BIOLOGICAL EVALUATION OF INVIVO DIURETIC, AND

ANTIUROLITHIATIC ACTIVITIES OF ETHANOLIC LEAF

EXTRACT OF SACCHARUM OFFICINARUM
M.N.Palaksha, K.Ravishankar, V. Girija Sastry
Corresponding author:
Dr. M.N.Palaksha M.Pharm, Ph.D.
Associate professor, HOD,
Department of Pharmacology,
Aditya College of Pharmacy, Surampalem,
E.G.Dist-533437. +91-9293785255,
Mail id: palaksha.mn@gmail.com.
Co-author: Dr.K.Ravishankar M.Pharm, Ph.D.
Professor and principal,
Aditya College of Pharmacy,
Surampalem, E.G.Dist-533437.

Co-author: Dr. V. Girija Sastry M.Pharm, Ph.D.

Professor,
University College of Pharmaceutical Sciences,
Visakhapatnam-530003.

1
ABSTRACT:
The present work was investigated to evaluate the diuretic and antiurolithiatic
activities of ethanolic leaf extract of Saccharum officinarum. The ethanolic
leaf extract was administered to experimental rats orally at a dose of 200mg/
kg and 400mg/kg. Furosemide (5mg/kg) was used as standard for diuretic
activity. The parameters measured for diuretic activity was total urine volume;
urine electrolyte concentrations such as sodium, potassium, chloride and
bicarbonates. In in-vitro antiurolithiatic activity, Calcium oxalate
crystallization was induced by the addition of 0.01M sodium oxalate solutions
in synthetic urine. The effect of extract (100, 200 and 500g/ml) was studied
by time course measurement of absorbance at 620 nm for ten minutes by
means of a spectrophotometer. Ethanolic extract showed inhibition at 120 sec
(40.31, 60.46 and 63.56 %), and maximum inhibition of the crystallization of
calcium oxalate at 600 seconds (55.24, 69.93 and 72.72%). In in-vivo
antiurolithiatic activity, urolithiasis was induced in male rats by administering
ethylene glycol (0.75% v/v) in drinking water for 28days and the parameters
such as oxalate, calcium and phosphate were estimated in urine. Serum
creatinine, calcium and uric acid were also estimated. Treatment with the leaf
extract of Saccharum officinarum restored all biochemical, urinary
parameters. The results obtained justified the importance of the leaf extract of
Saccharum officinarum as a diuretic and antiurolithiatic agent.

2
1. INTRODUCTION

1.1. Diuretics:
Diuretics are drugs which promote the excretion of electrolytes in
water. The saluretics are the agents which excrete more amounts of chloride
and sodium in urine (Barar, 2015). Diuretics play a very important role in
conditions like hypercalciuria, edema, in renal failure, liver cirrhosis and acts
as hypotensive agent. (Singh et al., 1991). Diuresis induced by drugs is
advantage in renal failure, heart failure, liver cirrhosis, hypertension,water
intoxication and toxaemia (Agunu et al., 2005). Diuretics act by increasing
the excretion of sodium in urine; this is the reason why diuretics are used in
heart diseases, liver cirrhosis, hypertension, water poisoning and certain
kidney disease (Samiulla and Harish, 2000).
1.1.1. Composition of urine:
Urine consists of the following components. They are:
Water
Electrolytes Na+ , K+, Cl- , HCO3-
End products of nucleic acid metabolism- uric acid
End products of protein metabolism are urea, creatinine, PO4-
3 ,SO4-2
Break down products of phosphoric acid and sulphuric acid
H+ ions excreted bound to buffers such as PO4, NH3 (Glann et
al., 2002)54

3
 1.1.2. Biological role of ions:
Sodium and its role:
Sodium is one of the important ion or electrolyte present in the
extracellular fluid. The aproximate daily requirement of sodium for healthy
indivdual is 500mg/kg.
Aids in muscle contraction
Regulate blood pressure
Maintain with the transmission of nerve signal
Maintain with acid-base balance
Participates in the active transport of glucose and some other
nutrient
Potassium and its role:
Potassium is the important ion or electrolyte present in the extracellular
fluid.
Maintain in nerve transmission
Aids in the immune function
Managing blood pressure by decreasing the effects of sodium
Chloride its role:
This is one of the anion or electrolyte present in the body
Maintain nerve transmission
Used to form hydrochloric acid (HCL) secreted into stomach
Improves the immune function
Bicarbonate and its role:
Bicarbonate is the major element in our body
. It is necessary for digestion
. Plays major role in acid-base balance (Fulgoni et al., 2011)

4
1.2. Urolithiasis:
Urolithiasis is one of the complicated disease identified and in
literature survey by the Roman and Greek physicians. Urolithiasis consists of
both ureteric and renal stones. The prevalence of urolithiasis is about 1 in
1000 every year in New Zealanders, about 15% males, 6% females in
America and 7% population of Australia in their life-time are affected by
urolithiasis. Kidney stones changes with sex,age, society and environment.
Urolithiasis is considered as life time disease because half of them are re
admitted within 5 years of treatment.
The process of stone formation, urolithiasis, is also called
nephrolithiasis. Calculi consist of calcium, uric acid, oxalate, phosphate,
magnesium, ammonia and cystine. Calcium is the main constituent of kidney
stones because 85% of stones contain calcium oxalate and calcium phosphate.
12% of stones are formed due to infection calculi, about 7% are uric acid
calculi, and cystine calculi are very rare. Radio-opaque stones are seen in
more than 85% cases of calculi are, and only few are radiolucent ones.
They are many marketed formulations which are having anti
urolithiatic activity; some of them are Cystone, calculi and chandraprabhabati.
These formulations are used to dissolve urinary calculi in the urinary system
include bladder and kidneys (Anubhav and Rajeev 2013). Cystone works by
relaxing detrusor muscle and increasing the diuresis by virtue of its high
content of mineral salts of natural. Cystone have also been found to be useful
in urolithiasis, crystalluria and urinary tract infections (Yadav 2011).

