloraceae) is a vigorous climber.
They cling to anything they can grab. The leaves are evergreen and alternate, 3 lobed leaves when
mature. They grow quickly and 15 20 feet per year once established. They should have strong
support. Their life cycle seems to be short in 5-7 years, but new plants can be planted and fruit can
happen the same year. Several species are grown in the tropics for edible fruits, the most widely grown
being P. edulis (McGuire, 1999).The pulp of the fruit is stimulant and tonic(Chopra et al., 1986).The
fruit has anticarcinogenic effect ( Neira et al., 2003).The flower extract of P. edulis has sedative and
hypnotic effect(Capasso et al., 2005). Glycosides, phenols and alkaloids are major constituents in P.
edulis (Dhawan et al., 2004). Identified constituents in the plant includes carotenoids (Goday &
Rodriguez, 1994), l-ascorbic acid (Wekesa et al., 1996), anthocyanins (Kidoey et al., 1997), -lactones
(Bernreuther et al., 1989), flavour components, volatile oil constituents (Dawes and Paul, 1961;
Kuhlmann, 1984; Arriaza et al., 1997), amino acids (Fang & Ling, 1981), carbohydrates (Fang and
Chang, 1984; Simpson et al., 1984), minerals (Nogueira et al., 1998), the cytoplasmic enzyme pyruvate
kinase (Guo & Li, 1993), cycloartane trierpenes, cyclo passiloic acids A-D, and their saponins,
cyclopassiflosides I-VI (Yoshikawa et al., 2000). The present study was undertaken to investigate the
cytotoxicity, antimicrobial activity and the preliminary antioxidant activities of the organic extractives
of P. edulis.

2. Materials and Methods

2.1. Plant Material
The plant, P. edulis was collected from Tangail in the month of March 2009 and identified by DR.
M.A. Razzaque Shah, Tissue Culture Specialist, BRAC Plant Biotechnology Laboratory, Bangladesh.

2.2. Plant Material Extraction

The fresh leaf and stem were collected, sun dried for seven days and ground. The dried powder of P.
edulis leaf (200gm) and stem (200gm) were soaked in 600ml of ethanol for 7 days and filtered through
a cotton plug followed by Whatman filter paper number. A portion (15 gm) of the concentrated ethanol
of leaf extract (16 gm) and stem extract (14 gm) were fractionated by the modified Kupchan
partitioning method (Van Wagenen et al., 1993) into petroleum ether, chloroform and aqueous soluble
fractions. The subsequent evaporation of solvents afforded petroleum ether (450 mg) and chloroform
594 Farhana Alam Ripa, Mahmuda Haque, Laizuman Nahar and Md. Monirul Islam

(700 mg) from leaf extract and petroleum ether (400 mg), and chloroform (650 mg) from stem extract
respectively.

2.3. Antibacterial Screening

The antibacterial assay was performed by disc diffusion technique (Bauer, et al., 1966; Rios et al.,
1988). Disc diffusion technique is highly effective for rapidly growing microorganisms. The
microorganisms were collected as pure cultures from the Institute of Nutrition and Food Science
(INFS), University of Dhaka, Bangladesh. The sample solution of the material to be tested was
prepared by dissolving a definite amount of material in appropriate solvent to attain a concentration of
50mg/ml. 10 l of such solution was applied on sterile disc (5mm diameter, filter paper) and allowed to
dry off the solvent in an aseptic hood. Thus, such discs contain 500 g of crude extracts. To compare
the activity with standard antibiotics, Kanamycin (30 g/disc) was used.
The extracts of P. edulis was tested against four Gram- positive (Bacillus megaterium, B.
subtilis, Staphylococcus aureus and Sarcina lutea) and eight Gram- negative (Salmonella paratyphi, S.
typhi, Vibrio parahemolyticus, V. mimicus, Escherichia coli, Shigella dysenteriae, S. boydii and
Pseudomonas aeruginosa) bacteria. Briefly, in this study the test discs and standard disc were placed in
a Petri dish seeded with particular bacteria and then left in a refrigerator at 4C for 12-18 hrs in order to
diffuse the material from the discs to the surrounds media in the Petri dishes. The Petri dishes were
then incubated at 37C for overnight to allow the bacterial growth. The antibacterial activities of the
extracts were then determined by measuring the respective zone of inhibition in mm.

