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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Tambe et al. World Journal of Pharmacy and Pharmaceutical Sciences


SJIF Impact Factor 2.786

Volume 3, Issue 10, 880-889. Research Article ISSN 2278 4357

PHYTOCHEMICAL SCREENING AND ANTHELMINTIC ACTIVITY


OF WOOD AND LEAVES OF HIBISCUS TILIACEUS LINN.

Vijay Tambe1*, Rajendra Bhambar2

1
Department of Pharmacognosy, Pravara Rural College of Pharmacy, A/P-Loni, Tal-Rahata,
Dist-Ahmednagar-413 736 Maharashtra, India.
2
Department of Pharmacognosy, MGVS College of Pharmacy, Panchvati, Nasik,
Maharashtra, India.

ABSTRACT
Article Received on
25 July 2014, Various helminthes have been common cause of concern and leads
Revised on 19 August 2014,
Accepted on 10 September many problems to human beings and other animals. From ancient time
2014
different plants are known to possess significant anthelmintic activity
against these invasive types of worms and can be effectively used in

*Correspondence for treatment of worm infections. The prime objective of the study was to
Author investigate different types of phytoconstituents present and
Vijay Tambe anthelmintic activity of Hibiscus tiliaceus wood and leaves. Extraction
Department of
was carried out by continuous hot percolation method in soxhlet
Pharmacognosy, Pravara
apparatus using petroleum ether, ethyl acetate, methanol as solvents for
Rural College of Pharmacy,
A/P-Loni, Tal -Rahata, Dist- wood powder and petroleum ether, chloroform, ethyl acetate, ethanol
Ahmednagar-413 736 as solvents for leaves powder respectively. The extractive values were
Maharashtra, India. reported. All the extracts were subjected to phytochemical screening
using standards methods for detection of type of phytoconstituents
present in them. In-vitro anthelmintic activity was carried out at three different concentrations
using Pheretima posthuma as test organism, the parameters like the time of paralysis and the
time of death were determined. All the extracts showed significant anthelmintic activity in the
dose dependent manner compared to standard drug albendazole. Amongst the extracts
petroleum ether extract of wood and ethyl acetate extract of leaves showed good activity.
This might due to the presence of secondary metabolites like tannins, saponins and alkaloids
in the plant parts which may be responsible for the activity.
Keywords: Phytochemical screening, Hibiscus tiliaceus, anthelmintic activity, Pheretima
posthuma.

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INTRODUCTION
The half of world suffering from bacterial and helminthes infection, due to poor sanitation,
poor family hygiene, malnutrition, and crowded living conditions.[1] Helminthes infections
are among the most widespread infections in humans, distressing a huge population of the
world. Although the majority of infections due to helminthes are generally restricted to
tropical regions and cause enormous hazard to health and contribute to the prevalence of
under nourishment, anemia, eosinophilia and pneumonia.[2] Due to discovery and
development of anthelmintic physician now have effective, and in some cases, broad
spectrum of agents that will cure or control most infections caused by flukes or intestinal
helminthes.[3] Development of resistance to most of the commercially available anthelmintic
is became a severe problem worldwide.[4] The gastro-intestinal helminthes becomes resistant
to currently available anthelmintic drugs therefore there is a foremost problem in treatment of
helminthes diseases. Hence there is an increasing demand towards natural anthelmintics.[5]
There is lot of the organic & inorganic and antibiotics are antihelmintics drug which has
some side effects such as constipation, vomiting, gastric irritation hypersensitivity, immune
suppression and allergic reaction. Therefore, there is a need to develop alternative
antimicrobial drugs for the treatment of infectious diseases from medicinal plants. Plants
have provided mankind with herbal remedies for several diseases for many centuries. In India
herbal medicines have been the bases of treatment and cure for various diseases in traditional
methods such as Ayurveda, Unani and Sidha.

Natural products extracted from plants which belong to the Malvaceae family are used in the
treatment of many diseases worldwide. One important genus in this family is Hibiscus spp.,
[6]
with more than 220 species distributed in tropical and subtropical regions. Hibiscus
tiliaceus L. is a typical plant of tropical climates found in the regions of mangroves in
[7]
significant quantities. An aqueous extract of wood and fresh flowers is a registered
[8, 9]
treatment for skin diseases. Recently it was shown that methanolic flower extract exerts
an antioxidant effect on the yeast Saccharomyces cerevisiae, protecting against hydrogen
peroxide (H2O2) and tert-butylhydroperoxide (t-BHP) cytotoxicities. In addition, the extract
was not mutagenic in Salmonella typhimurium or S. cerevisiae and showed a significant
[6]
antimutagenic action against oxidative mutagens in S. cerevisiae. It is also reported
traditionally, where the leaves are used to treat fevers and soothe coughs, the bark to treat
dysentery, and the flowers aid in treating ear infections and abscesses.[10] Previous
pharmacological investigations of the genus Hibiscus plants indicated the presence of species

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Tambe et al. World Journal of Pharmacy and Pharmaceutical Sciences

with useful biological activities. The studies conducted to date have demonstrated that plants
of the Hibiscus genus have the potential to provide biologically active compounds that are
able to deter the proliferation of malignant cells. Thus, the Hibiscus genus deserves additional
evaluation as a provider of chemopreventive agents. Indeed, there is a current need for
availability of new plant-derived bioactive molecules; for the development of new drugs and
may provide a cost-effective mean of treating cancers and other diseases in the developing
world. [11, 12]

Present work was undertaken to screen type of phytochemical constituents present in and
anthelmintic potential of various extracts of Hibiscus tiliaceus wood and leaves in adult earth
worms (Pheretima posthuma).