5
2. Literature survey
2.1. Saccharum officinarum:

Fig. 2.1. Saccharum officinarum plant.

Species: Saccharum officinarum
Family: Poaceae
Genus: Saccharum
Synonym: Noblecane, sugarcane
Saccharum officinarum (family: Paoceae), Sugarcaneis one of the important
crop and has rich source of sucrose in the wold. The sugar cane juice have
high amount of flavanoids (Abbas et al., 2013). The stems and roots of sugar
cane are used various diseases like skin, urinary tract, jaundice cough,
anemia, bronchitis, constipation, heart, blood pressure decrease milk
production (Mira et al., 2011).
Sugarcane is an antidote, demulcent, antiseptic, and cardio-tonic,
diuretic, laxative, and intoxicant, bactericide, pectoral, antivenomous, and
stomachic. In traditional therapy sugarcane used in dysentery, arthritis, boils,

6
bedsores, (Wealth of India,1966) fever, eyes, inflammation, hiccups,
laryngitis, skin opacity, sores, sore throat, cancer, spleen, colds, tumors,
cough, diarrhoea, gonorrhea and wounds. (Duke 1981). In India, the districts
of Mysore, Mandya and Tumkur rural parts of Karnataka, the plant parts are
used for kidney stones and abdominal tumors.
2.1.1. Botanical description
Saccharum officinarum is a perennial plant belongs to the family
poaceae or graminae that grows in lumps contains number of unbranched
stems. The stems of Saccharum officinarum colour varies include green, pink
or purple. The stems are jointed nodes being present at the baseof the
alternative leaves. The height of the plants varies between 5 to 16 feet. The
leaves have thick midrib and sawtoothed edges that grows about 25 to 50 cm
and width is about 5cm. The terminal inflorescence is a panicle up to 60 cm
(24 ins) long, a pinkish plume that tapering upwards and broadest downwards.
The Saccharum officinarum fruits contains single seeded and dried one
(Amandeep Singh etal., 2015).
2.1.2. Origin and geographic distribution:
The plant Saccharum officinarum is widely distributed across tropical,
subtropical, and warm temperate regions in Asia, India Pakistan, Bangladesh
Africa, Europe, Australia, the America. Several species of Saccharum are
cultivated in china, Pakistan, North America and in Philippines (Flora of
Pakistan1754; Biota of North America 2013; Davidse 1994; Phillips 1995;
Welker and Longhi-Wagner 2012).
2.1.3. Chemical constituents:
Sugarcane comprises of 70 % water, 13% sucrose, and 10 % fiber.
Sugarcane juice shows the prescence of flavanoids, Phenolic compounds and
Phenolic acids caffeic acid, hydroxycinnamic acid, and sinapic acid, along

7
with flavones such as luteolin, apigenin, and tricin.. Further, recently four new
flavones were isolated from Saccharum officinarum are swertisin. (Amandeep
Singh etal., 2015 ).
2.1.4. Uses:
Traditionally Saccharum officinarum is used as follows: (Duke 1981)
1. Antivenomous, 12.Laryngitis,
2. Refrigerant, and 13.Opacity,
3. Stomachic. 14.Skin problems,
4. Arthritis, 15.Sore throat,
5. Boils, 16.Spleen,
6. Bedsores, 17.Cancer,
7. Dysentery, 18.Colds,
8. Eye wash, 19.Cough,
9. Fever, 20.Diarrhoea,
10.Hiccups, 21.Tumors,
11.Inflammation,
22.And wounds.
23.Gonorrhea and vaginal discharges (duke 1981).

2.1.5. Other Species (Linnaeus and Carl von. 1753):

1. Saccharum arundinaceum Retz. Southeast Asia; New Guinea
2. Saccharum angustifolium (Nees) - South America
3. Saccharum alopecuroidum (L.). - Southeastern USA
4. Saccharum asperum (Nees) - South America
5. Saccharum coarctatum (Fern.) - southeastern USA
6. Saccharum beccarii (Stapf) - Sumatra
7. Saccharum bengalense Retz. - India, Pakistan, Iran, Afghanistan
8. Saccharum giganteum (Walt.)- southeastern USA
9. Saccharum baldwinii Spreng. - southeastern USA
10.Saccharum contortum (Baldwin ex Elliott). - southeastern USA