2.4. Cytotoxicity Screening

Brine shrimp lethality bioassay is widely used in the bioassay for the bioactive compounds (Meyer et
al., 1982; Zhao et al, 1992). Here simple zoological organism (Artemia salina) was used as a
convenient monitor for the screening.
The eggs of the brine shrimp were collected from an aquarium shop (Dhaka, Bangladesh) and
hatched in artificial seawater (3.8% NaCl solution) for 48 hr to mature shrimp called nauplii. The
cytotoxicity assay was performed on brine shrimp nauplii using Meyer method (Meyer, et al.,
1982).The test samples (extract) were prepared by dissolving them in DMSO (not more than 50 l in 5
ml solution) plus sea water (3.8% NaCl in water) to attain concentrations of 5g/ml, 10g/ml,
20g/ml, 40g/ml, and 80g/ml. A vial containing 50l DMSO diluted to 5ml was used as a control.
Standard Vincristine sulphate was used as positive control. Then matured shrimps were applied to each
of all experimental vials and control vial. The number of the nauplii that died after 24 hr was counted.
Then % of mortality was plotted against respective concentrations used and from the graph LC50 was
calculated.

2.5. Antioxidant activity

The antioxidant activity of leaf and stem extracts of P. edulis was determined using the 1, 1-diphenyl-
2-picrylhydrazyl (DPPH) free radical scavenging assay by the method of Blois (1958). DPPH offers a
convenient and accurate method for titrating the oxidizable groups of natural or synthetic anti-oxidants
(Cao et al., 1997).The crude extracts (leaf and stem) of P. edulis were mixed with 95% methanol to
prepare the stock solution (10 mg/100mL). The test samples were prepared from stock solution by
dilution with methanol to attain a concentration of 20g/ml, 40g/ml, 60g/ml, 80g/ml & 100g/ml
respectively. DPPH solution was prepared in methanol. Freshly prepared DPPH solution was added in
each of these test tubes containing P. edulis extract and after 20 min, the absorbance was taken at 517
nm using a spectrophotometer. Ascorbic acid was used as a positive control. The DPPH solution
without sample solution was used as control. 95% methanol was used as blank. Percent scavenging of
the DPPH free radical was measured using the following equation-
% DPPH radical scavenging (%) = [1-(As/Ac)] 100.
Antibacterial, Cytotoxic and Antioxidant Activity of Passiflora Edulis Sims 595

Here, Ac=absorbance of control, As=absorbance of sample solution.

Then % inhibitions were plotted against respective concentrations used and from the graph IC50
was calculated.

3. Results
3.1. The Results of Antibacterial Screening
The petroleum ether extract (500g/disc) of P. edulis leaf showed no activity against most of the tested
organisms, except B. megaterium and P. aeruginosa having positive effect. On the other hand
chloroform crude leaf extracts (500g/disc) showed moderate antibacterial activity with the average
zone of inhibition of 7-10 mm by disc diffusion method (Table 1). Among the tested bacteria, the
growth of S. dysenteriae and S. boydii (10 mm) were moderately inhibited. In case of stem, the
chloroform extract showed the highest activity against the growth of V. mimicus having the zone of
inhibition of 17mm. Besides this, the extract showed good activity against the growth of V.
parahemolyticus (16mm), S. dysenteriae (15mm) and S. boydii (14 mm). The petroleum ether extract
showed moderate zone of inhibition from 7 12 mm (Table 2).

