Antiurolithiatic Potential of Eleusine indica Linn.

(GOOSE GRASS) Root Extract on

ethylene Glycol Induced Nephrolithiasis in Rattus norvegicus
(ALBINO RATS)

Authors: Daniel O. Amoah., Maritan B. Joson and Marlon C. Pareja

Biological Sciences Department

College of Science and computer Studies

De La Salle University-Dasmariñas,

Dasmariñas-Cavite, Philippines
ABSTRACT

The study was mainly aimed to determine the antiurolithiathic potential of different
concentrations of E. indica root extract on ethylene glycol induced nephrolithiasis in Rattus
norvergicus (albino rats). It specifically pointed out the effects of the plant extract on serum
parameters of creatinine, BUN and uric acid. It also looked at its effect on nitrituria,
proteinuria, calcium oxalate excretion, and the histopathology of kidney tubules and
glomeruli of albino rats. In the study, roots of E. indica were harvested, dried and its
contents extracted using distilled water of 80°C -100°C. The actual experimental period
lasted for 4 weeks. The study involved 5 test groups; negative control group (T-), positive
control group or lithogenic group (T+), two prophylactic groups (T1 and T2) receiving
different concentrations of E. indica root extract (T1- 500mg/kg and T2- 800mg/kg) and one
curative group receiving 800mg/kg of E. indica root extract after being induced with kidney
stones during the first 2 weeks of the experimental period. All the test groups received 0.75%
ethylene glycol (stone inducing chemical) in water for 4 week ad libitum with the exception
of the negative control group. At the end of the study, there was elevated levels of serum
creatinine, BUN and uric acid among the lithogenic group. All these serum parameters were
normalized in all the three treatment groups. There was no significant difference between
the serum parameters of the E. indica treatment groups and that of negative control (P<
0.05). More so, there was nitrituria, proteinuria, high pH and high levels of calcium oxalate
excretion among Rats within the lithogenic group. Again, all these parameters were
normalized in the three E. indica treatment groups. E. indica root extract also maintained
the normal structure of kidney glomeruli and tubules among T1 and T2 (prophylactic
groups) and repaired kidney defects in T3 (curative group). E. indica root extract thus
possess antiurolithiathic potentials and can be used to prevent and cure nephrolithiasis in
albino rats.

Key Words: Antiurolithiatic, ethylene glycol, nitrituria, proteinuria, nephrolithiasis

2
Table of Contents
ABSTRACT ...................................................................................................................... 2

INTRODUCTION ............................................................................................................ 5

Medicinal plants .............................................................................................................. 5

Eleusine indica ................................................................................................................ 6

The kidney....................................................................................................................... 8

Nephrolithiasis ................................................................................................................ 9

Ethylene glycol ............................................................................................................. 11

MATERIALS AND METHODS .................................................................................. 12

Plant acquisition preparation and Extraction ................................................................ 12

Acquisition of Albino rats ............................................................................................. 12

Ethylene Glycol Induction and Treatments with Plant Extract .................................... 13

Data Gathering and Statistical Analysis ....................................................................... 14

RESULTS AND DISCUSSION .................................................................................... 15

Serum Analysis ............................................................................................................. 15

Serum creatinine ........................................................................................................ 15

Blood urea Nitrogen................................................................................................... 17

Serum Uric Acid ........................................................................................................ 18

Urinalysis ...................................................................................................................... 20

Histopathology .............................................................................................................. 22

Discussion ..................................................................................................................... 23

CONCLUSION ............................................................................................................... 32
RECOMMENDATIONS ............................................................................................... 32

ACKNOWLEDGEMENTS........................................................................................... 33

CITED REFERENCES ................................................................................................. 33

APPENDICES ................................................................................................................ 39

APPPEDIX A ................................................................................................................ 39

Photo documentation of Eleusine indica plant ......................................................... 39

APPENDIX B ............................................................................................................... 40

Histopathology Plates of Various Testgroups ........................................................... 40

4
INTRODUCTION
Urinary calculi disease or kidney stones is a pathological condition (-iasis) in which stones
(lith-) are formed in the kidneys (Nephro-); hence the name Nephrolithiasis. Many people
around the globe suffer from this disease and there is an increasing prevalence of it
through the twentieth century until today [1]. In the United States alone approximately 1
in every 11 individuals is affected with Nephrolithiasis [2]. Kidney problems is reported
to be Philippines 7th leading cause of death and some studies show that about 2.3% of the
total Filipino population has kidney stones [3]. This ancient disease has claimed the lives
of many individuals especially those in the Middle East and had a mortality rate of about
19,000 deaths per year between 1990 and 2010 [4]. About 85% of kidney stones are
caused by accumulation of calcium oxalate or uric acid crystals in the kidney tubules [5].
The widespread of diseases such as Nephrolithiasis in our world today has gotten many
researchers to probe into the good old traditional medicinal plants to find solutions since
most of these fatal modern diseases were not dominant during the ages where medicinal
plants were widely used [1]. More so, the emergence of drug resistant organisms and
increasing prices of medicines calls for the discovery of new and less expensive plant-
based medicines. The geological background and global position of the Philippines has
made it one of the most bio-diversified countries in the world and as such an excellent
center to pursue medicinal plant-based researches. This country is rich in many plants
species possessing special medicinal properties. One of these medicinal plants is the
Eleusine indica commonly known as goose or wiregrass.

Medicinal plants

Medicinal plants contain special chemical compounds, which enables them to have
similar properties as conventional pharmaceutical drugs [6]. Humans have used them
throughout history to either cure or lessen symptoms of an illness [7]. Plants have
medicinal uses because of the production of some secondary metabolites, which they use
5
mainly for defense purposes. Since plants are immobile and unable to migrate from one
point to another during harsh environmental changes or when being attacked by other
species of organisms, they produce these secondary metabolites as a protective
mechanism to defend them and to increase their chances of survival [8]. Secondary
metabolites are organic compounds that are not directly involved in the, normal growth,
development, or reproduction of an organism [6]. Unlike primary metabolites
(metabolites directly involved in normal growth, development, and reproduction) absence
of secondary metabolites does not result in immediate death, but rather in long-term
impairment of the organism's survivability. Plants secondary metabolites are generally
toxic in nature but can marvelously heal many human related diseases in low
concentrations [8][9]. It is the presence of some of these secondary metabolites that makes
Eleusine indica possess medicinal properties [10].