8
11.Saccharum griffithii Munro ex Aitch. - Yemen to Bangladesh
12.Saccharum fallax Balansa - Southeast Asia
13.Saccharum brevibarbe (Michx.) - Southeastern USA
14.Saccharum formosanum (Stapf)- Southern China
15.Saccharum narenga (Nees ex Steud.). - China, Indian Subcontinent.
16.Saccharum filifolium Steud. - Afghanistan, Himalayas
17.Saccharum kajkaiense (Melderis) - Oman, Iran, Afghanistan, Pakistan
18.Saccharum longesetosum (Andersson) ex Bor - China, Himalayas,
Indochina
19.Saccharum hildebrandtii (Hack.) - Madagascar
20.Saccharum kanashiroi (Ohwi) - Nansei-shoto
21.Saccharum spontaneum L. - Asia, Africa, Sicily, Papuasia
22.Saccharum perrieri (A.Camus) - Madagascar
23.Saccharum maximum (Brongn.) - Pacific Islands
24.Saccharum procerum. - China, Himalayas, Indochina
25.Saccharum ravennae (L.) L. - Europe, Asia, Africa
26.Saccharum officinarum L. - New Guinea; distributed in many warm
places
27.Saccharum rufipilum. - China, Indian Subcontinent, Indochina
28.Saccharum strictum (Host). - From Italy to Iran
29.Saccharum viguieri (A.Camus) - Madagascar
30.Saccharum stewartii - western Himalayas
31.Saccharum wardii (Bor)- Assam, Bhutan, Myanmar
32.Saccharum velutinum (Holttum) - Peninsular Malaysia
33.Saccharum robustum. - New Guinea
34.Saccharum villosum - South America, Mesoamerica
35.Saccharum sikkimense (Hook.f.). ex Bor - eastern Himalayas
9
 36.Saccharum williamsii (Bor) Nepal
2.1.6. Pharmacological activities:
2.1.6.1. Anti-inflammatory and Antioxidant activity:
Methanolic extract and ethanolic extracts of Saccharum officinarum
were evaluated for In-vitro and in-vivo anti-inflammatory activity and DPPH
averting activity, The Results reveal that they have potent antioxidant and
anti-inflammatory activity (Ghiware et al., 2012).
2.1.6.2. Antifertility activity:
Methanolic extract Saccharum officinarum Leaves were evaluated for
antifertility and antiestrogenic effects in female rats. The results from the
study indicates that methanolic extracts of S.officinarum treatment reduces the
weight of reproductive organ, circulating level of estrogen and reduces the
fertility, number of litters. (Balamurugan et al., 2009).
2.1.6.3. Antiulcer activity:
Ethanolic and methanolic leaf extracts of S.officinarum was studied for
antiulcer activity by using gastric ulcer models in rats. Ulcer induced by oral
administration of ethanol and by pylorusligated method. Total gastric acid
output was determined in the pylorus ligated rats. Histology of Gastric tissue
was examined. The extracts of Saccharum officinarum Linn leaves shows
significant protection. (Nitin ghiware et al., 2014).

2.1.6.4. Antihyperglycaemic, antihyperlipidaemic and antioxidant

potentials:
The extracts of Saccharum officinarum were evaluated for
antihyperglycaemic, antihyperlipidaemic and antioxidant potentials. The
Saccharum officinarum extracts showed significant antihyperglycaemic
10
activity by decreasing the total cholesterol, low density lipoproteins,
antihyperlipidaemic (glutathione, catalase) and antioxidant potentials,
superoxide dismutase (Oyesola et al., 2013).
2.1.6. 5. Antioxidant activity of phenolics compounds:
Juice of Saccharum officinarum was used to evaluate antioxidant
activity by HPLC analytical and photodiode array methods shows significant
antioxidant activity (Mauricio et al., 2006). Flowers of Saccharum
officinarum were screened for DPPH method gives % inhibition of Ethyl
acetate 15.063, Methanol 37.21, Aqueous 83.29% when compared to ascorbic
acid 88.22% (Bhore et al., 2012).

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3. AIM AND OBJECTIVE
3.1. Aim:
The folkloric observations revealed that the selected medicinal plant in the
tribal region of East Godavari mostly used to treat kidney disorders without
any scientific systematic evaluation. Hence the present investigations was
aimed to evaluate with a systematic scientific approach and evidence to add
knowledge to the existing literature.
3.2. Objective of research
3.2.1. To extract the Saccharum officinarum leaves.
3.2.2. Qualitative phytochemical screening of Saccharum officinarum leaf
extracts
3.2.3. To evaluate the pharmacological activities of Saccharum officinarum
leaf extracts: Diuretic, antiurolithiatic activities.

12
4. MATERIALS AND METHODS:

4.1. Phytochemical screening of leaf extracts

4.1.1. Plant material:

The fresh plants of Saccharum officinarum were collected near

Surampalem, East Godavari dist, Andhrapradesh. They were authenticated
and confirmed by Botanist Mr. T.V. Raghava Rao, Lecturer, Department of
Botany SRVBSJB Maharanee College, Peddapuram, East Godavari district,
Andhra Pradesh.

4.1.2. Preparation of extracts:

The Saccharum officinarum leaves were collected near Surampalem, East

Godavari district Andhrapradesh India. They were dried, powdered and
passed through 40mesh. The obtained powder weighed 100gram of
(Saccharum officinarum) was successively extracted with Chloroform (400,
250 and 150ml) followed by Ethanol (400,250, 150ml) in soxhlet apparatus
separately for 3 days at 500C.The filtrate was evaporated to obtained constant
weight of gummy exudates under reduced pressure at 45C. Chloroform
extract yields 8.12% and ethanol extract yields 9.5%. The obtained crude
extracts were then stored at 1015C.

4.1.3. Qualitative Phytochemical tests:

The extracts of Saccharum officinarum were used for various qualitative

phytochemical tests (Kokate, 1994).

13
4.1.3.1. Reagents used:

Alcoholic -napthol, ferric chloride,Ninhydrine, lead acetate solution, 1%

gelatin solution containing 10% sodium chloride, hydrochloric acid, zinc dust,
acetic anhydride, sulphuric acid, sodium hydroxide, bromine water, dilute
ammonia, Fehling's, Dragendroff's , Wagners, Hager's, Millons Mayer's test,
reagents.