Table 1: In vitro antibacterial activity of Passiflora edulis leaf and standard Kanamycin discs

Diameter of zone of inhibition

Test organisms
Gram positive bacteria
Bacillus megaterium
Bacillus subtilis
Staphylococcus aureus
Sarcina lutea
Gram negative bacteria
Escherichia coli
Pseudomonas aeruginosa
Salmonella paratyphi
Salmonella typhi
Shigella boydii
Shigella dysenteriae
Vibrio mimicus
Vibrio parahemolyticus
+ sign indicates positive effect and - sign indicates no activity.
Petroleum ether extract Chloroform extract
Kanamycin (30g/disc)
(500g/disc) (500g/disc)
+ 8 30
- 7 23
- 9 26
- 7 24
- 9 22
+ 10 25
- 7 25
- 8 25
- 10 25
- 10 25
- 7 28
- 7 26
596 Farhana Alam Ripa, Mahmuda Haque, Laizuman Nahar and Md. Monirul Islam
Table 2: In vitro antibacterial activity of Passiflora edulis stem and standard Kanamycin discs

Diameter of zone of inhibition

Test organisms Petroleum ether extract Chloroform extract
Kanamycin (30g/disc)
(500g/disc) (500g/disc)
Gram positive bacteria
Bacillus megaterium 9 13 30
Bacillus subtilis 7 12 23
Staphylococcus aureus 7 10 26
Sarcina lutea 9 13 24
Gram negative bacteria
Escherichia coli + 11 22
Pseudomonas aeruginosa 9 12 25
Salmonella paratyphi 10 8 25
Salmonella typhi 10 9 25
Shigella boydii 11 14 25
Shigella dysenteriae 12 15 25
Vibrio mimicus 10 17 28
Vibrio parahemolyticus 9 16 26
+ sign indicates positive effect and - sign indicates no effect.

3.2. The Results of Brime Shrimp Lethality Bioassay

Following the procedure of Meyer, the lethality of the crude petroleum ether and chloroform extracts
of P. edulis leaf and stem to brine shrimp was determined on A. salina after 24 hours of exposure the
samples and the positive control, vincristine sulphate. This technique was applied for the determination
of general toxic property of the plant extractive. The LC50 values for standard vincristine sulphate and
extracts of P. edulis were presented in table 3. The chloroform extract of stem showed the lowest LC50
value and petroleum ether extract of leaf showed highest value which was 6.63 g/ml and 11.17g/ml
respectively.

Table 3: LC50 data of test samples of Passiflora edulis and Vincristine sulphate

Samples LC50 (g/ml)

Vincristine sulphate 5.68
Petroleum ether extract of leaf 11.17
Chloroform extract of leaf 7.91
Petroleum ether extract of stem 6.89
Chloroform extract of stem 6.63

3.3. The Result of Antioxidant Activity

The antioxidant activity of the extracts was assessed by the DPPH free radical scavenging assay as
shown in table 4. All the four extract exhibited potential antioxidant activity. The chloroform extract of
stem scavenged 50% DPPH free radical at the lowest inhibitory concentration (IC50: 51.28g/ml). The
petroleum ether extract of stem also revealed strong antioxidant activity (IC50: 54.01g/ml). On the
other hand, petroleum ether and chloroform extracts of leaf showed antioxidant activity with IC50 of
58.88g/ml and 56.85g/ml respectively. These results denote the presence of antioxidant principles in
the extractives.
Antibacterial, Cytotoxic and Antioxidant Activity of Passiflora Edulis Sims 597
Table 4: IC50 data of test samples of Passiflora edulis and Ascorbic acid

Samples IC50 (g/ml)

Ascorbic acid 43.04
Petroleum ether extract of leaf 58.88
Chloroform extract of leaf 56.85
Petroleum ether extract of stem 54.01
Chloroform extract of stem 51.28

4. Summary and Concluding Remarks

The present study indicated that the chloroform and petroleum ether extract of P. edulis stem have got
profound antibacterial, cytotoxic and antioxidant effect than chloroform and petroleum ether extract of
leaf and may have potential use in medicine.
The stem extracts have got significant cytotoxic activity. Moderate cytotoxic effects indicate
that the plant can be selected for further cell line assay, because many scientists have shown a
correlation between cytotoxicity and activity against the brine shrimp nauplii using extract. Moreover
P. edulis also has got profound antioxidant activity.
It may be concluded from this study that P. edulis is active against the tested
pathogenicmicroorganisms and also have cytotoxic and antioxidant effects. In addition, the results
confirm the use of the plant in traditional medicine. The results of the investigation do not reveal that
which chemical compound is responsible for aforementioned activity. Now our study will be directed
to explore the lead compound responsible for aforesaid activity from this plant.

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