IN VITRO ANTI-UORLITHIATIC ACTIVITY OF SAMPA-SAMPALUKAN (Phyllanthus niruri)

ETHANOLIC LEAF EXTRACT TO CALCUIM OXOLATE CRYSTAL INDUCED MICE

(Mus musculus).
Jerico O. Julaton IV-Phoenix

RESEARCH PROPOSAL

INTRODUCTION
All over the world especially in developing countries, approximately 80% of population continues to
use traditional medicine in primarymedical problems. In the past decade, therefore, research has beenfocused
on scientific evaluation of traditional drugs of plant origin. There is an urgent need to systematically evaluate
the plants used intraditional medicine. Such research could lead to new drugdiscovery or advance use of
indigenous herbal medicines fortreatment. This revival of interest in plant derived drugs is mainly due to the
current widespread belief that green medicine is safe andmore dependable than the costly synthetic drugs
many of which have adverse side effects.
Urolithiasis is characterized by the formation of a stone in the kidneys or urinary tracts. A large
number of people, nearly 415% of the human populations are suffering from urinary stone problem all over
the globe.The crystals of calcium oxalate (CaOx) are the primary constituent of more than 60% of the
majority of human kidney stones; they exist in the form of CaOx monohydrate (COM) and CaOx dihydrate
(COD) [3]. The pathogenesis of calcium oxalate stone formation is a multi-step process and in essence
includesnucleation, crystal growth, crystal aggregation and crystal retention. The stone formation requires
supersaturated urine. Supersaturation also depends on urinary pH, ionic strength, solute concentration and
complexations. In spite of substantial progress in the pathophysiology and treatment of urolithiasis, there is no
satisfactory drug being used in clinical therapy. Endoscopic stone removal and extracorporeal shock wave
lithotripsy are prohibitively costly and recurrence is quite common with these procedures. Thus a drug for the
prevention of this disease or its recurrence would be of great interest. Phyllanthus niruri Linn. (Bhui amla) has
occupied an important place in Indian culture and folk medicines. It has been used in all most all the

traditional systems of medicine viz., Ayurveda, Unanai and Sidha. From the ancient time the tribal and rural
people of our country commonly used this herb in treating various disorders. P. niruri has also been used
traditionally for treating liver problems like hepatitis, elimination of mucous, kidney stones and diuretic
problems .
Keeping above knowledge in the mind, current study was done to find out the stone formation
inhibitor effect and stone dissolving effect of P. niruri extracts.
HYPOTHESIS
This study aims to find out the inhibitory activity of Ethanolic Leaf Extract from Sampa-sampalukan
(Phyllanthus-niruri) against calcium oxalate crystal induced mice. It sought to answer two specific questions.
a. Is enthanolic leaf extract of Sampa-sampalukan (P. niruri) is effective as a treatment for Urinary
stone associated with calcium oxalate crystal induced mice.
b. Is there a significant effect on the inhibiton of calcium oxalate crystal induced mice between the
trestments levels A,B,C,D after the application or intake of the extract of samapa-sampalukan at different
extract proportions.

Null (Ho)
There are no significant difference on the size of calcium oxalate crystals before and after the
application of the P. niruri ethanolic leaf extract on calcium oxalate induce mice.

Alternative (Ha)
There are significant difference on the size of calcium oxalate crystals before and after the
application of the P. niruri ethanolic leaf extract on calcium oxalate induce mice.

MATERIALS AND METHODS

a)
b)
c)
d)
e)
f)
g)
h)
i)
j)

Mice Cages
Syringes
P. niruri Leaves
Distilling flsk
Distilled water
CaOx crystals
Surgical knives
Beaker
Condenser
Vials

Materials and Methods

2.1 Plant Material
P .niruri leaves were collected from Himamaylan NHS. It grows into anherbal garden in our
school , Voucher specimen was dried in shade and stored in air tight container at 250C for further
study.
2.2 Extraction and Isolation
The leaves are pulverised and about 60 gms of powder was extracted with chloroform and
aqueous in soxhlet, also extracted successively with acetone, benzene, petroleum ether, Alcohol. All
extracts were concentrated on a water bath and residue was dried in a desiccator. All the prepared
extracts were subjected to qualitative chemical tests to detect the presenceof different classes of
phytoconstituents. TLC studies were done for identifying the presence of constituents which are
detected in chemical tests and to known how many extracts are present in each extracts . This
separated parameter is subjected for physical, chemical and spectral study (UV). And positive results
for two fractions were taken for pharmacological evaluation.
2.3 Evaluation for Anti-urolithiatic Activity
Step-1: Preparation of experimental kidneystones (Calcium oxalate stones) by homogenous
precipitation:
Equimolar solution of Calcium chloride dihydrate (AR) in distilled water and Sodium oxalate
(AR) in 10ml of 2N H2SO4 were allowed to react in sufficient quantity of distilled water in a beaker.
The resulting precipitate was calcium oxalate. Equimolar solution of Calcium chloride dehydrate
(AR) in distilled water and Disodium hydrogen phosphate (AR) in 10ml of (2N H2SO4), was
allowed to react in sufficient quantity of distilled water in a beaker. The resulting precipitate
wascalcium phosphate. Both precipipitates freed from traces of sulphuric acid by Ammonia
solution.Washed with distilled water and dried at 60 0C for 4 hours.
Step -2: Preparation of semi-permeablemembrane from farm eggs:
The semi -permeable membrane of eggs lies in between the outer calcified shell and the inner
contents like albumin & yolk. Shell was removed chemically by placing the eggs in 2M HCl for an
overnight, which caused complete decalcification. Further, washed with distilled water, and carefully
with a sharp pointer a hole is made on the top and the contents squeezed out completely from the
decalcified egg. Then egg membrane washed thoroughly with distilled water, and placed it in
ammonia solution, in the moistened condition for a while & rinsed it with distilled water. Stored
inrefrigerator at a pH of 7- 7.4.
Step-3:
Estimation of Calcium oxalate by Titrimetry:
Weighed exactly 1mg of the calcium oxalate and 10mg of the extract/compound/standard and
packed it together in semi evaluation. Permeable membrane by suturing as shown in Model design
Figure. This was allowed to suspend in a conical flask containing 100ml 0.1 M TRIS buffer. One
group served as negative control (contained only 1mg of calcium oxalate). Placed the conical flask
of all groups in an incubator, preheated to 37 0C for 2 hours, for about 7-8 hours. Removed the
contents of semi-permeable membrane from each group
into a test tube. Added 2 ml of 1 N sulphuric acid and titrated with 0.9494 N KMnO4 till a light pink
colour end point obtained.1ml of 0.9494 N KMnO4 equivalent to 0.1898mg of 4 Calcium. The
amount of undissolved calcium oxalate is subtracted from the total quantity used in the experiment

in the beginning, to know how much quantity of calcium oxalate actually test substance(s) could
dissolve.
Data Analysis
Data will be collected before and after the application of extract or the pre-test, posttest
experimental design. The initial weight must be measured and after the application. The initial and
final weight after application is compared with each other and the % of dissolution with a different
treatment levels.
Statistical Tools Used
One way around the problem is to compare the groups on differences between post-test and
pretest, sometimes called change scores or gain scores. [figure] The test can be carried out in a
number of equivalent ways:

t-test of the differences;

2-group ANOVA of the differences,

repeated measures analysis of variance.

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