4.1.3.2. Carbohydrates:

The prescence of carbohydrates were tested by using, Molischs and

Fehling's test (Kokate, 1994).

a. Molischs test: Treat the extract with few drops of alcoholic -napthol,
then add few drops of concentrated H2SO4 through sides of test tube;
purple to violet colour ring appears at the junction

b. Fehlings test: Mix equal volumes of Fehlings A and B solution, boil for
1 minute. Add 1ml of extract, heat on boiling water bath for 5-10 minutes.
Yellow, then brick red precipitation was observed (Kokate, 1994)

4.1.3.3. Tests for alkaloids:

The alkaloids have been tested by using Dragendroff's test, Mayer's test,
Wagners test and Hager's test

a. Dragendroffs test: Dragendroffs reagent gives reddish brown

precipitate

14
 b. Mayers test: Mayers reagent precipitates the Alkaloids and gives
cream colour precipitate .

c. Wagners test: Wagners reagent precipitates the Alkaloids and gives

reddish brown precipitate.

d. Hagers test: Hagers reagent precipitates alkaloids and gives yellow

precipitate.

4.1.3.4. Proteins and amino acids:

Millons-Biuret Test and Ninhydrin test were used to test proteins and
amino acids

a. Millons- Biuret test: Add about 2ml of Millons reagent to the extract,
white precipitate.

b. Ninhydrin test: Add Ninhydrine solution to the extract, boil violet

colour.

4.1.3.5. Tannins:

Tannins and phenolics were tested by using following methods.

a. Ferric chloride test: Add ferric chloride solution to the extract,

appears blue or green colour appears.

b. Lead acetate test: Add two drops of lead acetate to 3 ml of alcoholic

extract, it gives a white precipitate.

c. Gelatin test: Add 1% gelatin solution contain 10% sodium chloride to

the extract, Precipitate was formed.

15
 d. Bromine water test: Add few drops of bromine water to 3 ml of
alcoholic extract, which causes decolourisation of bromine water.

e. Potassium dichromate test: Add two drops of potassium dichromate

to 3 ml of alcoholic extract, which gives a red precipitate.

f. Acetic acid test: Add few drops of acetic acid to 3 ml of alcoholic

extract, which gives a red colour solution.

g. Dilute iodine solution test: Add few drops of dilute iodine solution to
3 ml of alcoholic extract, which gives a transient red colour.

4.1.3.6. Flavonoids:

Flavonoids were tested by means of Shinoda Test (Kokate, 1994)

a. Shinoda test: Add some magnesium turnings to the extract and

concentrated hydrochloric acid drop wise obsereve the change of colour
from pink scarlet, crimson red or occasionally green to blue after few
minutes.

b. Zinc hydrochloride test: Add a mixture of zinc dust and concentrated

hydrochloric acid to extract. Observe red colour after a few minutes.

c. Alkaline reagent test: Addition of sodium hydroxide solution to the

extract, shows yellow colour, to colourless on addition of dilute acid

4.1.3.7. Steroids:

The steroids were tested by using Salkowski and Libermann Burchard test.

a. Salkowski test: Add few drops of concentrated sulphuric acid to the

extract gives red colour at lower layer.

16
 b. Libermann burchard test: Add few drops of acetic anhydrid to the
extract boil the solution and cooled and add concentrated sulphuric acid
through the wall of the test tube, observe brown ring at the junction of
two layers and upper layers turns green

4.1.3.8. Saponins:

a. Foam test: dilute the extract 20 times with distilled water then shaken
in a graduated cylinder for 15 minutes. Observe the 1cm foam

4.1.3.9. Glycosides:

Glycosides were tested by using Borntrager's test [Kokate, 1994]

a. Borntrager's test: Add 1ml sulphuric acid to the extract with in a test tube,
boil for five minutes. Filter it on hot. Cool the filtrate and shake with 2ml of
chloroform. Separate the chloroform layer and shake it with 1ml of dilute
ammonia. Ammoniacal layer shows A rose pink to red.

17
4.2. Diuretic Activity:

4.2.1. Chemicals used:

Chemicals Manufacture

Furosemide Sri Krishna Drugs

and Pharmaceutical
Ltd.HYD

Cystone Himalaya drug house,

Makali, Bengaluru.

Sodium Chloride, Potassium Chloride, Silver

Nitrate, Methyl Orange, Sodium Phosphate, SD-FINE
Potassium Permanganate, Oxalic Acid, CHEMICALS LTD
Ethanol, Methanol.
4.2.2.Equipments used:

Fig: 4.2.1. Metabolic cages

18
 Fig: 4.2.2. ELICO CL-354 Flame Photometry

4.2.3. Experimental Animals:

Fourty two healthy adult male albino rats weighs approximately 180 g were
procured from the Animal House used for Diuretic activity. The experimental
protocols and animal care were followed according to CPCSEA guidelines.
4.2.4. Housing:
Rats were kept in a clean cages six in a group in each cage. The bedding
material is replaced every third day with fresh material to keep the animals
clean and dry. Drinking tubes were examined routinely to ensure their proper
function.
4.2.5. Experimental design:
Diuretic activity of Saccharum officinarum determined by using
Lipschitz method (Lipschitz et al., 1943). The pellets provide to rats were
stopped before 18hrs and stop water prior to the experiment.The rats were
grouped in to six groups [3rats in each]. An initial dose of 25ml/kg of Normal
saline was given to all rats.

