Vector
control activity studies on the leaf of Zyziphus
mauritiana
Sandhya
S.*, Jafferi SAH, Vinod KR, David Banji
and Narender Prasad D
Nalanda
ABSTRACT:
The present study was undertaken to evaluate
anthelmintic and larvicidal activity of crude ethanolic leaf extract of Zyziphus mauritiana belonging to family
Rhamnaceae. Through the preliminary chemical tests it was found that the plant
is rich in flavonoids, phenols, steroids and tannins. Pheretima posthuma was
used as the test worms. Various concentrations of ethanolic extracts were
tested in the anthelmintic screening, which involved determination of time of
paralysis (P) and time of death (D) of the worms. Piperazine citrate was
included as standard reference and distilled water as control. In the case of
larvicidal activity the study was conducted on Culex quniquefasicatus species
of mosquito larvae and the rate of larval mortality was calculated. The results
indicated that the crude ethanolic extract significantly demonstrated paralysis
and also caused death of the helminth especially at higher concentration of 50
mg/ml, as compared to standard reference piperazine citrate. Similarly very
optimistic results were observed for Culex quniquefasicatus species of mosquito
larvae and LC50 value was calculated as 54.9658.
KEY
WORDS: Antihelmintic;
larvicidal; Zyziphus mauritiana;,
Pheretima posthuma; Culex quniquefasicatus.
INTRODUCTION:
There has always been a closed association
between plants and human beings through ancient time and till date. In this
respect the intuition observation’s and experimentation made by the primitive
societies need to be acknowledged by present day workers of science. The
tribes’ of
The use of Ziziphus mauritiana in
In Ayurveda the fruits are applied on cuts and
ulcers. They are employed in pulmonary ailments, fevers and are usually mixed
with salt and chilly peppers to cure indigestion. The dried ripe fruit is a
mild laxative. The seeds are sedative and are taken, sometimes with buttermilk,
to halt nausea, vomiting, and abdominal pains in pregnancy. The leaf helps
check diarrhea. Mixed with oil, they are rubbed on rheumatic areas to reduce
inflammation. The leaves are applied as poultices and are helpful in liver
troubles, asthma and together with catechu, are administered when an astringent
is needed on wounds5.
It is commonly used to treat worms in children. The root has a purgative
action. A root decoction is given as a febrifuge and emmenagogue, and the
powdered root is dusted on wounds. Juice of the root bark is said to alleviate
gout and rheumatism. Strong doses of the bark or root may be toxic. The bark paste is applied on sores. An infusion of the
flowers serves as an eye lotion6.
The entire plant is a rich source of phenolic
compounds and flavonoids. Through various studies it has been found out that
even in hybrid varieties of Z.mauritiana, the flavonoidal peaks were traceable.
Thus the impact of hybridity on
flavonoidal spectrum in Z.mauritiana hybrid species was minimal7.The
plant showed significant amounts of total phenolic content in the fruit .Fruits
showed antioxidant activities since it possess high phenolic and flavonoid
contents. There was a strong correlation between total phenolic and flavonoid
levels and antioxidant activities8.The various part of the plant has
proved to be good in antidiarreal activity, antidiabetic activity and
fermentation prospects for the
development of starter cultures to produce masau wine and beverage9,10,11 .
Ziziphus mauritiana is an excellent source of essential fatty acid
linoleic acid and several of the metals including iron, calcium, magnesium and
zinc. Its content of other essential nutrients, however, was rather low12.
This work is an attempt to credentiate and presents the
vector control potency of Zyziphus
mauritiana leaf extract, thus scientifically proving its traditional
claims.
MATERIALS
AND METHODS:
Plant collection and authentication:
The plant was collected in the month of November and
December from the surrounding areas of Nalgonda and Ranga Reddy district, A.P,
Plant extraction:
The leaf of the plant was dried for several days under
shade and powdered with the help of an electric grinder and extracted exhaustively
with ethanol in a soxhlet apparatus for 22 hrs. The liquid extract was
concentrated using rotary vacuum evaporatorand the percentage yield obtained
was found to be 15.65%w/w.
Preliminary chemical screening:13,14
The extract was subjected to various chemical tests to
identify the chemical nature of the constituents. It was observed that the
extracts gave highly positive result for the Shinoda test, Libermann-Burchards
test, ferric chloride test. From this it was concluded that the extract is rich
in flavonoids, steroids and phenolic compounds.
Collection of helminth and larvae:
Indian earthworm,
Pheretima posthuma (Annelida)
were collected from the cultured water logged areas of soil at the Nizam
College of Science,
Preparation of test sample for the anthelmintic and
larvicidal activities:15,16
Samples for in-vitro anthelmintic study were
prepared by dissolving and suspending 2.5 gms of each crude ethanolic extract
fractions in 25 ml of distilled water to obtain a stock solution of 100 mg/ml.
