Indo American Journal of Pharmaceutical Research, 2013
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ISSN NO: 2231-6876
INDO AMERICAN
JOURNAL OF
PHARMACEUTICAL
RESEARCH
Phytochemical evaluation and anthelmintic activity of ethanolic leaves extract of
Passiflora foetida Linn.
N.M.A. Rasheed1, Tarannum Fatima2 & M.A. Waheed1
1
Central Research Institute of Unani Medicine, Opp. E.S.I. Hospital, Hyderabad 500038
Sultan-Ul-Uloom College of Pharmacy, Road No. 1, Banjara Hills, Hyderabad 500038
2
ARTICLE INFO
Article history
Received 25/06/2013
Available online
31/07/2013
Keywords
Passiflora foetida,
Phytochemical
screening, HPTLC,
Anthelmintic activity.
ABSTRACT
Passiflora foetida Linn. (Fam-Passifloraceae) is traditionally used by the tribes and
native medical practitioners for the treatment of various ailments including liver
disorders, tumors, asthma, itches and dressing for wounds. Folklore claims
reported its use in diarrhoea, throat and ear infections, liver disorders, tumours,
skin diseases. The present study is an attempt to explore the phytochemical
constituents and in-vitro anthelmintic activity of ethanolic extract of leaves.
Preliminary phytochemical screening carried on ethanolic extract of leaves showed
the presence of alkaloids, glycosides (cyanogenetic glycosides), tannins, phenolic
compounds, flavonoids, etc. High Performance Thin Layer Chromatography
(HPTLC) analysis was also carried for determination of number of components
present. Further, Anthelmintic activity of ethanolic extract was evaluated on Indian
earthworms; Pheretima postuma showed significant activity upon comparison to
the standard drug albendazole at 10 mg/ml concentration.
Corresponding author
N.M.A. Rasheed
[email protected]
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Please cite this article in press as N.M.A. Rasheed et al. Phytochemical evaluation and anthelmintic activity of ethanolic leaves
extract of Passiflora foetida Linn. Indo American Journal of Pharm Research.2013:3(7).
Vol 3, Issue 7, 2013.
N.M.A. Rasheed et al.
ISSN NO: 2231-6876
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Materials and Methods
The plant material of Passiflora foetida L. leaves were collected and authenticated by Dr. Mohammad Kashif
Hussain, Research Officer (Botany) at Central Research Institute of Unani Medicine, Hyderabad. The leaves
were shade dried and coarsely powdered. The powdered plant material was subjected to continuous Soxhlet
extraction by using ethanol as a solvent at 70°C for 72 hrs. The ethanolic extract of the leaves of Passiflora
foetida L. was evaporated at 40°C and the sticky blackish green substance was stored in dessicators for further
phytochemical investigations and anthelmintic activity.
Biological study
Healthy adult Indian earthworms, Pheretima postuma, due to its anatomical and physiological resemblance with
the intestinal roundworm parasites of human beings[10-12] were used in the present study. All earthworms were
of approximately equal size. They were collected from local place, washed and kept in water.
Drugs
Ethanolic extract of Passiflora foetida Linn. was tested in various doses in each group. Normal saline water was
used as control. Albendazole was used as the standard drug for the study with ethanolic extract.
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Introduction
Passiflora foetida Linn also called as stinking passionflower or wild water lemon belongs to Passifloraceae
family. It is a fetid, herbaceous, hairy, perennial vine, scrambling or climbing to 5m or more by axillary,
unbranched, coiling tendrils with soft to hard, yellow to brown hairs, distributed and found wild in several parts
of India. It is native to the Southwestern United States (Southern Texas and Arizona), Mexico, the Caribbean,
Central America, and much of South America. It has been introduced to tropical regions around the world, such
as Southeast Asia and Hawaii[1,2]. The stems are thin and wiry, covered with minute sticky yellow hairs. Older
stems become woody. The leaves are alternate, palmately three-five lobed and viscid-hairy. The flowers are
white to pale cream coloured, about 5-6 cm in diameter and with an epicalyx of pinnatifid bracteoles. The fruit
is globose up to 2.5 cm in diameter, hairy, yellowish when ripe and has numerous black seeds embedded in the
pulp[3]. Leaves have a mild aroma and ripened fruit is edible. The plant is reported to contain alkaloids,
phenols, glycosides, flavonoids, cyanogenic compounds, passifloricins, polypeptides and alpha-pyrones.[4]
Passiflora foetida Linn. reported to possess sedative, hypnotic, antispasmodic and anodyne properties[5]. Tea of
its leaves is used as an expectorant and for nervous disorders. Traditionally it is used for diarrhoea, intestinal
tract, throat, ear infections, fever and skin diseases. It is also used in eczema, inflammation and pain[1,2].