19
Group I: (Control) group receives only vehicle, 0.5% acacia orally.
Group II: (Standard) group receives Furosemide (5mg/kg p.o) dissolved in
vehicle.
Group III & IV: (Test-1) group receives Chloroform extract of Saccharum
officinarum (200 mg/kg and 400mg/kg p.o respectively).
Group V & VI: (Test-2) Treated group receives Ethanolic extracts of
Saccharum officinarum (200 mg/kg and 400mg/kg p.o respectively).
After receiving the respectd dose of the drug the animals were
immediately kept in metabolic.(Vogel and vogel 1997). The volume of urine
was collected and measured after 5hrs. The urine was used to estimate the
respective ions by using Flame photometry -sodium, potassium ions (Jeffery,
1989) and by titrimetric analysis- chloride, bicarbonate were determined
(Beckette & Stenlake, 1997) after 24 hrs.
4.3. Antiurolithiatic activity:
4.3.1. Requirements:
Citric acid, Sodium hydroxide, Potassium chloride, Ascorbic acid,
Sodium chloride, Urea, Sodium bicarbonate, Potassium phosphate,
Creatinine, Sulfuric acid and Deionized water.
4.3.2. Chemicals used:
Ethylene glycol was received from Mumbai, S D fine chemicals laboratories,.
CYSTONE tablets from Bengaluru,(The Himalaya Drug Company), used as
standard antiurolithiatic drug.
4.3.3. Equipment used:
ELICO SL210 Double Beam UV spectrophotometer
Shimadzu Electronic balance
Metabolic cages

20
Figure 4.3.4: Biochemistry Analyser

4.3.4. In-vitro Antiurolithiatic activity:

4.3.4.1. Experimental Protocol:
The In-vitro Antiurolithiatic activity of Saccharum officinarum extracts were
determined by calcium oxalate crystallization method. Which measures
turbidity changes due to the crystallization on addition of sodium oxalate
solution to artificial urine. The calcium oxalate Precipitates at 37C and pH
6.8 has been read at 620 nm (Burns and Finlayson 1980).

4.3.4.2. Synthetic urine Preparation :

Ingradients Quantity
Potassium chloride, 3.8gm
Sodium chloride 8.5gm
Urea 24.5gm
Citric acid 1.03gm
Ascorbic acid 0.34gm
Potassium phosphate 1.18gm
Creatinine 1.4gm
Sodium hydroxide 0.64gm
Sodium bicarbonate 0.47gm
Sulfuric acid 0.28ml

21
 For preparation of synthetic urine add above ingradients were added in
500ml of deionized water and stirred for 1hour and the synthetic urine was
stored at -40c til further use (Bensatal and Ouahrani, 2008).

4.3.4.3. Study without inhibitor:

1ml of artificial urine was taken in the cuveette and add 0.5ml distilled
water to it and take the blank reading. The 0.5ml of sodium oxalate was
added, to the 1ml of artificial urine and measured the readings for a period of
ten minutes.
4.3.4.4. Study with inhibitor:
The extracts were dissolved in distilled water filtered through
membrane filter and the concentration of 100, 300 and 500g/ml were
obtained. A mixture of 1ml of artificial urine and 0.5ml of extracts solution
were taken in the cell. The blank reading was taken and then 0.5ml of sodium
oxalate solution was added to the artificial urine and record the absorbance
for a period of the 10 minutes with 2 min interval at 620nm (Bensatal and
Ouahrani 2008).
The % of inhibition was calculated as:

% inhibition 100

22
4.3.5. In-vivo Antiurolithiatic activity:
4.3.5.1. Experimental animals:
The animals were selected in such a way that they were free from illness,
injury, disease. Only those animals which are healthy having weights 150-200
g were selected and maintained at standard laboratory conditions.
4.3.5.2. Preparation and administration of doses:
2% tragacanth was used to prepare doses in 1 ml/100g of body weight. The
extracts were given by gastric intubation tube in a single dose after fasting for
3 to 4 h.
4.3.5.3. Observations:
After giving drug carefully observe animals up to first 24 h. Additional
ANS and CNS are also required along with skin colouration, convulsions, eye
movements, tremors etc.,.
4.3.5.4. Collection and analysis of urine:
One day before starting the experiment and after 28 days treatment, the
urine samples were Animals were kept in metabolic cages 24 hours urine
collected using metabolic cages and estimated for the prescence stone
forming constituents using biochemical analyser.
4.3.5.5. Experimental Procedure: (Babita and Vishnudev 2013)
Ethylene glycol-induced hyperoxaluria method was used to study
the antiurolithiatic activity in rats. Ethylene glycol (0.75%v/v) was given
in drinking water to all groups for producing kidney stones for 28 days
and for the group IV to VII extract treatment was started from 15 th day to
28th day. The details were given in below table 4.3.5.1.

23
 Table 4.3.5.1. Experimental design of Antiurolithiatic activity:
Group Dose
Group I Control group received normal drinking water
Induction of renal calculi as Positive control received only ethylene
Group II glycol (0.75%v/v) dissolved in drinking water for 28 days.
Received ethylene glycol 1st to 28th day and standard antiurolithiatic
Group III
drug Cystone (750mg/kg ) from 15thday to 28th day.
Received ethylene glycol 1st to 28th day and Saccharum officinarum
Group IV
Chloroform Extract (200mg/kg) from 15th day to 28th day.
Received ethylene glycol 1st to 28th day and Saccharum officinarum
Group V
Chloroform Extract (400mg/kg) from 15th day to 28th day.
Received ethylene glycol 1st to 28th day and Saccharum officinarum
Group VI
Ethanolic Extract (200mg/kg) from 15th day to 28th day.
Received ethylene glycol 1st to 28th day and Saccharum officinarum
Group VII
Ethanolic Extract (400mg/kg) from 15th day to 28th day.
4.3.5.6. Estimation of Parameters:
The following parameters were analysed by using biochemical analyser and
diagnostic kits:
a. Calcium(Miller, 1994)
b. Oxalate (Hodgkinson and Williams 1972)
c. Phosphorus (Daly, 1972) and
d. Creatinine (Thomas,1998)

24
5. RESULTS:
5.1. Phytochemical screening on leaf extract of Saccharum officinarum
5.1.1. Qualitative Determination:
The qualitative Phytochemical Screening of Saccharum officinarum
leaf extracts shows the prescence of phytoconstituents like carbohydrates,
glycosides, Tannins, Saponins, Flavonoids and are given below. (Table-
5.1.1.)