Eleusine indica

E. indica is a tufted annual grass which is normally found spreading or erect with a diploid
chromosomal number of eighteen (2n=18) [11].It is found in Kingdom Plantae, Division
Magnoliophyta, Class Liliopsida, Order Poales, Family Poaceae, Genus Eleusine and
Species E. indica. The genus Eleusine contains about 8 species which are E. africana, E.
coracana, E. kigeziensis, E. indica, E. floccifolia, E. intermedia, E. multiflora and E.
jaegeri. The Species within this genus have little morphological differences between them
[12]. E. indica is closely related to the African crop finger millet; Eleusine coracana[11].
E. indica has a well-developed strong root system earning it the name \“jongos grass”
among some tribes in South Africa implying that it requires a young ox to uproot it [11].
It has flat to V-shaped leaves which measures about 8 mm in width and 15 cm lengthwise.
Its inflorescence contain a cluster of three (3) to eight (8) spikes arranged digitatively,
each with a length of about 5cm to 10 cm. All the narrow spikes radiate from the apex of

6
the stem, with the exception of one arising shortly below the below apex. In spite of the
uncertain geographical origin of E. indica, many scientist usually consider it to have
originated from Africa or Asia [11]. This small xerophyte is distributed mostly throughout
the warmer areas of the world and it is found in all the habitable continents present in
about 150 countries across the globe [13]. It is distributed almost throughout the tropical
world and also found in the sub-tropics, especially in North America and Europe [14]. It
has a broad tolerance to a wide range of environmental conditions and can thrive up to
2000 meters above sea level in the tropics. It is mainly propagated by means of seeds as
its vegetative growth is significantly reduced during dry seasons [15]. A single plant may
produce more than 50,000 small seeds, which can be easily dispersed by wind, water, or
animals. In the Philippines, E. indica is one of the most abundant weed found in waste
places, along river banks, roads, and settled areas [16]. It has been among the predominant
weed species ranking as the second most abundant in the Philippines [17]. It is considered
as one of the most invasive and most disturbing species of weed and has reduced rice
yields by 80% in some parts of the Philippines [17]. However, in spite of all the odds
about this grass, it is used traditionally as a medicinal plant to treat many diseases [18].
A review paper published by the institute of the Philippine medicinal plant in 2015 on E.
indica shows that the entire plant especially the root is used traditionally to enhance
diuresis [19]. Phytochemical screening carried out on the plant showed that it is rich in
alkaloids, steroids, flavonoids, tannins, and glycosides, which may be the reason behind
its incredible diuretic properties [20].

In spite of the fact that this plant is traditionally known to have some potentials to enhance
the discharge of urine, no vivid scientific study has been conducted on the potential it has
on the aversion of kidney stones [10];[20].This boosted the interest of researchers to
pursue this study. Since E. indica is easily accessible and has a wide distribution, a study
of this sort will be advantageous to people from all around the globe. More so, the study

7
will economically reduce the millions of money which is spent in controlling this weed
since the plant will become an important asset to the world’s health sector.

The kidney

The kidneys are two bean-shaped organs, each about the size of a fist supplied with two
main blood vessels; the renal artery and renal vein. They are located just below the rib
cage, one on each side of the spine [21]. Every day, the two kidneys filter about 120 to
150 liters of blood to produce about 1 to 2 liters of urine, composed of wastes and extra
fluid [21]. Each kidney is made up of about a million filtering units called nephrons which
filters a small amount of blood. The nephron includes a filter, called the glomerulus, and
a tubule. The nephrons work through a two-step process, ultra-filtration of selective
reabsorption. Ultra-filtration is the process whereby waste substances and fluid from the
blood are filtered out from the glomerulus into the bowman’s capsules of the nephron.
Since the glomerulus is made up of thin capillaries with squamous epithelial walls,
substances are able to pass across the walls. It allows fluid and waste products to pass
through it but prevents blood cells and large molecules, mostly proteins, from passing.
The filtered fluid then passes through the tubules to be passed out however needed
minerals and water are reabsorbed back into the bloodstream. The final product becomes
urine which is passed to the collecting ducts, the ureter, bladder and finally out through
the urethra. The kidneys are very important because they keep the composition, or makeup
of the blood stable, ensuring homeostasis and proper body function. They prevent the
build-up of wastes and extra fluid in the body, keep levels of electrolytes stable, such as
sodium, potassium, and phosphate and make hormones that help regulate blood pressure
make red blood cells bones stay strong [21]. Nephrolithiasis is one the major diseases
that affect and reduce the function of the kidneys.

8
Nephrolithiasis

Nephrolithiasis (kidney stones) is a common kidney disease prevalent among both men
and women in many countries. There is no specific factor that increases ones risk of
having the disease. Nephrolithiasis occurs when stones are formed due to high
concentration of many crystal-forming substances such as calcium, oxalate and uric acid
in the urine. The absence of other important chemical substances that prevent crystals
from sticking together also creates an ideal environment for kidney stones to form [22].
Calcium oxalate stones, uric acid stones, struvite stones and cystine stones are some of
the different types of kidney stones found among human [23]. Among these, Calcium
oxalate crystals is the major cause of nephrolithiasis. About 75% of kidney stones contain
calcium oxalate (CaC2O4) as the major constituent [5]. These stones are formed by
complex processes involving crystal nucleation, crystal growth, crystal aggregation, and
crystal-cell interaction [24].The presence of high calcium (hypercalciuria) and high
oxalate (hyperoxaluria) levels present in urine increases the risk to form calcium oxalate
stones. Common sources of oxalate include dietary excess of refined sugar, chocolate,
cranberries, rhubarb, spinach, high fructose corn syrup, apple juice and grapefruit juice
[25]. Hypercalciuria is normally caused by increased absorption of calcium in the
intestines, excessive parathyroid hormone levels (hyperparathyroidism) and renal calcium
leak leading to the excessive entrance of calcium into the urine [2]. Urinary citrate and
magnesium has the ability to prevent calcium oxalates from forming. Therefore Low
levels of citrate and magnesium, coupled with to high levels of oxalates and inadequate
water in urine increases the risk of crystallization of calcium oxalates. Renal tubular
acidosis is an inherited condition in which the kidneys are unable to excrete acid. This
condition significantly reduces urinary citrate and increases urinary acidity which
increases the risk to form stones especially phosphate stones [26]. The crystals formed
from supper saturation of calcium oxalate in urine combine with small amounts of some
proteins and accumulate within the kidney tubules. Some studies proved that Calcium