Literature study shows that the fruits of Passiflora foetida Linn. possess hepatoprotective activity may be
attributed to flavonoids present in the fruits of Passiflora foetida Linn[6]. The antibacterial properties of leaf
and fruit (ethanol and acetone) extracts were screened against four human pathogenic bacteria i.e. Pseudomonas
putida, Vibrio cholerae, Shigella flexneri and Streptococcus pyogenes by well-in agar method. The results
showed the leaf extract having remarkable activity against all bacterial pathogens compared to fruits[7]. Besides
these, the amount of harmaline, a betacarboline alkaloid in Passiflora foetida was estimated by comparing the
peak area of standard and that present in the leaf extract. The harmaline content present in the extract was
estimated to be 0.75% w/w[8]. Antinociceptive, antidiarrhoeal and cytotoxic activities of ethanolic extract of
whole plant were also studied. The extract produced significant (P<0.001) writhing inhibition in acetic acidinduced writhing in mice. The extract also showed antidiarrhoeal activity on castor oil induced diarrhoea in
mice and cytotoxic activity against brine shrimp Artemia salina.[9] There are no reports on systematic and
scientific study of anthelmintic activity of leaf extract for passiflora foetida Linn. In the present study, we report
the anthelmintic activity of ethanolic extract of the leaves of Passiflora foetida Linn.
Vol 3, Issue 7, 2013.
N.M.A. Rasheed et al.
ISSN NO: 2231-6876
Preliminary Phytochemical Analysis
The ethanolic extract of dried leaves of Passiflora foetida Linn. was subjected to preliminary phytochemical
screening for determination of nature of functional group[13].
Tests for Reducing Sugar
Benedict’s Test: 0.5 ml of the extract was placed in a test tube and then 5 ml Benedict’s solution was added to
it, boiled for 5 min and allowed to cool spontaneously. Red, yellow or green solution is considered as an
indication for the presence of reducing sugars.
Fehling’s Test: 2 ml of the extract was added in 1 ml of a mixture of equal volumes of Fehling’s solutions A
and B, and was boiled for few min. Brick red precipitate is considered as an indication for the presence of
reducing sugars.
Tests for Tannins
Ferric Chloride Test: 5 ml of the extract was placed in a test tube and then 1 ml of 5% Ferric chloride solution
was added to it. Deep blue colour is considered as an indication for the presence of tannins and phenolic
compounds.
Potassium dichromate test: 5 ml of the extract was placed in a test tube and then 1 ml of 10% potassium
dichromate solution was added. Red precipitate is considered as an indication for the presence of tannins and
phenolic compounds.
Test for Flavonoids
5 ml of 95% ethanol, few drops of concentrated hydrochloric acid and 0.5 gm magnesium turnings were added
to 5 ml of the extract. Pink colour is considered as an indication for the presence of flavonoids.
Test for Saponins
1 ml of the extract was placed in a graduated cylinder and was diluted to 20 ml with distilled water and shaken
gently for 15 min. Persistent foam is considered as an indication for the presence of saponin glycosides.
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Tests for Alkaloids
Mayer’s test: 2 ml of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube and 1ml of
Mayer’s reagent was added to it. Formation of a yellow coloured precipitate indicates the presence of alkaloids.
Dragendroff’s test: 2 ml of the extract and 0.2 ml of dilute hydrochloric acid were placed in a test tube and then
1 ml Dragendroff’s reagent was added. Formation of orange brown precipitate indicates the presence of
alkaloids.
Wagner’s test: 2 ml of the extract and 0.2 ml of dilute hydrochloric acid were placed in a test tube. Then 1 ml of
iodine solution (Wagner’s reagent) was added. Formation of brown/reddish precipitate indicates the presence of
alkaloids.
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Tests for Steroids
Libermann-Burchard test: 1 ml of the extract was placed in a test tube and then 2 ml Libermann-Burchard
reagent was added to it. Green colour solution is considered as an indication for the presence of steroids.
Salkowski reaction: 2 ml of the extract was placed in a test tube and then 2 ml of chloroform and 2 ml of
concentrated sulphuric acid were added to it and shaken well. Chloroform layer appearing red and acid layer
showing greenish yellow fluorescence is considered as an indication for the presence of steroids.
Vol 3, Issue 7, 2013.
N.M.A. Rasheed et al.
ISSN NO: 2231-6876
Hager’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were placed in a test tube. Then
1 ml of picric acid solution (Hager’s reagent) was added. formation of yellow coloured precipitate indicates the
presence of alkaloids.