Table: 5.1.1. Qualitative determination of Saccharum officinarum leaf

extracts

S. No. Phytoconstituents Ethanolic Extract Chloroform

1 Carbohydrades + +
2 Glycosides + +
3 Tannins + +
4 Phytosterols - -
5 Saponins + -
6 Flavonoids + +
7 Alkaloids - -
8 Proteins - -
+ = Presence, - = Absence

5.1.2. Quantitative Determination:

The crude extracts of Saccharum officinarum was subjected to Quantitative
Determination Phytochemical Screening for the detection of total Phenolics,
total flavanoids and total alkaloids. The total phenolic contents of Saccharum
officinarum in terms of mg Gallic acid equivalent (GAE) per 100mg dried
extract, The flavonoid concentration of Saccharum officinarum in terms of
mg Quercetin equivalent (QE) per 100mg dried extract and total alkaloids

25
Saccharum officinarum in terms of mg Atropine equivalent(AE) per 100mg
dried extract were given in (Table 5.1.2.)

Table No. 5.1.2. Quantitative Determination of Saccharum officinarum

leaf extracts
S. Name of the Total Total Total
No extract Phenolics flavanoids alkaloids
GAE/100mg QE/100mg AE/100mg
1 Ethanol extract 12.060.13 4.320.43 5.060.42
2 Chloroform 7.120.72 3.040.24 8.180.56
extract

5.2. Diuretic activity:

The ethanolic extract of Saccharum officinarum at dose 200, 400
mg/kg body produced diuresis and volume of urine are 1.1, 1.55ml after 5hrs.
The excretion of sodium by both doses of extract was found to be 109 and
157 moles/kg. Similarly the excretion of potassium, chlorides and
bicarbonates were markedly increased in extract treated groups. The volume
output and the electrolytes excretion with the standard drug Furosemide
(group II) was found to be excellent. However the ethanolic extract of
Saccharum officinarum both low and high doses produced significant diuretic
effect compared to control were tabulated in table 5.2.1. Natriuretic Saluretic,
& Diuretic indices were were tabulated in table 5.2.2.

Table No. 5.2.1. Diuretic effect of Saccharum officinarum leaf extract

26
 Urine
S. Volume Sodium Potassium Chloride Bicarbonate
Groups
No. (mL/Hr) moles/Kg moles/Kg moles/Kg moles/Kg
after 5hrs
1 Vehicle (Control) 0.6 0.05 96 1.73 92 1.15 60.5 9 37.5 0.28
2 Furosemide 5mg/kg 1.6 0.05** 167 0.57** 188 1.15** 152 0.66** 74 2.51**
(standard)
3 S. officinarum 200mg/kg 1.1 0.15** 109 0.57** 108 0.57** 65 0.86* 42.5 0.76
ethanol extract
4 S. officinarum ethanol extract 1.55 0.02** 157 1.15** 180 1.52** 150 1.15** 62 **
(400mg/kg)
5 S. officinarum chloroform 0.210.26 162.140.26** 1180.22** 960.26** 090.36
extract (200mg/kg)
6 S. officinarum chloroform 0.330.44 172.140.57** 1280.54** 1010.31** 110.71
extract (400mg/kg)
Mean Values are expressed in SEM; n=3 (animals in each group); *p<0.05,
**p<0.01.

27
 Fig. 5.2.1. Diuretic effect of Saccharum officinarum leaf extract

Table No.5.2.2. Saluretic, Natriuretic & Diuretic indices of Saccharum officinarum

leaf extract
Sodium + Sodium/ Diuretic
S. No. Group
Chloride Potassium Index
1 Group-I (Control) 156.5 0.24 1.04 0.01 -
2 Group-II (standard) 384 0.07** 0.88 0.02** 2.67
Group-III ethanol (S.officinarum- 174 0.15** 1.03 0.12 1.83
3
200mg/kg)
Group-IV ethanol (S.officinarum- 307 0.27** 0.87 0.04** 2.58
4
400mg/kg)
5 Group-V chloroform(S.officinarum- 2580.16 1.370.11 0.330.24

28
 200mg/kg)
Group-VI chloroform (S.officinarum- 2680.28** 1.450.36 1.830.22
6
400mg/kg)
MeanValues are expressed as SEM; n=3 (animals in each group);
**p<0.01.
Diuretic Index = {volume of urine in test group/volume of urine in
control}
Natriuretic Index = Sodium/ Potassium Saluretic index= Sodium +
Chloride

Fig. 5.1.2. Saluretic, Natriuretic & Diuretic indices of Saccharum officinarum leaf
extract

5.3. Anti-Urolithiatic activity of Saccharum officinarum

5.3.1. In-vitro Anti-Urolithiatic activity:
Saccharum officinarum of leaf extract at 100, 200 and 400 mg/ml was
subjected to invitro anti-urolithiatic activity and the leaf extracts have shown

29
 dose and time dependent % of inhibition and the results are given in table
5.2.1.