From this stock solution, different working dilutions were prepared to get
concentration range of 10, 25 and 50 mg/ml
Samples for larvicidal activity were prepared by making
a stock solution which was serially diluted in water to prepare test
concentrations of 25, 50, 100, 150 and 200 mg/l.
Anthelmintic assay:15
The anthelmintic
assay was carried as per the method of Ajayieoba E. O. et al with minor
modifications. The assay was performed on adult Indian earthworm Pheretima
posthuma, due to its anatomical and physiological resemblance with the
intestinal roundworm parasites of human beings.
50 ml of 10, 25
and 50 mg/ml of the leaf extracts were prepared and placed in different
petriplates. Six earth worms of same size were placed in each one of them. Time
for paralysis was noted when no movement of any sort could be observed, except
when the worms were shaken vigorously. Time of death was recorded after
ascertaining that the worms neither moved when shaken vigorously nor when
dipped in warm water at 500 C. Piperazine citrate of strength 10
mg/ml was used as reference standard and distilled water as the control.
Larvicidal assay: 16
The larvicidal
assay was carried as per the W.H.O guidelines with minor modifications. Batches of approximately 25, third or fourth
instar larvae were transferred by means of screen loops to small disposable
test cups or vessels, each containing 100–200 ml of water along with drug
concentrations. The test containers were
held at 25–28oC under
a photoperiod of 12 hr light followed by 12 hr dark (12L: 12D).
After 24 hr exposure, larval mortality was recorded.
Moribund larvae were counted and added to dead larvae for calculating
percentage mortality. Dead larvae were those that could not be induced to move
when they were probed with a needle in the siphon or the cervical region.
Moribund larvae were those incapable of rising to the surface or not showing
the characteristic diving reaction when the water was disturbed. The results
were recorded where the LC50, LC90 and LC99 values, and slope were also
plotted.
Data analysis:
Data from all replicates were pooled for
analysis. LC50 and LC90 values were calculated from a log dosage–probit
mortality regression line using a biological statistical program Biostat 2008,
Professional package by Analyst soft .Inc,
RESULTS:
Ethanolic extracts of Z.mauritiana exhibited anthelmintic activity in dose-dependant
manner giving shortest time of paralysis (P) and death (D) with 50 mg/ml
concentration. The extract caused paralysis in 14.38min and time of death of
51.04 min respectively (table no.1 & fig no.1). The reference drug piperazine
citrate at 10mg/ml concentration showed paralysis and time of death as 19.26
and 63.25 minutes, respectively. As R2 is closer to one for the
extract it shows good co-relation among paralysis time taken at different
concentration (fig no.2) and death time taken at different concentration (fig
no.3).
Fig no.1: Paralyzed worms in
50mg /ml conc. in ethanolic leaf extract of Z.mauritiana
Table.no.1: Anthelmentic time
profile for ethanolic leaf extract of Z.mauritiana
|
Concentration(mg/ml) |
Time for paralysis (min) |
Time of Death (min) |
|
10 |
29.07±0.02 |
98.39±0.45 |
|
25 |
21.04±0.03 |
78.04±0.03 |
|
50 |
14.38±0.04 |
51.04±0.03 |
|
Piprazine
citrate (10 mg/ml) |
19.26±0.62 |
63.25.±0.58 |
Results are expressed as Mean±SD from three set of
observations.
Fig no.2:
Paralysis time for ethanolic leaf extract of Z.mauritiana
Fig no.3 Death
time for ethanolic leaf extract of Z.mauritiana
The larvicidal activity of the plant was found to be
satisfactory (table no.2). The LC50 value was calculated and was found to be
54.9658 (Table no.4). The percentile mortality values of instar larvae treated
with different concentration of the leaf extract at the end of 24 hr are
represented in Table no.4. The regression equations (based on probit analysis)
between the concentration of leaf extract and 24 h percent mortality of 3rd
and 4th instar larvae of C.
quinquefasciatus are represented in Fig no 5.The end larvicidal activity of
the extract is shown in fig 4.
Fig no .4 Image of dead larvae
at the 24 hrs in ethanolic leaf extract of Z.mauritiana
Table no.2 Efficiency of ethanolic leaf extract of Z.mauritiana on larvae of Cx.quiniquefasicatus
|
Concentration (mg/l) |
No. Exposed |
No. Dead |
|
Water (
Control) |
25 |
2 |
|
25 |
21 |
6 |
|
50 |
23 |
11 |
|
100 |
25 |
16 |
|
150 |
23 |
18 |
|
200 |
25 |
21 |
DISCUSSION:
Tribals of Andhra Pradesh use Ziziphus mauritiana for its anthelmntic property. This traditional
claim has been proved by this experiment where the plant has shown potent
anthelmintic activity (fig no.1). As shown in the Table no.1 Z.mauritiana has
exhibited anthelmintic activity in a dose dependent manner taking shortest time
for paralysis (P) and death (D) at 50mg/ml concentration. Phytochemical
analysis of crude extract reveled presence of phenols, flavonoids and steriods
.It has been reported that some synthetic phenols interfere with energy
generation in helminth parasites by uncoupling oxidative phosphorylation
(Martin 1997).Hence it is possible that these two extracts could also produce
similar effects17-19.