Tests for Glycosides
A small amount of extract was taken in 1 ml water. Then few drops of aqueous sodium
hydroxide were added. Yellow precipitate is considered as an indication for the presence of glycosides. in a
boiling water bath. Brick red precipitate is considered as an indication for the presence of glycosides.
Tests for cyanogenetic glycosides
Guignard reaction or sodium picrate test: 2ml of the extract was taken in a conical flask In another test, a small
amount of extract was taken in 1 ml water and boiled with 5 ml Fehling’s solution in a boiling water bath.
Brick-red precipitate is considered as an indication for the presence of glycosides.
In another test, a small amount of extract was boiled with few drops of dilute sulfuric acid, neutralized with
sodium hydroxide solution and boiled with 5 ml Fehling’s solution and corked. Filter paper strip soaked in 10%
picric acid and in 10% sodium carbonate was placed in the slit in the cork. The filter paper turning into brick red
or maroon is considered as an indication for the presence of cyanogenetic glycosides.
HPTLC Analysis:
Preparation of ethanolic Extract
Five grams powder of finely powdered leaves were dissolved in 100 ml of ethanol using a soxhlet
extraction and the extract was filtered through Whattmann No. 41 filter paper and concentrated over the water
bath to 20 ml. The solution obtained was used as sample for the determination of components.
Development and determination of the solvent system
Sample applied
:
Sample drug solution of about 10µl.
Solvent system
:
Toluene: Ethyl acetate: Methanol (7: 2: 1)
Scanning wavelength :
366nm
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Development of HPTLC technique
After developing, TLC plates were dried completely and detected with the suitable detection system like UV
Cabinet system in order to examine number of spots at 366nm as shown in figures 2. Further it was scanned
with the Densitometer CD60 of DESAGA Sarstedt Gruppe system under the UV range of 366nm appearing a
maximum number of components. A corresponding densitograms was obtained as shown in the figures 2a in
which peaks are appeared for the corresponding spots being detected in the densitometer while scanning and the
peaks area under the curve corresponds to the concentration of the component in the sample for the
concentration we applied on the TLC plate.
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The sample applied as a band of 10mm with the help of Automatic TLC applicator system of the
DESAGA Sarstedt Gruppe on Precoated Aluminium Sheets of Silica Gel 60 F254 (Merck). After trying with
various solvent systems with variable volume ratios, the suitable solvent system as stated above is selected in its
proportional ratio and developed in the Twin through chamber of TLC to the maximum height of the plate so
that it can be able to separate the components on the polar phase of silica gel and that of mobile phase of solvent
system[14-16].
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N.M.A. Rasheed et al.
ISSN NO: 2231-6876
Pharmacological Study
Anthelmintic Activity
Nargund[17] method was followed for the screening of anthelmintic activity on adult Indian earthworm,
Pheretima postuma. Earthworms were divided into eight groups consisting of 4 in each. Group 1 served as
normal control which received saline water only. Group 2 received the standard drug, Albendazole at a dose
level of 10 mg/ml. Groups 3 to 8 received doses of ethanolic extract of 10mg/ml, 20mg/ml, 30mg/ml, 40mg/ml,
50mg/ml and 60mg/ml respectively. Observations were made for time taken to cause paralysis and death of
individual worms in two hours. Paralysis was said to occur when the worms do not revive even in normal saline
water. Death was concluded when the worms lost their motility followed with fading away of their body
colours.
Statistical analysis
The data on biological studies were reported as mean ± Standard deviation (n = 4). For determining the
statistical significance, standard error mean and analysis of variance (ANOVA) at 5% level significance was
employed. P < 0.05 was considered significant[18].
Results and Disscussion
Chemical Group Test
Results of different chemical group tests on the ethanolic extract of dried leaves of the plant Passiflora foetida
Linn. showed the presence of Alkaloids, Flavonoids, Tannins and Glycosides.
Table 1: Results of different chemical group tests of the ethanolic extract of dried leaves of Passiflora foetida
Linn.
Ethanloic extract of dried leaves of
Passiflora foetida Linn.
Presence
Reducing sugar
Steroids
Alkaloids
Tannins
Flavonoids
Glycosides
Saponins
+
+
+
+
-
Key: + = Presence, - = Absence
HPTLC Analysis: Developed chromatograms at UV 366nm wavelength for ethanolic extracts of drug is given
in figure 1 and peak list and Rf values are given in table 2 respectively. It is evident from TLC plate that the
number of spots at UV 366nm wavelength is five.
