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Bioassay Guided Phytometabolites Extraction for Screening of Potent

Antimicrobials in Passiflora foetida L.
Article in Journal of Applied Pharmaceutical Science · September 2012
DOI: 10.7324/JAPS.2012.2927

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Journal of Applied Pharmaceutical Science Vol. 2 (9), pp. 137-142, September, 2012
Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2012.2927
ISSN 2231-3354

Bioassay Guided Phytometabolites Extraction for Screening of Potent

Antimicrobials in Passiflora foetida L.
Patil A.S.* and Paikrao H.M
Department of Biotechnology, Sant Gadge Baba, Amravati University, Amravati (MS), 444602 India.

ARTICLE INFO ABSTRACT

Article history: Passiflora is a genus belonging to the family Passifloraceae having varied species with highly therapeutic
Received on: 11/08/2012 values. Tribes with traditional medicinal knowledge suggested Passiflora foetida L. as a source of high value
Revised on: 29/08/2012 pharmaceutical plant. Present study deals with the Isolation, Purification, characterization and bioassays of
Accepted on: 08/09/2012 antimicrobial secondary metabolites. In spite of traditional soxhlet extraction, Cold percolation proved
Available online: 28/09/2012 suitable extraction scheme. Bioassay guided TLC characterization, and purification led the effective
collection of bioactive natural products. Disc diffusion method shows a potent inhibitory activity of
Kay words: Passiflora foetida compounds against multi drug resistant pathogenic microorganisms like S. aureus(OARS). For structural
L., Phytochemicals, TLC- characterization bioactive products were analyzed using UV. The results confirmed the presence of
bioautography, antimicrobial polyacetylenes as active constituents in the plant.
activity, polyacetylenes

INTRODUCTION P. amethystina Mikan, found in the rain forest of the Brazilian

south-eastern coast (Fouqué, 1972), is a wild passion fruit species.
The genus Passiflora belongs to Passifloraceae family
It has purple-blue flowers, which are very aromatic. The genus also
and includes the passion fruit, is the largest and the most
contains some species of ornamental use and medicinal properties
widespread genus of tropical flora. About 400 species of this
as sedatives, antispasmodics and antibacterial (Nhut et al., 2007).
genus are grouped into 21 subgenera (Cronquist, 1981). More than
Several species have edible fruits and attractive flowers, about 40
350 species have been found in tropical regions and rain forests of
species have been cultivated, but fewer than 6 are fruit crops in the
South America (Killip, 1938) and 60 of them are edible species.
neotropics and only one, P. edulis (and its varieties, such as the
Passion fruit is an important fruit crop in many exotic and
yellow favicarpa), is economically important. A few species, such
subtropical countries due to its edible fruits, ornamental use and
as P. foetida and P. lonchocarpa, are extremely foul smelling
medicinal properties. Some species (P. edulis, P. quadrangularis
(Benson et al., 1976). Eleven species, including P. foetida and P.
and P. ligularis). are chiefly cultivated for the production of fruit tripartita (= P. mollissima) are recorded as weeds in distinctive
juice. P. incarnata is reputed for its sedative properties, and parts of the world (Swarbrick 1981). Both P. foetida and P.
several other species are known for their ethnobotanical uses. tripartita are closely related taxonomically, whereas, P. edulis
* Corresponding Author
belongs to a different subgenus (Waage et al., 1981) and is the only
Email: [email protected] , Fax no. 91- 0721 – 2662135; 2660949 economic crop at risk from oligophagous insects attacking
Telephone (O) 91-721-2662208 ext 267, 091- 9881735354 (Mobile)
138 Patil and Paikrao / Journal of Applied Pharmaceutical Science 2 (09); 2012: 137-142