9
oxalate kidney stones generally contain a variety of proteins at low levels [27]. Some of
the proteins identified in Calcium oxalate kidney stones include Tamm-Horsfall protein
(THP or uromodulin), osteopontin, prothrombin, calgranulin A (S100A8), calgranulin B
(S100A9), serum albumin and defensin [28]. Amino acids of gamma-carboxyglutamic
acid, aspartic acid and glutamic acid have also been found to have a very high affinity
binding with calcium oxalate stones [29]. Proteins might therefore be the facade involved
in the onset of crystallizations of these stone. The combination of calcium oxalates with
these proteins leads to the formation of complex crystals. The crystals formed throughout
the nephrons begin to accumulate together as clusters. This aggregation process also
involves the formations of very tight bonds along with glycoprotein additions that seal
and bind them further [30]. Crystals start to interact with cells attaching themselves unto
apical microvilli of surrounding renal cells. Endocytosis may then occur within some
renal tubular epithelial cells leading to crystal growths within. Some cells are able to
dissolve the crystals while it remains undissolved in others. Cells which are unable to
dissolve these crystals start to undergo cellular changes in some important parts of the
cell such as cytoskeleton and microfilament. After mitosis these crystals continue to
present in newly formed daughter cells [30]. Some renal proximal tubule cells undergo
acute inflammations after endocytosis eventually leading to cell apoptosis. Crystals that
just adhere to apical cell surfaces continues accumulating and forming bigger crystals.
Inflammations are the results of these adhesions which when continued can lead to
membranolysis of renal proximal tubular cells and cell death [30]. Uric acid stones are
formed from uric acid, a product formed from the digestion and metabolism of meat. At
very high urine acidity, uric acid may not dissolve and uric acid stones are likely to be
formed. Only about 10% of nephrolithiasis patients develop this type of stone [26].
Struvite stones, also called an infection stone or triple phosphate stone are formed when
bacteria present in the urinary tract neutralize urinary acid to enable their growth. This
condition on promotes struvite stone development. Struvite stones can grow to become

10
very large and are usually developed as jagged structures called "staghorns” [26]. Cystine
stones are stones which has some relation with the genetic make-up of a person. Upon
birth one may inherit a rare congenital condition that results in large amounts of cystine,
a type of amino acid in the urine. These crystallize and lead to the formation of stone.
Cystine stones are rare but very difficult to be treated [26].

Kidney stones is also associated with Colicky pain as the lower back due to scrapping of
stones on renal walls, which is described as the worst pain ever. Other symptoms include
hematuria; blood in the urine due to minor damage to inside wall of kidney and Pyuria;
pus in the urine. Dysuria; burning sensation when passing out stones and Oliguria;
reduced urinary volume caused by obstruction of the bladder or urethra by stone are also
major problems experienced by kidney stone patients [23].

Ethylene glycol

Ethylene glycol is an organic compound with the chemical formula CH₂(OH)₂. It has
many uses, including as antifreeze in cooling and heating systems, in hydraulic brake
fluids, and as a solvent. A short term exposure of humans to ethylene glycol through
ingestion causes central nervous system (CNS) depression, cardiopulmonary effects, and
renal damage [31]. When rats and mice are exposed to ethylene glycol in their diet, they
exhibit signs of kidney toxicity and liver effects. Small concentration of ethylene glycol
mixed with water and introduced into the body of a rat through ingestion, is initially
metabolized by alcohol dehydrogenase into glycoaldehyde in the liver which is further
oxidized to form glycolic acid [31]. The accumulation of glycolic acid lead to metabolic
acidosis, which provide a good atmosphere for the Oxalic acid to be formed and
crystallize upon the binding with calcium leading to the formation of calcium oxalate
[31]. Calcium oxalate crystals are deposited in the kidney as the kidney tries to filter

11
blood. They accumulate obstruct the renal tubules leading to kidney stones formation
(nephrolithiasis).

This study therefore sort to find out whether E. indica root extract can prevent and/or treat
nephrolithiasis among albino rats. Thus the main objective of the study was to establish
the antiurolithic potential of different concentrations of E. indica root extract on ethylene
glycol induced nephrolithiasis in Rattus norvegicus.

MATERIALS AND METHODS

Plant acquisition preparation and Extraction

Eleusine indica plants were collected from Indang-Cavite, Philippines within the months
of November and December, 2016. The plant was identified and authenticated by a
Botanist at De La Salle University-Dasmariñas. During collection, the plants were
uprooted and the whole root up till an inch of the stem were harvested on the spot. They
cut roots were then washed thoroughly with water to remove all the soil particles and were
air dried at room temperature of 25°C (± 2) [32].
The dried roots were boiled in distilled water at low heat for about 15 minutes after which
the solvent was filtered out and the residue discarded [33].The filtrate containing the
desired extracts was then cooled, poured into a clean bottle and stored in a refrigerator. 1
mL of the cooled liquid sample was placed on a pre-weighed petri dish. The water in the
sample was evaporated and the exact weight of extract per milliliters was determines.