MATERIALS AND METHODS


Plant material: Hibiscus tiliaceus Linn. wood and leaves part was collected from Costal side
of Ratnagiri district of Maharashtra and authenticated By Dr. J. Jayanthi, Scientist C for
Director, Botanical Survey of India, Pune vide letter No. BSI/WRC/TECH/2010 Voucher No.
HIBCA1VIT. The herbarium is kept at BSI, Pune for further reference.

Experimental worms [13-16]


Indian adult earthworms were used to study anthelmintic activity since its anatomical and
physiological resemblance with the intestinal roundworm parasites of human being. Adult
earthworm Pheretima posthuma were collected from moist soil, obtained from agricultural
fields nearby. The earthworms were washed with normal saline to remove all adhering soil,
fecal matter. Adult earthworms were used for all experimental protocol.

Preparation of extracts
For the present study, the extracts were obtained by continuous hot extraction method using
soxhlet apparatus. 300 gm of wood powder was passed through sieve no. 60 and packed in
soxhlet apparatus and extracted using petroleum ether, ethyl acetate, methanol as solvents.
Similarly powder of leaves was subjected to soxhlet extractor using solvents petroleum ether,
chloroform, ethyl acetate and ethanol. The filtrate was concentrated in rotary evaporator and
the extracts were calculated for their extractive value. Preliminary phytochemical analysis
was carried out to find out nature of chemical constituents present in extracts.

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Phytochemical Screening
Qualitative chemical test were carried out for all the extracts. It revealed positive result of
carbohydrates, proteins, steroids, alkaloids, saponins, tannins, glycosides, Amino acids.
Phytochemical screening of the extract was carried out according to the standard method. [17]

Standard solution
Albendazole (10 mg/ml 20 mg/ml and 40mg/ml) was administered as standard solution.

Test solution
The different concentration (10, 20 and 40 mg/ml) of petroleum ether (PEW), ethyl acetate
(EAW), methanol extracts (MW) of wood of Hibiscus tiliaceus were prepared. Extracts of
leaves were also prepared in same concentrations as petroleum ether (PEL), chloroform (CL),
ethyl acetate (EAL) and ethanol (EL) extract. All the extracts and the standard drug
albendazole solution were freshly prepared before starting the experiments.

Experimental design [18]


The experiment was carried out separately for wood and leaves extracts. The anthelmintic
activity was carried out in 13 groups for wood extracts; each group consists of 6 worms and
worms were released into 10 ml of desired formulations. Group 1 were the control worms
placed in normal saline; Group 2-4 received petroleum ether (PEW) extracts of wood of
Hibiscus tiliaceus at 10, 20 and 40 mg/ml concentrations; Group 5-7 were treated with ethyl
acetate (EAW) extract of wood at 10, 20 and 40 mg/ml concentrations. Group 8-10 were
treated with methanol extracts (MW) of wood of Hibiscus tiliaceus at 10, 20 and 40 mg/ml
concentrations. Group 11-13 serves as standard and was treated with Albendazole (10, 20 and
40 mg/ml). All the extract solutions were prepared in normal saline containing 0.5% DMF.
Final volume was adjusted to 10 ml in each petridish. The anthelmintic activity was carried
out in 16 groups for leaves extracts using similar groups and concentrations like for wood
extracts. The observations were made for the time taken for paralysis and death of worm.
Time for paralysis was noted when no movement of any sort could be observed except when
the worms were shaken vigorously. Death was concluded when the worms lost their motility
followed with their body colors fading away.

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RESULTS AND DISCUSSIONS


Table.No.1. Extractive value determination
Plant
Plant part Extractive value (% w/w)
Name
Ethyl
Petroleum Chloroform Methanol Ethanol
acetate
ether extract extract extract extract
extract
Hibiscus Wood 9.3 -- 3.5 6.8 --
tiliaceus
Leaves 11.5 7.5 5.0 -- 8.0
Linn.

