In-vitro Antiprotozoal Activity of Zizyphus jujuba Mill and Lamk.
Veeresh*,
Kambhoja S, Nagraj MS, Manjunath and Vishesh
Department of
Pharmacognosy, The Oxford College of Pharmacy, Hongasandra, Bangaluru-560068.
ABSTRACT:
Zizyphus jujuba Mill and Lamk. Is also called as Baer tree,
belongs to the family Rhamnaceae. The dried bark was powdered and extracted
with various solvents by successive soxhlet hot extraction process with
increasing order of polarity. On phytochemical investigation, the different
extracts has shown the presence of carbohydrates, alkaloids, steroids,
flavonoids and tannins. The different extracts were screened for antiprotozoal
activity on Entamoeba histolytica and
Giardia lamblia using metronidazole
as standard drug. Both acetone and methanol extract showed significant
antiprotozoal activity compared to standard drug metronidazole.
KEYWORDS: Antiprotozoal activity, Zizyphus jujuba, metronidazole, Entamoeba
histolytica, Giardia lamblia.
INTRODUCTION:
Zizyphus jujuba Mill and Lamk. is also called as Badari,
Baer, Bogari, Barihannu belonging to family Rhamnaceae. The plant is
distributed throughout India, Iran, Afganistan, and in China1.It is a small subdeciduous tree with dense
spreading crown, commonly 0.6 m. girth and 6 m. high. The bark is blackish to
grey or brown, rough, regularly and deeply furrow, the furrowed, the furrows
are at about 1.2 cm apart. Blaze 9-13mm., Branches usually armed with spines,
mostly in pairs, one straight, the other curved. Leaves 3-6.3 by 2.5-5 cm.,
oblong or ovate, usually minutely serrulate or apex distinctly toothed, obtuse,
base oblique and 3-nerved, nerves depressed on the glabrous shining upper
surface. Petiole 2.5-10 mm long. Flowers 3.8-5 mm. In Diam., greenish, in dense
axillary tomentose cymes or fascicles 1.2-1.9 cm Long. Drupe 1.2-2.5 cm. Diam.,
globose, first yellow then orange and finally reddish brown, containing a
single stone surrounded by fleshy pulp2.
A survey of literature on Zizyphus jujuba Mill and Lamk. revealed a few pharmacological
reports on the plant like antioxidant and antilisterial effect3,
antisteroidogenic activity4, antiobesity activity5,
sedative and hypnotic6, anxiolytic7, anticancer8.
The plant is reported to
contain alkaloid jubanine-E9. It contains three
flavone-C-glucosides-6’’-sinapoylspinosin, 6’’-feruloylspinosin
and 6’’-p-coumaroylspinosin. The leaves and stems of Zizyphus
jujuba Mill and Lamk.
contains saponins
3-o-[2-o-α-L-fucopyranosyl-3-o-β-D-glucopyranosyl-α-L-arabinopyranosyl]jujubogenin.The
fruits of Zizyphus jujuba Mill
and Lamk. contain Zizyphus saponins I,
II, III and jujuboside B10, jujuboside D11 and jujuboside
E12. The bark of Zizyphus
jujuba Mill and Lamk. contains 7%
tannin13.
Protozoal attacks are common in different parts of the
world population and account for the major cause of mortality in developing
countries14.
On considering protozoal disease, amoebic dysentery or
amoebiasis caused by Entamoeba
histolytica infects more than 50 million people around the world and up to
110,000 die every year.15. Giardiasis caused by Giardia lamblia, Balantidiasis
caused by Balantidium coli, and
Malaria caused by plasmodium species are some of the other common protozoal
diseases of the tropical countries. Trichomonas
vaginalis, a protozoa causing STD- Trichomoniasis, also shows the medical
significance of protozoa16,17.
Although a good number antiprotozoal drugs are
available, most of them are toxic or less efficient18,19. Therefore,
efforts should be made to invent new and more efficient drug from natural
sources. Based on this objective, crude extract of Zizyphus jujuba was tested for its antiprotozoal activity. In the
present study, different extract of stem bark was tested for antiprotozoal
activity by a microscopic count method20, using Entamoeba histolytica and Giardia
lamblia protozoas.
MATERIAL
AND METHODS:
Plant Material: The dried stem bark of Zizyphus jujuba Mill and Lamk. belonging to the
family Rhamnaceae, was
collected from Raichur and authenticated by
Dr. Siddamallayya N, NADRI, Jayanagar, Bangalore. The bark was dried
under sun for seven days and powdered.
Preparation of extract: The dried stem bark was powdered in a mixer
grinder. 380 gram of the dried powder was successively extracted with 1500ml
each petroleum ether, benzene, chloroform, acetone, methanol and distilled
water using a soxhelet hot extraction process. From different extract stock,
aliquot was taken and 5ml of distilled water was added, gently heated until the
samples were completely dissolved. The final concentration of the sample
prepared was 20mg/ml. This solution of different extracts was used for testing
antiprotozoal activity.