Acquisition of Albino rats

Thirty (30) healthy male albino rats of Rattus norvegicus species which were 5-6 weeks
old weighing between 200-250 grams and fed with standard dry pellets and water ad
libitum prior to study was bought from the Food and Drugs Administration (FDA),
Alabang- Muntinlupa City Philippines [33]. They were then grouped into five (5) test
groups (T-, T+, T1, T2 and T3) with each group containing six (6) albino rats. As advised
12
by veterinarian two albino rats were be put in a single highly ventilated standard rat cage
having dimensions of 35.5 cm long, 25.4 cm wide and 12.7 cm high. The cages was then
tagged with their test group labels (T-, T+,T1, T2 ,T3) and arranged in a random blocking
set up to ensure equitable distribution of other environmental factors [33]. The rats were
then kept under standard condition (25°C ± 2°C) in 12 hours dark and light cycles [34].
They had access to standard dry pellets and water ad libitum. They were kept in the
farmhouse for 2 weeks before the start of the experimental period to ensure that they are
well acclimatized to their new environment. An approval of a study protocol was sort
from the Institutional Animal Ethics Committee according to the regulation of Committee
for the Purpose of Control and Supervision of Experiments on Animals before the
inception of all the experimental period. The experiment was conducted in accordance
with accepted standard guidelines for the care and use of animals in scientific research
[33].

Ethylene Glycol Induction and Treatments with Plant Extract

From the commencement of the experimental period, each of the groups were
administered different supplies which were maintained throughout the entire
experimental period. Albino rats in T- (negative or vehicle control) was maintained on
standard dry pellets and drinking water. All the other groups; T +,T1, T2 and T3 received
calculi inducing treatment of 0.75% v/v ethylene glycol in drinking water ad libitum
within the entire experimental period [33]. T+ which served as the lithogenic group was
not given any other treatment apart from the 0.75% v/v ethylene glycol in water. T1, T2
and T3 served as the treatment groups who received different concentration of E. indica
roots extract. 500mg/kg and 800mg/kg of root extract were administered to albino rats in
T1 and T2 respectively from 1st day to the 28th day of ethylene glycol induction and served
as the prophylactic groups [35]. T3 on the other hand served as the curative group and was
be administered 800mg/kg of E. indica root extract at the beginning of week 3 (day 15)
13
of the experimental period. The concentration of E. indica given to each of the groups did
not exceed 3.45g/kg body weight, the LD50 of E. indica in albino rats [18]. The entire
treatment period lasted for 4 weeks.
Data Gathering and Statistical Analysis

At the end of the experimental period, all animals were kept in individual metabolic cages,
and a urine and blood samples were collected from some randomly selected rats in each
group [35]. The blood and urine were then taken to the diagnostic laboratory for Serum
analysis and urinalysis. The urine volume, pH, general urinalysis parameters, calcium
oxalate crystals and nitrites levels were determined from the urine samples. Serum
creatinine, Blood Urea Nitrogen (BUN) and uric acid levels were determined in serum
analysis [33]. Blood was collected from the jugular vein near the heart of rat under light
ether anesthesia. The serum in the blood was then be separated by centrifugation at 10,000
× g for 10 min and analyzed for the creatinine, uric acid, urea, and blood urea nitrogen
(BUN) at the diagnostic laboratory (Hi-precision diagnostic laboratory, Dasmariñas-
Cavite, Philippines).
In addition to the collection of the urine and blood, one (1) randomly selected rat
from each group was killed through the gas chamber. An incision was made through the
abdominal cavity and the kidneys were then removed. The kidneys were stored in 37%
formaldehyde and taken to the diagnostic laboratory (Hi-precision diagnostic laboratory,
Dasmariñas-Cavite) for histopathological analysis of the kidney. A longitudinal section
of the kidneys were obtained, stained and observed under the microscope. The degree of
damage to the glomerulus and other kidney tubules were used as the baseline for
establishing the effect of E. indica extract on kidney stones.
All data was expressed as mean ± SD. Differences and was statistically analyzed
using one way analysis of variance (ANOVA) to find out the statistical significant
difference of the result [36].

14
RESULTS AND DISCUSSION
Serum Analysis

Antiurolithic potential of different concentration of E. indica root extract on induced

nephrolithiasis in albino rats was determined based on serum analysis, urinalysis and
histopathological procedures.

Serum analysis involved the extraction of blood from rats on the last day of the
experimental period. The serum was then analyzed creatinine, blood urea nitrogen and
uric acid.

Serum creatinine
Table 1 shows the average creatinine levels for each group expressed mg/dL and the
standard deviation, and significant differences among test groups.

Table 1; serum creatinine levels of various test groups

Test Groups Average (mg/dL) ± SD

T- 0.26 ± 0. 001 X

T+ 0.37 ± 0.013 Y

T1 0.22 ± 0.008 X

T2 0.25 ± 0.014 X

T3 0.25 ± 0.018 X

Legend: Different superscript (x,y) denotes significant difference. Same superscript

denotes no significant difference

From table 1, it can be seen that group T1 (prophylactic group 1) had the lowest amount
of blood creatinine levels in serum at 0.22mg/dL. On the other hand T+ (lithogenic group)
had the highest amount of creatinine levels in serum at 0.37mg/dL. There was no
15
significant difference (represented by same superscript letter) between the creatinine
levels of groups T- (negative control), T1 (prophylactic group 1), T2 (prophylactic group
2) and T3 (curative group). However there was a significant difference between T+ and
all the other groups. Figure 1 shown below shows the summary of the results shows on
Table 1.

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
Test groups

T- T+ T1 T2 T3

Figure 1. Effects of E. indica on the serum creatinine levels of albino rats

Figure 1 shows a bar chart of the serum creatinine levels of the various test groups used
in the study. From the figure it can be observed that, there is a higher level of serum
creatinine among the T+ (Positive control or lithogenic group). However these creatinine
levels are normalized among the various E. indica treatment groups. The serum creatinine
of the various treatment groups as seen from the figure is almost at the same level like
that of the negative control group.