Y-Pos
14.8
24.8
36.2
48.8
64.0
Area
1924.37
45.31
56.41
794.38
77.27
Area (%)
66.4
1.6
1.9
27.4
2.7
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Height
527.41
13.25
13.67
107.05
14.93
Rf values
0.02
0.16
0.32
0.49
0.70
Page
Peak no
1
2
3
4
5
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Table 2: Peak list & densitogram of Passiflora foetida Ethanolic extract at UV 366nm with Rf values of
the spots
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N.M.A. Rasheed et al.
ISSN NO: 2231-6876
Page
Figure 2: HPTLC densitogram of Passiflora foetida Ethanolic extract scanned at UV 366nm
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Figure1: TLC chromatogram of Passiflora foetida Ethanolic extract at UV 366nm.
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N.M.A. Rasheed et al.
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Evaluation of Anthelmintic Activity
The ethanolic extract of the leaves of Passiflora foetida L. produced a significant anthelmintic activity in dose
dependent manner as shown in Table 2. The anthelmintic activity of ethanolic extract was compared with that of
standard drug, albendazole. The normal saline water was used as a control.
The ethanolic extract of the leaves of Passiflora foetida L. showed a dose dependent activity like
shortest time of paralysis (p) and death (d) with 60mg/ml concentration. The ethanolic extract of Passiflora
foetida L. caused paralysis and death in 53.7 min and 62.3min at 10 mg/ml concentration while the standard
drug albendazole showed the same at 65.6 min and 73.4 min. respectively.
Phytochemical analysis showed the presence of tannins as one of the chemical constituent, these are said to
possess anthelmintic activity. Chemically tannins are polyphenolic compounds, synthetic anthelmintics
possessing phenolic group eg. Oxyclozanide bithionol etc are shown to interfere with energy generation by
uncoupling oxidative phosphorylation. There is a possibility that the tannins in Passiflora foetida L. produced
similar effects. Another mechanism of action of tannins is that they can bind to free protein in GIT of host
animal or glycoprotein on the cuticle of the parasite and cause death[19]. Hence, the folklore claims have been
confirmed and supports by the study with leaves extract of Passiflora foetida L. as an anthelmintic activity
against the earthworms used in the study. Further studies are required to isolate and reveal the active
compounds present in the crude extract of Passiflora foetida L. and to establish the mechanism of action
responsible for anthelmintic activity.
Table 3: Anthelmintic activity of ethanolic leaf extract of Passiflora foetida L.
1
Control
(Normal saline water)
Standard
(Albendazole)
Ethanolic extract
Ethanolic extract
Ethanolic extract
Ethanolic extract
Ethanolic extract
Ethanolic extract
2
3
4
5
6
7
8
10
65.6 ±0.31
73.4 ±0.82
10
20
30
40
50
60
53.7 ± 0.74
47.5 ± 0.57
41.7 ± 0.51
34.3 ± 0.96
17 ± 0.82
10.8 ± 0.94
62.3 ± 0.5
50.5 ± 0.62
43.8 ±0.80
44 ± 0.83
31.3 ± 0.96
15.5 ± 1.2
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Treatments
Time taken for Time taken for
paralysis (min)
death (min)
(X ± S.D.)
(X ± S.D.)
-
Page
Groups
Dose
(mg/ml)
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Vol 3, Issue 7, 2013.
N.M.A. Rasheed et al.
ISSN NO: 2231-6876
Standard drug, Albendazole
Ethanolic extract of leaves of
Passiflora foetida L.
Figure 3: In-vitro anthelmintic activity of standard and extract.
Series 1 - Paralysis time
Series 2 - Death time
80
70
Time [min]
60
50
Series1
40
Series2
30
20
10
0
1
2
3
4
5
6
7
8
Groups
Page
Conclusion
The phytochemical screening on quantitative analysis shows that the leaves of the Passiflora foetida Linn. are
rich in popular phytochemical constituents such as alkaloids, glycosides ( cyanogenetic glycosides), flavonoids
and tannins. The present study has confirmed that the ethanolic extract of leaves of Passiflora foetida Linn.
possess better anthelmintic activity when compared with the standard drug, albendazole. Further studies are
required to isolate and reveal the active compound present in the crude extract of Passiflora foetida L. and to
establish the mechanism of action responsible for anthelmintic activity. An account of the finger print HPTLC
of ethanolic extract of leaves will definitely help in future for quality check and ensure the genuineness of drug.
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Figure 4: Evaluation of In vitro Anthelmintic Activity of Ethanolic Extract of Leaves of
Passiflora foetida L.
Acknowledgement
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We the authors are thankful to Dr. J. Venkateswara Rao, Principal Sultan- Ul -Uloom College of Pharmacy for
providing necessary facilities and encouragement during the research work.
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