P. foetida. In recent years, a significant revival of interest in were purchased from Sigma-Aldrich, St. Louis, MO, USA. The
natural products as a potential source for new medicines has been UV spectrophotometer-1800 (Shimadzu, Japan), and UV
observed throughout the world. Several modern drugs ~40% in use Transilluminator (Cleaver, Korea) was used for study and analysis.
have been developed from natural products. Nowadays, multiple
drug resistance in human pathogenic microorganisms develops due PRELIMINARY PHYTOCHEMICAL SCREENING
to indiscriminate use of commercial chemical antimicrobial drugs
The extracts of Passiflora foetida were analyzed for the
commonly used in the treatment of human diseases. Over the last
presence of Alkaloids, Saponins, Tannins, Cardiac Glycoside,
three centuries, intense efforts have been made to discover
Anthraquinones, Steroid, coumarin, carbohydrates and flavonoids,
clinically useful antimicrobial drugs (Ahmed et al., 1998; Sarker
according to standard methods (Odebiyi and sofowora, 1978;
et al., 2006; Werner et al., 1999). The increasing interest on
Sofowora, 1982; Williamson et. al., 1996; Banso and Ngbede,
traditional ethno medicine may lead to discovery of novel
2006; Ngbede et. al., 2008). For the study of phytochemical
therapeutic agents. The World Health Organization (2000)
analysis, the ethanol extract of the plant leaves was prepared
estimates that 80% of the population of developing countries still
according to standard methods (Sofowora, 1982). The plant leaves
relies on traditional medicines, mostly plant drugs, for their
were air dried and powdered. Transferred the powdered material
primary health care needs. Herbs are supposed to be safe, but
many unsafe and fatal side effects have recently been reported into solvent extractor and extracted it with 95% ethanol for 72 h.
(Ikegami et al., 2003; Izzo, 2004). The extract was obtained as a brown gummy solid. The extract was
Traditionally, fresh or dried whole plants as well as their stored and used for phytochemical screening.
preparations are accepted for medicinal use in America, Germany,
Screening for alkaloids
France, and other European countries for the treatment of nervous
Three grams of extract was stirred with ethanol
anxiety (Blumenthal, 1997; Speroni and Minghetti, 1988).
containing 3% tartaric acid. The filtrate was shared into 3 beakers
Pharmacological studies show that passion flower has
and tested for alkaloids as follows-
antispasmodic, sedative, anxiolytic, and hypotensive activity
In to the first beaker, Hagar’s reagent was added into the second
(Akhondzadeh et al., 2001; Abascal and Yarnell, 2004; Dhawan et
beaker, Mayer’s reagent was added and into the last beaker
al., 2001a; 2001b; Dhawan et al., 2003; Weiss, 1988; Wolfman et
al., 1994). Marquins reagent was added. Precipitations in any of 3 tests
A passion flower (Passiflora foetida), an exotic and fast- indicate the presence of an alkaloid (Odebiyi and sofowora, 1978;
growing perennial vine, is found in the Western USA and in Asian Banso and Ngbede, 2006)
countries like India. Ethnobotanical reviews of P. foetida report
Screening for saponins
the decoction of leaves and fruits to treat asthma and biliousness
About 0.5 g of the plant extract was shaken with water in
(Ambasta, 1986); and leaf paste is applied on the head for
a test tube, frothing, which persists on warming was taken as
giddiness and headache (Chopra et al., 1956). In Brazil, the herb is
preliminary evidence for the presence of saponins.
used in the form of lotions or poultices to treat erysipelas and skin
Few drops of olive oil were added to 0.5 g of extract and
diseases with inflammation (Chopra et al., 1944). The major
vigorously shaken; formation of soluble emulsion in the extract
phytoconstituents of this plant are alkaloids, phenols, glycoside
flavonoids, and cyanogenic compounds, passifloricins, indicates the presence of saponins (Odebiyi and sofowora, 1978).
polyketides, and alpha-pyrones (Dhawan et al., 2004; Echeverri et
Screening for tannins
al., 2001). One chemical component of a passion flowers Passicol,
Into 10ml of freshly prepared 10% potassium hydroxide
a polyacetylenic compound has antimicrobial activity (Birner and
(KOH) in a beaker, 0.5 g of extract was added and shaken to
Nicolls, 1973; Nicolls, 1970; Nicolls et al., 1973), which is still
dissolve. A dirty precipitate observed indicated the presence of
not reported in P. foetida L. The majority of the active components
in this plant are C-glycosyl flavones based on apigenin and tannin (Odebiyi and sofowora, 1978; Willimson et al., 1996).
luteolin; Harman alkaloids are found in trace amounts along with
Screening for steroid
sucrose and trace amounts of volatile oil (Bradley, 1992; Leung
Total 100mg of Passiflora foetida extract was dissolved
and Froster, 1996; Newall et al., 1996).
in 2 ml of chloroform. Sulphuric acid was carefully added to form
MATERIALS AND METHODS a lower layer. A reddish-brown color at the interface is indicative
of the presence of steroidal ring (Sofowora, 1982)
Leaves and fruits of P. foetida L. was collected from the
Melghat forest area, Amravati, India. The plant was authenticated Screening for flavonoids
using standard flora and cross-checked with herbarium records at About 2 g of the powdered leaves was completely
the NBRI, Lucknow, India as Passiflora foetida L. with an detanned with acetone. The residue was extracted in warm
accession number- 98181. Silica gel-GF 254 was purchased from water after evaporating from the acetone in water bath. The
Himedia Laboratories, India and all other reagents and solvents
mixture was filtered while still hot. The filtrate was cooled and
used.
 Patil and Paikrao / Journal of Applied Pharmaceutical Science 2 (09); 2012: 137-142 139