Table 5.3.1. In-vitro Anti-Urolithiatic activity of Saccharum officinarum

extracts

Test Concentration % inhibition at time in seconds

S.No.
0sec 120sec 240 sec 360 sec 480 sec 600 sec
1 S.officinarum Ethanol extract 37.02 0 40.31 0 45.52 48.9 0 52.14 55.24
100 g/ml .34 .44** 0.34** .24** 0.13** 0.16**
2 S.officinarum Ethanol extract 55.34 0 60.46 0 63.43 65.69 67.85 69.93
200 g/ml .14 .12** 0.18** 0.34** 0.56** 0.15**
3 S.officinarum Ethanol extract 60.42 0 63.56 0 66.41 68.61 70.71 72.72
400 g/ml .35 .26** 0.57** 0.14** 0.44** 0.16**
4 S.officinarum chloroform 22.510. 24.510. 27.68 29.74 31.70 33.59
extract in 100g\ml 451 49** 0.554 0.595 0.63** 0.63**
5 S.officinarum chloroform 27.020. 29.420. 33.22 35.69 38.05 40.31
extract in 200g\ml 348 42**4 0.515 0.467 0.55** 0.61**
6 S.officinarum chloroform 38.280. 41.720. 46.55 49.39 53.31 56.85
extract in 400g\ml 18 48** 0.18 0.18 0.18** 0.18**
Mean Values are expressed as SEM; n=3 (number in each group);
**p<0.01.

30
Fig. 5.3.1. In-vitro Anti-Urolithiatic activity of Saccharum officinarum
extracts

5.3.2. In-vivo Anti-Urolithiatic activity:

The Saccharum officinarum leaf extract exhibit marked decrease in the
levels of oxalate, phosphate and calcium in urine. But the levels of uric acid,
calcium and creatinine in serum increased. The oxalate, phosphate and
calcium levels in calculi induced rats were found to be15, 39 and 7.6mg/ml.
The rats treated with Saccharum officinarum with a dose 200, 400 mg/kg
decreased level of oxalate, phosphate and calcium to a greater extent. All
these results are significant and comparable to standard drug Cystone.
Similarly Saccharum officinarum leaf extract markedly decreased the levels
of calcium and creatinine levels in serum. The maximum uric acid, calcium
and creatinine levels were found to be calculi induced rats (group II) 7.6, 38
and 16mg/dl.
31
 The ethanolic extract of Saccharum officinarum in the dose 200, 400
mg/kg body wt has markedly decreases the creatinine and uric acid levels.
Both the biochemical parameters were decreased significantly by the extract.
All these results have shown positive anti Urolithiatic activity as they are
comparable to standard drug Cystone (group III) in table 5.3.2.
Table 5.3.2. In-vivo Anti-Urolithiatic activity of Saccharum officinarum
leaf extract
S.N Urine(mg/ml) Serum(mg/dl)
Group Calcium Phosphate Oxalate Calcium Creatinine Uric acid
o.
1 Group I (Control) 3.5 1.15 18 2.08 4.2 1.04 8 1.52 0.6 0.14 3.2 1.04
2 Group II (Calculi Induced) 15 0.14 39 0.14 7.6 2.08 16 2.08 38 1.52 7.6 1.15
3 Group III 3.3 1.04 15 0.14* 3.1 2.08* 3.3 1.15 0.8 0.14 3.1 0.14
Cystone-750mg/kg ** * ** ** *
4 Group-IV S. officinarum 3.4 2.08 30 1.15* 6.6 2.08 11.2 1.1 0.6 2.08 6.6 1.52
ethanol (200mg/kg) ** * 5** **
5 Group-V S. officinarum 7 1.15** 25.5 0.14 3.6 1.04* 11.2 2.0 0.8 0.14 3.7 1.15
ethanol (400mg/kg) ** 8** ** *
6 Group-VI S. officinarum 6.880.0 30.830.2 4.570.15 12.700. 16.800. 3.930.20
chloroform extract 200mg/kg 94 89 3 520 354 8
7 Group-VII S. officinarum 4.400.1 23.630.4 2.990.09 8.310.2 10.500. 2.570.08
chloroform extract 400 mg/kg 00 46 8 72 500 4
Mean Values are expressed as SEM; n=3 (animals in each group);
**p<0.01; *p<0.05.

32
Fig. 5.3.2. In-vivo Anti-Urolithiatic activity of Saccharum officinarum leaf
extract

33
6. DISCUSSION:
6.1. Diuretic activity
In diuretic activity, the ethanolic extract of Saccharum officinarum at
200,400 mg/kg body weight dose produced diuresis and the volume of urine
are 1.1, 1.55 ml after 5hrs. The excretion of sodium by both doses of extract
was found to be 109 and 157 moles/kg. Similarly the excretion of potassium,
chlorides and bicarbonates were markedly increased in extract treated groups.
The volume output and the electrolytes excretion with the standard drug
Furosemide (group II) was found to be excellent. However the ethanolic
extract of Saccharum officinarum both low and high doses produced
significant diuretic effect when compared with that of control group.
Saluretic, Natriuretic & Diuretic indexes were calculated.
The diuretic potential of the extract can be evaluated based on volume of
urine output, concentration of electrolytes in the urine such as sodium,
potassium, chloride & bicarbonate were considered. From the experimental
results, the diuretic activity shows increase urine excretion (after 5hrs) and
increase elimination of electrolytes potassium, sodium, bicarbonate and
chloride in urine were given in Table 5.2.2.1. It was observed that ethanolic
leaf extracts showed significant diuretic effect compared to that of control.
The present study indicates that Ethanolic leaf extracts of Saccharum
officinarum showed dose dependant diuretic activity.