16
Blood urea Nitrogen
Table 2 below shows average Blood Urea Nitrogen (BUN) levels, range value, Standard
deviation and the significant differences among various test groups.
Table 2; Blood Urea Nitrogen levels of various test groups

Test Groups Average (mg/dL) ± SD

T- 12.32 ± 0.65X

T+ 23.44 ± 0.49 Y

T1 11.14 ± 0.15 X

T2 16.13 ± 0.19X

T3 14.34 ± 0.19 X

Legend: Different superscript(x,y) denotes significant difference. Same superscript

denotes no significant difference

From Table 2, it can be seen that group T1 (prophylactic group 1) had the lowest average
amount of BUN levels in serum at 11.14 mg/dL. On the other hand T+ (lithogenic group)
had the highest amount of Blood Urea Nitrogen levels in serum at 23.44mg/dL. There
was no significant difference between the BUN levels of groups T- (negative control), T1,
T2 and T3 (P-value; 3.97E-16). However there was a significant between T+ and all the
other groups (P-value > 0.05). The figure below summarizes the data presented on table
2. It shows a graph of Blood Urea Nitrogen levels of various test groups.

17
 25

20

15

10

Test groups

T- T+ T1 T2 T3

Figure 2. Effects of E. indica root extract on Blood urea nitrogen levels of albino rats.

Figure 2 is a bar chart showing the levels of Blood Urea Nitrogen (BUN) among the
various test group. From the chart is can be clearly seen that the BUN levels of T+
(lithogenic group) is highly elevated as compared to the other groups. The BUN levels of
the various E. indica treatment groups (T1, T2, T3) is almost at par with the BUN levels
of the negative control group.

Serum Uric Acid

Table 3 below shows average Uric acid levels, range values, standard deviation and the
significant differences among various test groups.

18
Table 3; Uric acid levels of various test groups

Test Groups Average (mg/dL) ± SD

T- 0.99 ± 0.011 X

T+ 2.83 ± 0.014 Y

T1 0.82 ± 0.010 X

T2 0.83 ± 0.010 X

T3 0.99 ±0.019X

Legend: Different superscript (x, y) denotes significant difference. Same superscript

denotes no significant difference

From table 3, it can be seen that group T1 (prophylactic group 1) had the lowest amount
of Uric acid levels in serum at 0.82 mg/dL. On the other hand T + (lithogenic group) had
the highest amount of Uric acid levels in serum at 2.83 mg/dL. There was no significant
difference between the Uric acid levels of groups T- , T1, T2 and T3 (P value; 1.48E-15).
However there was a significant difference between serum uric acid levels of T+ and all
the other groups. The figure below shows the summary of the data shown on Table 3.

19
 3

2.5

1.5

0.5

Test groups
T- T+ T1 T2 T3

Figure 3. Effects of E. indica root extract on serum uric acid levels of albino rats.

Figure 3 is a bar chart showing the serum uric acid levels among various test groups.
From the figure, it can be observed that the uric acid levels in the lithogenic group is
highly elevated. The uric acid levels of the various E. indica treatment groups and that of
the negative control group is at par. The levels of uric acid are lowered among the E.
indica treatment groups (T1, T2, T3).

Urinalysis

Urinalysis involved obtaining 24 hours urine sample from the rats a day after the
experimental period. The rats were put in different cages after the treatment period and
their urine for the next 24 hours were collected. The urine sample was taken to the
diagnostic laboratory and regular urinalysis were performed on the urine to determine the
pH, nitrite excretion and calcium oxalate crystals.

20
Table 4 shows the results for Urine pH, proteins, nitrites and calcium oxalate crystals
observed in the urine of rats. Table 4 pH, proteins, nitrite and calcium oxalate (CaOX)
found in urine
Test Groups pH Protein Nitrite CaOX
Excretion Excretion
crystals
T- 8.0 + Negative Few

T+ 9.0 ++++ Positive Many

T1 8.0 + Negative Few

T2 8.0 + Negative Few

T3 8.0 + Negative Few

Reference values: pH (normal;6.00-8.00), proteins( normal; +) , nitrite (normal;

negative) CaOX (normal; few)

From Table 4, it can be seen that all the test groups with the exception of T + has a pH
of 8.0. T+ on the other hand has a higher urine pH of 9.0. urine proteins found in all test
groups were found to be minimal represented by (+) while that of the lithogenic group,
T+ was found to be very high represented by (++++). All test groups tested negative for
excretion of nitrate with the exception of T+ which tested negative. More so many
calcium oxalate (CaOX) crystals were found in the urine of the lithogenic group (T +)
while all other groups had just few CaOX crystals.

21
Histopathology

The pictures of H&E stained kidney section of the test groups are shown below.

22
Figure 4. Histopathology of H&E stained kidney sections of the various test groups

Labels A-B (negative control or T+), C-D (positive control or T-), E-F (prophylactic
group 1 or T1), G-H (prophylactic group 2 or T2), I-J (curative group or T3)

Legend:“g” represents glomerulus, “p” represents proximal convoluted tubules, “d”

represents distal convoluted tubules, “cs” represenst capsular space, “rbc” represents red
blood cells.

From the histopathology sections above it can be clearly seen that cells in the positive
control or lithogenic group (T+) are darkly stained, while those of the negative control as
well as the E. indica treatment groups (T1,T2,T3) are stained pale. Most of the nucleus of
the lithogenic group seems to be darkly stained and some of them are separated entirely
from the cells cytoplasm (refer to “D” from figure 4). However this is not the case in the
negative control and the various treatment groups (T1, T2,T3) as the nucleus of the cells
present in the various histopathology sections of the groups appears pale and with intact
cytoplasm. From the sections it can also be seen that the glomerulus on the negative
control and treatment groups remain intact. Most of the Red blood cells are found in the
glomerulus with few at some vintage points among the tubules showing the presence of
blood capillaries that intertwines the nephrons. More so, it can be seen from the
histopathology sections that there is a clear well defined capsular space in the negative
control and treatment groups. However the capsular space of the lithogenic group is not
clearly defined, the glomerulus appear to be deformed and there are a lot of Red blood
cells outside of the glomerulus; within the capsular space, and the various tubules.