Sodium hydroxide test TLC Characterization

Five ml of 20% sodium hydroxide was added to equal The presence of active metabolites in P. foetida L. extract
volume of the detanned water extract. A yellow solution indicates was evaluated by TLC plates. The preparative normal phase TLC
the presence of flavonoid. was performed on 15x10 cm glass plates coated with 1
mm layer of silica gel, dried in room temp. for 30 minutes and
Screening for anthraquinones activated in the oven at 110o C for next 30 minutes. The solvent
Bontrager’s test- About 0.5 g of the extract was taken in system chloroform: ethyl acetate: formic acid: methanol (5:3:2:0.4)
order to dry test tube and 5 ml chloroform was added and shaken 4 were used (Harborne, 1973). 10 µl of extract was spotted on the
for 5 min. The extract was filtered, and the filtrate shaken with an silica plates. The experimental plates were developed in a
equal volume of 100% ammonia solution. A pink violet or red chromatographic chamber, saturated by 100 ml of the solvent
colour in the ammoniacal layer (lower layer) indicates the presence system. Plate development required about 15 to 20 minutes; it was
of free anthraquinones. then visualized at 365 nm under UV transilluminator (Cleaver). Rf
values of bands were recorded.
Screening for cardiac glycoside
(Keller Killiani test)- Total 100 mg of extract was TLC Bioautography
dissolved in 1 ml of glacial acetic acid containing one drop of In order to find out the bioactive fraction from TLC plate
ferric chloride solution. This was then underlayerd with 1 ml of the TLC Bio-autography by agar overlay method was performed
concentrated sulphuric acid. A brown ring obtained at the interface (Hostettmann et al, 1990). In N.A medium, a suspension of test
indicates the presence of de-oxysugar characteristics of pathogen Oxacilin resistant S. aureus (OARS) i.e. 10mg/10mm
cardenolides. zone was added aseptically in the molten agar, further spread on
the TLC plate. The plate kept in moist chambers and incubated at
PREPARATION OF TANNIN FREE BIOACTIVE 370 C for overnight. Next the solution of tetrazolium salt MTT (3-
EXTRACT (4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide,
2mg/ml) was sprayed on the plate.
The schematic representation of procedure was given
below as proposed by Wall et al., (1996). Spectroscopic Analysis
The bioactive fractions were scrapped from the TLC
50 gm dried leaf powder suspended in methanol and rotated on an plate and Centrifuged in methanol, and the Purified fractions were
orbital shaker for 4 days. UV analyzed in the range of 200-400nm while the baseline was
corrected keeping methanol as blank.
Filtered and concentrated under vacuum.
Determination Of Minimum Inhibitory Concentration
Methanol extracts
1. Suspended in 90 % Methanol The antibacterial potency of the bioactive fraction in
2. Partition with Hexane(for defatting) variable concentrations was determined in terms of minimum
inhibitory concentration (MIC) using the filter paper disc diffusion
90 % Methanol partition hexane extracts method (Pelczar et al., 1998; Patil, 2010). The lawn of the log
1. Concentrate phase of Staphylococcus aureus, Salmonella typhi, Klebsiella
2. Suspend in Distilled water pneumoniae, and Pseudomonas aeruginosa were prepared using a
3. Partition with Chloroform (CHCl3 ) glass spreader. The variable concentrations of bioactive fraction
were prepared in 1 % DMSO (dimethyl sulphoxide) so that a
single disc contained 0.5, 1, 2, 3, 4 and 5 µg/ml. The filter paper
Crude Chloroform Partition Aqueous Extract discs (3 mm) were presoaked in their respective dilutions for 30
min and then kept in the test bacterial lawn. The plates were left on
1. Wash with 1% NaCl in H2O to remove
tannins a bench for 1 hr before incubation at 37°C for 24 h to allow
2. Evaporate to dryness. prediffusion of the extract (Esimone et al., 1998), control was
maintained by soaking paper disc in 1% DMSO. The zone of
inhibition was measured in mm using the zone measuring scale
and compared with the control set (i.e. disc with solvent). The MIC
Chloroform Partition (free of tannins) 1 % NaCl solution (Waste) value was taken as the lowest concentration of the extract showing
complete lack of growth after the incubation period (EUCAST,
2000) or maximum inhibition of test microorganisms each
Thin Layer Chromatography experiment was performed in triplicates.
140 Patil and Paikrao / Journal of Applied Pharmaceutical Science 2 (09); 2012: 137-142