34
 To be a diuretic, the diuretic index of the drug must be greater than 1.The
extracts and the standard at the administered dose possessed diuretic index
greater than 1. The diuretic activity of these extracts may be due to
vasodilation, rise in blood flow or inhibits the tubular absorption leads to rise
in urine volume (Stanic & Samarzija, 1993). These mechanisms related to its
diuretic effect of the extracts. The potassium, sodium, and bicarbonate and
chloride excretion by the extracts is seen in diuresis.
The Phytochemical studies have reported that the ethanolic extract contains
alkaloids, carbohydrates saponins, flavonoids, tannins. Flavanoids are act by
inhibiting Adenosine A1 Receptor and produces diuresis.(Yuliana et al.,
2009). The diuretic activity of extract may be through any of these possible
mechanisms since it is rich in alkaloids and flavanoids. The presence of
saponins is responsible for saluretic activity by altering the sodium excretion
in kidney (Haruna et al., 2002; Abdala et al., 2012). Qualitative
phytochemical tests of the extracts Saccharum officinarum and revealed the
presence of flavonoids and steroids. These substances might be responsible
for the observed diuretic activity and these phytococonstituents may act
individually or synergistically to produce diuresis. Earlier studies had reported
that many constituents are involved in diuretic effect of plants. (Maghrani et
al., 2005).
6.2. Antiurolithiatic Activity:
In-vitro Anti-Urolithiatic activity, the results of In-vitro Anti-
Urolithiatic activity of chloroform and ethanolic leaf extracts of Saccharum
officinarum exhibits dose and time dependent % inhibition. The maximum
inhibition with chloroform (500 g\ml) was observed at 66.71%. But when
compared with chloroform extracts the ethanolic extract showed maximum
inhibition at 67.16 %.
35
 In In-vitro Anti-Urolithiatic activity, Saccharum officinarum of leaf
extract at a concentration of 100, 200 and 400 mg/ml was subjected to in-
vitro anti-urolithiatic activity and the leaf extracts have shown dose and time
dependent % of inhibition 55.24, 69.93 and 72.72 at 600seconds (10 mins).
In In-vivo Anti-Urolithiatic activity, the ethanolic extract of
Saccharum officinarum exhibit marked decrease in the levels of oxalate,
phosphate and calcium in urine. But the levels of calcium, uric acid and
creatinine in serum increased. The oxalate, phosphate and calcium levels in
calculi induced rats were found to be 15, 39 and 7.6 mg/ml. The rats treated
with Saccharum officinarum with a dose of 200, 400 mg/kg decreased level of
oxalate, phosphate and calcium to a greater extent. All these results are
significant and comparable to standard drug Cystone750mg/kg. Similarly
Saccharum officinarum leaf extract markedly decreased the levels of calcium
and creatinine levels in serum. The maximum calcium, uric acid and
creatinine levels were found in calculi induced rats (group II) 16, 38 and 7.6
mg/dl. The ethanolic extract of Saccharum officinarum in the dose 200, 400
mg/kg body wt has markedly decreases the creatinine and uric acid levels.
Both the biochemical parameters were decreased significantly by the extract.
All these results have shown positive anti urolithiatic activity as they are
comparable to standard drug cystone.
Kidney is main target for Ethylene glycol induced toxicity. Ethylene
glycol is metabolized in liver by alcohol dehydrogenase to glycolaldehyde,
and is oxidized to glycolic acid (Mitra et al., 1998). The glycolic acid is
converted to glyoxylic acid and oxalic acid thus promoting hyperoxaluria. An
increase urinary calcium excretion, and decreases serum calcium levels leads
to urolithiatic rats (Karadi et al., 2006). An increased concentration of urinary
calcium leads to precipitation of Calcium oxalate or phosphate and formation
36
of crystal growth (Suresh Kumar et al., 2009). Not only). Ethanolic leaf
extract treatment decreased level of oxalate, phosphate and calcium excretion
compared to chloroform extract.
In this work male rats were used because they are resembled to human
beings but in female rats stone formation is rare. Now a-days researchers
shows wide interest towards herbal drugs for treating urolithiasis but they
required scientific and systematic evaluation Claim many promising remedies
in urolithiasis. (Vermeulen, 1962).
Ethanolic leaf extracts of Saccharum officinarum lowered the oxalate,
phosphate and calcium levels in urine (Group VI &VII). The level of serum
calcium, uric acid and creatinine were found to increase in calculi-induced
animal (Group II). Treatment with the ethanolic leaf extracts of Saccharum
officinarum decrease the levels of calcium, uric acid and creatinine.
The Phytochemical screening of Saccharum officinarum leaf extracts
revealed the presence of tannins and flavanoids (Palaksha et al., 2016). These
constituents may be responsible for the diuretic property of the plant
Ethanolic leaf extract of Saccharum officinarum, which favours the
antiurolithiasis by hastening the process of dissolving or by flushing of the
preformed stones. The possible mode of action of Ethanolic leaf extract of
Saccharum officinarum may be due to excessive secretion or decrease in the
urinary concentration of the urinary salts that prevent super saturation of the
crystallizing salts, based on In-vitro antiurolithiatic activity results.

37
7. CONCLUSION:
The diuretic activity of Saccharum officinarum extract might be due to
the flavanoids bound to Adenosine A1 Receptor. The presence of Saponins
acts by modulating renal sodium excretion and produces saluretic activity.
Treatment with the ethanolic leaf extract of Saccharum officinarum decreased
level of uric acid, calcium and creatinine serum.

38
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