Discussion

Nephrolithiasis is associated with the formation of stones in the kidneys. Ethylene Glycol
used in the study leads to the creation of calcium oxalate stones in kidney tubules. These
stones obstruct the kidney tubules and destroy the glomerulus of the nephrons.
Glomerular filtration rate decreases due to the obstruction to the flow of urine. As a result,
23
waste products from the blood which should be filtered out in the kidneys remain and
accumulate in the blood. Prominent among these waste products that are retained in the
blood include nitrogenous wastes such as creatinine, BUN, and uric acid [37].

Creatinine is a major metabolic waste produced from muscle metabolism. The kidneys
maintain the blood creatinine in a normal range by filtering it out of the body. As such
when there are kidney dysfunctions, especially with ultra-filtration, creatinine levels will
be elevated in the blood. Creatinine has therefore been found to be a fairly reliable
indicator of kidney function. A rise in serum creatinine level is a late marker, observed
only with marked damage to functioning nephrons and as such signifies impaired kidney
function or kidney disease [38]. The higher levels of creatinine seen among the T+ shows
that the kidney function had been highly compromised. This problem was corrected by E.
indica root extract in T1, T2, and T3. The lowered levels of serum creatinine indicated that
normal kidney function has been restored. It can be deduced from Table 1 that there was
no significant difference between the creatinine levels of the treatment groups and that of
the negative control group. That is to say that E. indica was able to maintain the normal
levels of serum creatinine among the treatment groups. It prevented the ethylene glycol
from inducing its kidney destructive effects on the kidneys of rats by preventing the
formation of stones in the kidney tubules. The normal levels of serum creatinine as seen
in all the treatment groups is a clear indicator of the antiurolithiatic effect of the plant
extract.
Blood Urea Nitrogen is also another indicator for determining kidney function among
mammals. Blood urea nitrogen (BUN) test measures the amount of nitrogen in
the blood that comes from the waste product urea. One of the main functions of the kidney
is to filter out nitrogenous waste which comes from proteins. Since proteins cannot be
stored in the body, excess proteins undergo deamination in the liver to be converted into
urea which is then excreted out of the body [39]. When there is a kidney disease like

24
nephrolithiasis which lead to kidney injury, this process is impeded and these nitrogenous
wastes are retained in the blood in high amounts. As such high BUN values signifies
kidney dysfunction and is one ways to diagnose the nephrolithiasis [40]. The normal
values of BUN in 6- 10 week old albino rats is 10-20 mg/dL [41]. In the study it can be
seen that the negative control and all the treatment groups had their BUN levels within
this range showing normal kidney function. Thus there was no significant difference
between the BUN levels of the negative control and the treatment groups (P < 0.05). The
lithogenic group on the other hand had a really high levels of BUN. E. indica root extract
was able to reduce and maintain the BUN levels among the treatment groups (T 1, T2, and
T3) suggesting preventive function and curative function of E. indica root extract. Other
studies which looks at the phytochemicals of E. indica suggest that the plant contains
enormous amount of tannins [42]. Tannins have been found decrease blood urea nitrogen
content [43]. The normalized BUN levels seen among the treatment groups may therefore
be due to the presence of tannins in the plant.

Uric acid is another parameter that is used to detect kidney function. Uric acid is naturally
produced in the body out of breakdown of purine containing dietary substances. But since
uric acid is a metabolic waste it is required to be excreted out of the body. This work of
excretion is done by the kidneys. When ethylene glycol induced in albino rats forms
calcium oxalate stones in the kidneys, glomerular filtration is highly affected and as such
uric acid excretion is compromised. This leads to high levels of uric acid in serum as seen
in the positive control (T+). Serum uric acid is commonly elevated in subjects with
chronic kidney disease (CKD). Uric acid also have the tendency to form crystals in
kidneys leading to nephrolithiasis. Raising the uric acid level in rats was found to induce
glomerular hypertension and renal disease as noted by the development glomerular injury
[44]. In line with this, the lowered serum uric acid levels in E. indica root extract treatment
groups (T1, T2 and T3) suggests normal kidney function and aversion of nephrolithiasis.

25
Thus there was no significant difference between the uric acid levels of the treatment
groups and the negative control (T-).

Urinalysis parameters can also be used to determine the presence of kidney stones. Urine
pH is an important parameter in diagnosing the presence or absence of kidney stones. The
formation of various types of kidney stones is strongly influenced by urinary pH. A highly
alkaline pH favours the crystallization of calcium and phosphate containing stones,
whereas and acidic urine pH promotes uric acid or cystine stones. [45]. The high alkaline
Urine pH of the positive control group suggests the possible presence of calcium oxalate
stones which is induced by ethylene glycol. The alkaline levels are reduced among the
treatment groups as that in the control suggesting the possible prevention or reduction
stones.

Proteinuria is one indicator of kidney damages among kidney stone victims. It refers to
the condition where there are enormous amount of proteins in the urine. The healthy
kidney do not allow the proteins to pass out; thus prevents protein filtration. However
when kidney function is compromised due to the presence of kidney diseases like
nephrolithiasis, the glomerular filters allow proteins like albumin to leak out from the
blood to the urine. Proteinuria may accelerate kidney disease progression to end-stage
renal failure [46]. The results of this study showed a high levels of proteins present in the
urine of the positive control group (lithogenic group) which is not present in the treatment
groups. The aversion of proteinuria in the treatment groups serves as major indicator that
E. indica root extract does have preventive and curative properties against nephrolithiasis.