Statistical Analysis control, 1 ml of bromine water was added drop wise, and the color
Data acquired was statistically analyzed using Microsoft change was observed; the disappearance of bromine water color
Excel 2007. indicated unsaturation confirming the presence of polyacetylenes
in fractions (Jamode et al., 1998).
RESULTS AND DISCUSSION
TLC Analysis
Phytochemical Analysis The TLC analysis of extract resulted in showing two
The preliminary phytochemical analysis of P. foetida prominent green coloured band named as PA1 and PA2, i.e.
resulted in presence of all the principal classes of compounds Polyacetylene 1 and Polyacetylene 2 with Rf value 0.32 and 0.70
discriminated in Table.1. respectively (Fig. 2).
Table. 1: Preliminary phytochemical screening of ethanol extract of Passiflora
foetida L.
Phytochemical constituents Results
Saponins +
Tannins +
Cardiac Glycosides +
Alkaloid +
Anthraquinones +
Steroid +
Flavonoid +
Key + = present

Extraction Of Material
The initial weight of leaf powder before extraction was
50 gm, after extraction the pure crude extracts was of 1. 11gm. The
extraction scheme resulted in efficient method as it removed the
tannins, Saponins and other interfering compounds.
Fig. 2: TLC characterizations of bioactive fractions.

Spectroscopic Analysis
TLC Bioautography
The UV analysis of bioactive fractions showed multiple
The Bioautography of TLC plate revealed the presence of
absorption maxima in the range of 230-370nm, confirming their
bioactive fractions as PA1 and PA2 both appeared as a white band
polyacetylenic nature (Birner and Nicolls, 1973; Sorensen, 1968;
against the violet background proving potent antimicrobial against
Bohlman et al., 1961). The finger like appearance of spectrum
multiple drug resistant S. aureus (Fig. 3).
(PA1) confirmed the presence of polyacetylenes (Fig.1).

Fig. 3: TLC Bioautography by agar overlay assay of bioactive fractions PA1

Fig. 1: UV spectrum of bioactive fraction PA1. and PA2.

Confirmative Test for Unsaturation Disc Diffusion Assay

To confirm the polyacetylenic nature of fraction, a Strains of Staphylococcus aureus, Salmonella typhi,
bromine water test was performed, in which a bioactive spot from Klebsiella pneumoniae, and Pseudomonas aeruginosa were
TLC plate was scrapped off and eluted with 2 ml distilled water; selected to study the inhibitory effect of Fractions extracted from
plain distilled water with an equal amount of scrapped silica was P. foetida L. The antibacterial activity was analyzed by disc
kept as the control.. To 1 ml of the test sample along with the diffusion techniques of both fractions eluted from the PA1 and
 Patil and Paikrao / Journal of Applied Pharmaceutical Science 2 (09); 2012: 137-142 141

Table. 2: Inhibitory effect of pure PA1 and PA2 fractions from P. foetida L. extracts.
S.No Conc. of Passicol Zone of Inhibition (mm)
µg /ml S. aureus S. typhi K. pneumoniae P. aeruginosa
1 PA1 PA2 PA1 PA2 PA1 PA2 PA1 PA2
Control 3 3 3 3 3 3 --- ---
2 0.5 12±0.16 12±0.16 13±0.16 12±0.16 12±0.16 11±0.24 --- ---
3 1 12±0.16 12±0.16 11±0.24 12±0.16 12±0.16 12±0.16 --- ---
4 2 12±0.16 12±0.16 12±0.16 12±0.16 12±0.16 12±0.16 --- ---
5 3 14±0.08 13±0.16 12±0.16 13±0.16 12±0.16 11±0.24 --- ---
6 4 14±0.08 21±0.08 14±0.08 13±0.16 12±0.16 11±0.24 6±0.24 6±0.24
7 5 15±0.08 11±0.24 14±0.08 13±0.16 11±0.24 13±0.16 6±0.24 6±0.24
aValues expressed as fractions concentration µg /ml.
b Values expressed as zone of inhibition in mm ± S.D

PA2 concentration, ranging from 0.5, 1, 2, 3, 4 and 5 µg/ml per assistance under FAST TRACK scheme to corresponding author.
disc. The PA2 was found to be significantly effective at S. aureus Also we are thankful to Dr. P.V. Thakare, Reader, Department of
since the higher zone of inhibition (21 mm) was observed at 4 Biotechnology, Sant Gadge Baba, Amravati University, Amravati
µg/ml concentration compared to zone (15 mm) at 5 µg/ml inPA1. for providing Oxacilin resistant bacterial cultures.
PA1 inhibited slightly more (14 mm) at 4 µg/ml, compared to PA2
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Ngbede J., Yakubu R. A., Nyam D.A. Phytochemical screening Patil A.S. and Paikrao H.M. Bioassay Guided Phytometabolites
for active compounds in Canarium schweinfurthii (Atile) leaves from Jos Extraction for Screening of Potent Antimicrobials in Passiflora
North, plateau state, Nigeria, Research Journal of Biological Sciences.
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