Table.No.2. Preliminary Phytochemical screening of Hibiscus tiliaceus wood extracts


Test for
PEW EAW MW
constituent
Alkaloids ++ ++ --
Glycosides -- -- ++
Tannins ++ ++ ++
Flavonoids -- ++ --
Carbohydrates ++ ++ ++
Steroids ++ ++ ++
Amino acids ++ ++ ++
Saponins ++ ++ --
Proteins ++ ++ ++
++ indicates presence --indicates absence

Table.No.3. Preliminary Phytochemical screening of Hibiscus tiliaceus leaves extracts


Test for
PEL CL EAL EL
constituent
Alkaloids ++ ++ -- ++
Glycosides -- -- ++ --
Tannins ++ ++ ++ ++
Flavonoids ++ ++ -- ++
Carbohydrates ++ ++ ++ ++
Steroids -- ++ -- ++
Amino acids ++ ++ ++ ++
Saponins ++ ++ -- ++
Proteins ++ ++ ++ ++
++ indicates presence --indicates absence

From these two table it is clear that the all the extracts of Hibiscus tiliaceus contains rich
presence of tannins and flavonoids which are responsible for the anthelmintic activity.

In vitro Anthelmintic activity: In anthelmintic study, the all extracts showed paralysis and
death of worms. The effect was dose dependant and shortest time taken for paralysis and

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Tambe et al. World Journal of Pharmacy and Pharmaceutical Sciences

death was observed in case of petroleum ether extract of wood and ethyl acetate extract of
leaves 40 mg/ml concentration with potent activity against Indian adult earthworms
(Pheretima posthuma). Both the extracts showed significant results compared to standard
drug as displayed in Table No.4 and 5.

Table 4. Anthelmintic Activity of Hibiscus tiliaceus wood extracts


Concentration Time taken for Time taken for
Treatment
mg/ml paralysis (min) death (min.)
Control
-- -- --
(Normal Saline)
10 45.070.43 77.520.71
PEW 20 31.40 1.01 58.170.65
40 29.000.53 46.500.16
10 58.401.12 59.371.09
EAW 20 46.541.27 43.441.65
40 33.520.77 49.171.10
10 50.000.73 81.370.67
MW 20 42.33 1.01 61.170.09
40 31.000.74 52.500.79
10 48.230.60 73.501.52
Standard
20 33.000.54 56.421.87
(Albendazole)
40 29.420.54 42.000.35
All Values represents Mean SEM; n=6 in each group

Table 5. Anthelmintic Activity of Hibiscus tiliaceus leaves extracts


Concentration Time taken for Time taken for
Treatment
mg/ml paralysis (min) death (min.)
Control
-- -- --
(Normal Saline)
10 49.210.42 79.470.67
PEL 20 41.21 1.01 63.240.09
40 36.001.02 55.200.42
10 55.431.24 76.371.58
CL 20 49.241.27 61.041.05
40 33.520.77 49.171.10
10 46.12 0.43 74.220.46
EAL 20 31.06 1.87 53.410.74
40 28.06 0.53 44.570.43
10 51.240.73 80.140.41
EL 20 39.47 1.52 66.470.58
40 34.250.43 48.250.46
10 48.530.60 73.501.52
Standard
20 33.000.54 56.431.87
(Albendazole)
40 29.530.54 42.000.35
All Values represents Mean SEM; n=6 in each group

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70 10 mg/ml
60 20 mg/ml
40 mg/ml
50
40
30
20
10
0
PEW EAW MW Standard

Graph-1. Time taken for paralysis after treatment with Hibiscus tiliaceus wood extracts
Where PEW Petroleum ether Extract, EAW- Ethyl acetate Extract, MW-Methanol
Extract, Standard- Albendazole. The number represents time in minutes.

60 10 mg/ml
50 20 mg/ml
40 mg/ml
40

30

20

10

Graph-2. Time taken for paralysis after treatment with Hibiscus tiliaceus leaves extracts
Where PEL Petroleum ether Extract, CL- Chloroform Extract, EAL- Ethyl acetate
Extract, EL-Ethanol Extract, Standard- Albendazole. The number represents time in
minutes.

As displayed in Graph -1 and Graph -2 petroleum ether extract of wood and ethyl acetate
extract of leaves exhibited significant anthelmintic activity in dose dependent when compare
with reference standard Albendazole. Ethyl acetate extract of leaves in concentration 40
mg/ml was found to be paralysis and death of warm in 28.06 and 44.57 minutes time
respectively which is potentially much effective as compare with standard reference drug
Albendazole. The compound constituents responsible for anthelmintic activity were not

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Tambe et al. World Journal of Pharmacy and Pharmaceutical Sciences

investigated however preliminary phytochemical screening of extracts give positive test for
steroids, alkaloids, terpenoides, saponins, tannins, flavonoides. The role of flavonoides,
[19-21]
phenolic compound and steroids as anthelmintic activity. The comparison between
treated groups with standards was carried out using one way ANOVA test. All the results
were found to be significant with P value less than 0.0001 (P< 0.0001)

CONCLUSION
From the present study it was concluded that both wood and leaves of Hibiscus tiliaceus
showed potential anthelmintic activity. Further research work is needed to isolate and identify
the phytoconstituents responsible for anthelmintic activity.

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