Under normal circumstances, urine is a sterile substance, so the presence of any

anomalous components is a potential sign of infection. A positive test for nitrites in the
urine is called nitrituria. The purpose of nitrite test in urine is to detect whether bacteria

26
are present, as they convert nitrate to nitrite in urine [47]. Nephrolithiasis has the
possibility of increasing the developing of urinary tract infection due to the destruction of
the mucosal walls of the urinary organs making it easy for other bacteria present to
proliferate and cause urinary tract infection. It can be seen from the table that both the
negative control group and the treatment groups tested negative for nitrite. The lithogenic
group which tested positive for nitrite is a clear indicator of a possible urinary tract
infection. This problem is not seen in the E. indica treatment groups which suggest the
absence of urinary tract infection [48]; [49].

The presence of Calcium Oxalate crystals in urine is another trademark indicator to

determine the presence of nephrolithiasis. Calcium oxalate crystals in the urine are the
most common constituent of human kidney stones. Kidney stones form when the urine
contains more crystal-forming substance such as calcium and oxalate than the fluid in
your urine can dilute [50]. Naturally, urine contains calcium oxalate crystals but the
presence of numerous amount of these crystals suggest the possible presence of stones in
the kidneys. The ability of E. indica root extract to reduce the number of calcium oxalate
crystals among treatment groups shows the antiurolithiathic property of this plant.

Histopathology is one of the ideal ways to detect the presence of nephrolithiasis and its
effects on the kidney. In histopathology, what actually happens in the kidney tubules is
clearly observed and understood. The glomerulus is a tuft of small blood vessels
called capillaries located within Bowman's capsule within the kidney. This tuft of
capillaries is supported by a matrix of collagen and other components that are called
mesangium. In addition, the tuft is surrounded by a layer of flat cells that forms something
like a tapestry that envelope it; the visceral epithelial cells or podocytes. The network of
capillaries, the mesangium, and the podocytes form the glomerular tuft. Glomerular
capsule is made up of outer basement membrane and inner epithelium. Between

27
glomerular capsule and visceral epithelial cell layer is capsular space. Abnormalities can
be in basement membrane, epithelium, and capsular space [51]. A normal kidney
histopathology will have a well-defined capsular space, basement membrane and the red
blood cells will be mostly observed in the glomerulus, with few spread out at some points
among the tubules. More so, Haematoxylin and Eosin (H&E) staining for normal kidney
yields the cytoplasm of the cuboidal cells of distal and proximal convoluted tubules as
pale pink while the nucleus is stained purple with distinct and visible nucleolus. The
nuclei of the various cells are not stuck together and each cell is seen as separated from
the other cell.

The kidney tubule cells of the lithogenic group (T+) in this study was found darkly stained
as compared to the negative control and treatment groups. The entire nucleus of the
lithogenic group appeared darkly stained purple and the nucleolus was not clearly distinct
and visible (figure 4). In nephrolithiasis, the formation of CaOX stones reacts with certain
stone forming proteins and creates a complex that reacts with the kidney tubules [30].
These complexes (crystals) attaches themselves unto apical microvilli of surrounding
renal cells leading to inflammations. Some cells of the tubules transport these complexes
through endocytosis in order to digest them. If they are unable to dissolve these crystals,
cells start to undergo changes in some important parts such as cytoskeleton and
microfilament. The inflammations created by crystal interaction with cells and
proliferation of these microfilaments in the cells make the cytoplasm appear darkly
stained. Inflammations also creates adhesions which when leads to membranolysis of
renal proximal tubular cells and ultimately cell apoptosis [30]. Apoptotic cells undergo
cell shrinkage, condensation and deep eosinophilia of the cytoplasm and pyknosis;
irreversible condensation of chromatin in the nucleus [52];[53]. This is what made the
cells in the lithogenic group appear with darkly stained cytoplasm and deep purplish
nucleus. All these symptoms are not seen in all the three E. indica root extract treatment

28
groups (T1, T2, T3). The cytoplasm of the kidney tubules of the groups were pale pink, the
nucleolus were distinct and visible as that of the negative control. All these evidences
clearly shows the antiurolithic property of the plant.

From the histopathology sections (refer to figure 4), it could be clearly seen that the
glomerular capsule of the lithogenic group which were affected with nephrolithiasis were
deformed. The bowman’s space (urinary space) was not clearly defined. The squamous
epithelial and basement membrane of the Bowman’s capsule were also deformed. The
nucleus of the podocytes was overlapping and there was a lot of red blood cells seen
outside the glomerulus. In the normal kidney, Red blood cells are too large to pass through
the small holes in the glomerular capillaries. Thus the kidney filtrate do not contain red
blood cells [54]. If they are found to proliferate outside the glomerulus, it suggest that the
capillaries has been enlarged and the ultrafiltration process has been compromised.
Crystals formed in nephrolithiasis cause expansions of the capillaries and lesions in the
glomerulus which leads to glomerulonephritis. This condition in turn leads to the seeping
out of RBC in to the capsular space and kidney tubules which leads to haematuria [55].
This is greatly observed in the H&E sections of the lithogenic group (refer to ‘C’ and ‘D’
from figure 4). However, the situation is averted in the E. indica root extract treatment
groups. All the three treatment groups showed normal structure of the glomerulus. The
capsular space, single squamous epithelial cells and basement membranes of the
bowman’s capsule could be observed as intact in the treatment groups like that of the
negative control group. More so, no red blood cells were found within the tubules and
capsular space of T1 and T2 (the prophylactic groups) which proves that E. indica root
extract is an excellent preventive treatment for nephrolithiasis and as such a good
nephron-protective plant. Few red blood cells were found in the capsular space T3
(curative group) which suggested that the kidneys were still in the process of healing
(refer to ‘I’ and ‘J’ from figure 4).

29
Studies which looked into the phytochemicals present in E. indica shows that the plant is
rich in flavonoids [42]. Flavonoids has also been found to prevent calcium oxalate crystal
deposition in the kidney by preventing hyperoxaluria-induced peroxidative damage to the
renal tubular membrane surface (lipid peroxidation) which in turn can prevent calcium
oxalate crystal attachment and subsequent development of kidney stones [30]. More so,
the presence of high levels of flavonoids in a plant makes it possible for it to have
preventive properties on the development of nephrolithiasis and the prevention of renal
tubular inflammations caused by this pathologic condition. [56]. Therefore, flavonoids
present in E. indica may therefore be a possible reason for it ability of prevent
inflammations in the kidney tubules and to prevent nephrolithiasis among albino rats.