Antiprotozoal test:
The strains of the protozoa used in the tests were Entamoeba histolytica HM1-IMSS and Giardia lamblia IMSS:0989:1. The strains
of trophozites were collected from LGC promochem india Pvt.Ltd, Bangalore. The
antiprotozoal test was made by microscopic count method, 1ml of solution of
different extracts was added to 4ml of protozoal inoculum, to get a final
concentration of 4 mg/ml. After two minutes 0.04ml was transferred on to the
glass slide, in control experiment only 1 ml distilled water is added instead
of extract solution and in standard experiment 1ml of metronidazole (20 mg/ml)
was added to 4ml of inoculum. Both the test, standard and controlled samples
were examined under a compound microscope and motile and non-motile organisms
were counted. Non-motile organisms were
considered as non-viable due to its susceptibility towards the extract and
motile were considered as resistant to the extracts. Test were repeated three
times and average number of motile/non-motile organisms was recorded.
RESULTS
AND DISCUSSION:
Table-1, shows the antiprotozoal activity of the
crude extracts of Zizyphus jujuba
Mill and Lamk. against Entamoeba
histolytica and Table-2 shows the activity against Giardia lamblia. Analysis of results showed that in the acetone,
methanol, aqueous extract almost all protozoas were non-motile, while in the
control sample they were alive. This indicates that Entamoeba histolytica and
Giardia lamblia were completely susceptible to the acetone, methanol,
aqueous extract of Zizyphus jujuba
even at a crude concentration of 4 mg/ml. The other extracts of Zizyphus jujuba Mill and Lamk. showed
significant antiprotozoal activity. The antiprotozoal effect of acetone,
methanol, aqueous extracts at 20 mg/ml concentration is comparable with that of
the effect produced by the standard drug metronidazole. The acetone extract
showed the effect at 20 mg/ml concentration that is comparable with the
standard drug metronidazole.
The present study suggest that the acetone extract was
more effective than the other extracts even though all the extract were endowed
with antiprotozoal property. The activity of the extracts was found to be
inversely proportional to the number of motile and non-motile organisms.
The active principles responsible for antiprotozoal
activity is due to the presence of flavonoids, steroids, alkaloids, tannins, in
the extracts. The isolation and characterization of a particular active
principle responsible for antiprotozoal activity is under progress.
Table 1. Antiprotozoal effect
of the different crude extracts of Zizyphus
jujuba against Entamoeba histolytica.
|
Sl. no |
Type of sample |
Concentration (mg/ml) |
Total no. of protozoa counted |
Observation of protozoa after 2 min sensitivity/
resistance |
|
|
No. of motile resistant organisms |
No. of non-motile sensitive organisms |
||||
|
1 |
Pet. Ether |
20 |
14 ± 1 |
6 |
8 |
|
2 |
Benzene |
20 |
13 ± 2 |
8 |
6 |
|
3 |
Chloroform |
20 |
12 ± 1 |
6 |
6 |
|
4 |
Acetone |
20 |
14 ± 2 |
0 |
13 |
|
5 |
Methanol |
20 |
13 ± 1 |
2 |
10 |
|
6 |
Aqueous |
20 |
12 ± 1 |
5 |
7 |
|
7 |
Std.Metronidazole |
20 |
12 ± 2 |
0 |
12 |
|
8 |
Control |
20 |
13 ± 1 |
13 |
0 |
Table 2. Antiprotozoal effect
of the different crude extracts of Zizyphus
jujuba against Giardia lamblia
|
Sl. no |
Type of sample |
Concentration (mg/ml) |
Total no. of protozoa counted |
Observation of protozoa after 2 min sensitivity/
resistance |
|
|
No. of motile resistant organisms |
No. of non-motile sensitive organisms |
||||
|
1 |
Pet. Ether |
20 |
12 ± 2 |
6 |
7 |
|
2 |
Benzene |
20 |
12 ± 1 |
5 |
7 |
|
3 |
Chloroform |
20 |
13 ± 1 |
7 |
6 |
|
4 |
Acetone |
20 |
14 ± 1 |
0 |
14 |
|
5 |
Methanol |
20 |
12 ± 1 |
2 |
10 |
|
6 |
Aqueous |
20 |
13 ± 2 |
3 |
11 |
|
7 |
Std. Metronidazole |
20 |
12 ± 2 |
0 |
12 |
|
8 |
Control |
20 |
12 ± 1 |
13 |
0 |
ACKNOWLEDGMENT:
The authors are thankful to the Chairman, Director and
Principal of The Oxford College of Pharmacy, Bangalore, for providing necessary
facilities to carry out the research work. The authors are also thankful to Dr.
Siddamallayya N, NADRI, Jayanagar, Bangalore, for identifying and
authentication of the plant Zizyphus
jujuba Mill and Lamk.
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Received on 11.12.2010
Accepted on 25.12.2010
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Research J. Pharmacology and
Pharmacodynamics. 3(1): Jan. –Feb. 2011, 34-36