The control of mosquito-borne diseases can be achieved
either by killing, preventing mosquitoes to bite human beings (by using
repellents) or by causing larval mortality in a large scale at the breeding
centers of the vectors in the environment. Since the discovery of DDT, control
of disease-causing mosquito species has been almost completely based on
synthetic organic insecticides. Following DDT, conventional pesticides such as
malathion and pyrithroids are generally used for mosquito control. But the
extensive use of synthetic organic insecticides during the last five decades
has resulted in environmental hazards. Besides, this also caused the
development of physiological resistance in the major vector species. This has
necessitated the need for search and development of environmentally safe,
biodegradable, low cost and indigenous methods for vector control, which can be
used with minimum care by individual and communities in specific situation20.
The extract of Zyziphus mauritiana could be used for spraying in stagnant water
bodies which are known to be the breeding grounds for mosquitoes acting as
vector for a multitude of infectious diseases.
Table no – 3 Finneys probit
analysis for the larvicidal activity of ethanolic leaf extract of Z.mauritiana
|
Log10[Dose (Stimulus)] |
Actual Percent (%) |
Probit Percent(%) |
N |
R |
E(R) |
Difference |
Chi-square |
|
1.3979 |
0.2857 |
0.2796 |
21 |
6. |
5.8707 |
0.1293 |
0.0028 |
|
1.699 |
0.4783 |
0.472 |
23 |
11. |
10.8563 |
0.1437 |
0.0019 |
|
2. |
0.64 |
0.6714 |
25 |
16. |
16.7846 |
-0.7846 |
0.0367 |
|
2.1761 |
0.7826 |
0.7717 |
23 |
18. |
17.7486 |
0.2514 |
0.0036 |
|
2.301 |
0.84 |
0.8309 |
25 |
21. |
20.7722 |
0.2278 |
0.0025 |
|
Chi-square |
0.0475 |
Degrees Of Freedom |
3 |
p-level |
0.9973 |
|
|
Table no - 4 Finneys
percentile results for the larvicidal activity of ethanolic leaf extract of Z.mauritiana
|
Percentile |
Probit (Y) |
Log10[Dose (Stimulus)] |
Standard Error |
Dose (Stimulus) |
Standard Error |
|
1 |
2.6732 |
0.3772 |
0.4515 |
2.3837 |
2.9488 |
|
5 |
3.3548 |
0.7765 |
0.3382 |
5.9766 |
5.1384 |
|
10 |
3.7183 |
0.9894 |
0.2786 |
9.7579 |
6.6983 |
|
20 |
4.1585 |
1.2472 |
0.2082 |
17.6698 |
8.8005 |
|
25 |
4.3258 |
1.3452 |
0.1824 |
22.1414 |
9.5763 |
|
30 |
4.476 |
1.4332 |
0.16 |
27.1127 |
10.2171 |
|
40 |
4.7471 |
1.5919 |
0.1227 |
39.079 |
11.1875 |
|
50 |
5. |
1.7401 |
0.0951 |
54.9658 |
12.1283 |
|
60 |
5.2529 |
1.8882 |
0.082 |
77.3109 |
14.6794 |
|
70 |
5.524 |
2.047 |
0.0913 |
111.4325 |
23.6052 |
|
75 |
5.6742 |
2.135 |
0.1052 |
136.4519 |
33.3756 |
|
80 |
5.8415 |
2.233 |
0.125 |
170.9826 |
49.9137 |
|
90 |
6.2817 |
2.4908 |
0.1881 |
309.62 |
138.3627 |
|
95 |
6.6452 |
2.7037 |
0.2453 |
505.5088 |
300.8903 |
|
99 |
7.3268 |
3.1029 |
0.3566 |
1,267.4811 |
1,161.6197 |
Fig no. 5 Probit analysis
graph for the larvicidal activity of ethanolic leaf extract of Z. mauritiana.
ACKNOWLEDGEMENT:
The authors are grateful to Nalanda College
of Pharmacy for providing all necessary information resource, electronic data
processing and moral support for the present work. Also they wish to express
their thanks to Nizam College of Science and Dept. of Zoology, Osmania Uty. for providing
helminth and larvae respectively.
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Received on 22.12.2009
Accepted on 15.02.2009
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Research J. Pharmacology and
Pharmacodynamics 2(1): Jan. –Feb. 2010: 71-74