From all the results explained above, it is clearly seen that E. indica root extract exhibit a
great effect on Serum creatinine, Blood Urea Nitrogen (BUN) and uric acid levels of
albino rats. The plant extract reduces these nitrogenous waste substances in the blood to
normal levels in all the three E. indica treatment groups (T1, T2, T3) as that observed in
the negative control group. The presence of normal levels of these parameters suggests
the preventive and curative properties of the plant extract against nephrolithiasis. The root
extract also exhibit effects on the excretion of urinary nitrite, proteins and calcium oxalate
crystals of albino rats. It prevents the formation of urinary nitrites, a major indicator of
urinary infections among all treatment groups. The plant extract also prevents proteinuria
(the presence of high levels of proteins in urine) among all E. indica treatment groups
which suggest normal glomerular filtration. More so the levels of calcium oxalate crystals
seen in the urine of treatment groups are just few as compared to the lithogenic group. All
these suggest the absence of nephrolithiasis among treatment groups supporting the fact
that E. indica root extract indeed has antiurolithic properties and as such can be used to
prevent and cure nephrolithiasis. E. indica root extract also exhibit effects on the structure

30
of the glomerulus and kidney tubules of albino rats. The plant extract prevents the
disruption of normal structure of the glomerulus and tubules of rat’s kidney among the
prophylactic groups (T1, T2). It also shows restoration of already disrupted glomeruli and
kidney tubules back to the normal structure among curative treatment group (T3). The
plant extract maintains the normal structure of glomerular capillaries, podocytes,
mesangium, basement membrane, capsular space, and the entire Bowman’s capsule
among all the treatment groups. Presence of red blood cells in capsular space and kidney
tubules which can lead to hematuria are totally prevented among the prophylactic groups
(T1, T2) and highly reduced in curative group (T3). Cell lesions, membranolysis and
apoptosis which are highly present among lithogenic group was prevented and corrected
in all E. indica treatment groups. All these are clear indications of the antiurolithic
property of E. indica root extract.

In spite of the fact that, all concentration of E. indica root extract used were able to
maintain these parameters within the normal range, the concentration used for T1
(500mg/ml) showed the best results. This concentration reduced serum creatinine, BUN
and uric acid drastically to the best levels. It also prevented the excretion of urinary nitrite,
proteins calcium oxalate crystal. More so, it fully maintained the normal structure of the
kidney tubules and glomerulus like that observed in the negative control group.

31
CONCLUSION
E. indica root extract exhibit a great effect on Serum creatinine, Blood Urea Nitrogen
(BUN) and uric acid levels of albino rats as there was no significant difference between
negative control and treatment groups (P value < 0 .05). E. indica root extract also exhibit
effects on the excretion of urinary nitrite, proteins and calcium oxalate crystals of albino
rats. It prevents nitrituria, proteinuria and oxaluria among treatment groups. The
formation of urinary nitrites, a major indicator of urinary infections among all treatment
groups. E. indica root extract also exhibit effects on the structure of the glomerulus and
kidney tubules of albino rats. The plant extract prevents the disruption of normal structure
of the glomerulus and tubules of rat’s kidney among the prophylactic groups (T1, T2). It
also shows restoration of already disrupted glomeruli and kidney tubules back to the
normal structure among curative treatment group (T3). There is a slight difference in the
effects that different concentration of E. indica root extract posed on serum, urine and
histopathological parameters utilized in the study. In spite of the fact that, all
concentration of E. indica root extract used were able to maintain these parameters within
the normal range, the concentration used for T1 (500mg/ml) showed the best results as
it reduced the concentration serum creatinine, BUN and uric acid drastically to the best
levels and prevented nitrituria, proteinuria and oxaluria among treatment groups.

All the above, clearly proves that E. indica root extract has antiurolithiatic effects against
ethylene glycol induced nephrolithiasis in Rattus norvergicus (albino rat)

RECOMMENDATIONS
The following are some of the recommendations of the researchers of this study. We
recommend future researchers;

1. To replicate the same study and should make use of many curative study groups
and should extend the time of administration of treatment to the curative group.
32
 2. To conduct further study to determine the exact phytochemicals responsible for the
antiurolithic property of E. indica root extract.
3. To replicate the study among higher mammals like guinea pigs and rabbits to
determine if the plant extract will pose the same antiurolithic effects on
nephrolithiasis induced in these mammals.
4. To look into the effects different part E. indica have on the aversion of
nephrolithiasis.
5. To conduct further on the same topic making use of a different extraction solvent
like ethanol or methanol.

ACKNOWLEDGEMENTS
The researchers wish to express their utmost gratitude and appreciation to The Almighty
God for granting us the strength and knowledge to write this research paper; Mr. Marlon
Pareja for his enormous assistance as the thesis advisor for this research paper; Dr. Johnny
Ching for his guidance provided as the thesis professor in conducting this research; Dr.
Jonthan S. Rubio, Dr. Janet Luistro and Ms. Hazel Ann Tabo for their advice and
criticisms as the research panel to make this study a success.

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17(1- 3):1-3.

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APPENDICES
APPPEDIX A

Photo documentation of Eleusine indica plant

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APPENDIX B

Histopathology Plates of Various Testgroups

Negative Control (T-)

Plate 1a Plate 1c

Plate 1b Plate 1d

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Positive Control (T+)

Plate 2a Plate 2b

Plate 2c Plate 2d

41
T1 (prophylactic group 1)

Plate 3a Plate 3b

Plate 3c Plate 3d

42
T2 (prophylactic group 2)

Plate 4a Plate 4b

Plate 4c Plate 4d

43
T3 (Curative group)

Plate 5a Plate 5b

Plate 5c

44