1 International Journal of Toxicology and Applied Pharmacology 2011, 1 (1) 1-5

Original Article

Evaluation of Coleus barbatus and Trigonella foenum graecum extracts and

their combination on kidney stones in rats

Javed Khan Pathan* 1

, Neelam Khan2, H.M. Sohel

1

1. Swami Vivekanand College of Pharmacy, Indore

2. Indore Institute of Pharmacy, Indore

Received 21 march 2011; accepted 28 March 2011

Abstract

In the present study the effect of calcium oxalate urolithiasis urinary risk factor of extract of Coleus barbatus and Trigonella

foenum graecum Roots and their combination have been studied in albino rats. By this study it is deduced that the possible

effect of the extract of Coleus barbatus and Trigonella foenum graecum Roots and their combination have positive effect on

the main urolithiasis risk factors. © 2011 Universal Research Publications. All rights reserved

Key words: Urolithiasis, Ethylene glycol (EG), Coleus barbatus and Trigonella foenum graecum

1. Introduction Kidney stones, one of the most painful of the

urologic disorders. Urolithiasis is the medical term used to

describe stones occurring in the urinary tract. (Robbins et al

2005). Calcium containing stones, especially calcium oxalate

monohydrate (whewellite),calcium oxalate dehydrate

(weddellite) and basic calcium phosphate (apatite)are most

commonly occurring ones to an extant of 75-90% followed by magnesium ammonium phosphate (struvite) to an extant

of 10-15%,uric acid 3-10% and cystine 0.5-1%.( Dennis L.

Kasper.,2002)The vast Ayurvedic literature claims a number

of plants to be useful in urinary stones; still many plants need

to exploited for their pharmacological actions.Inspite of

intensive research to establish the mechanism of stone

formation, dietary management, evaluation of medicinal

plants and other agents in the treatment of urinary stones.

(Prasad, K.V.S.R.G., 2007)

2. Material and method

2.1. Plant material The plant materials, roots of Coleus barbatus

(Laminaceae) and Trigonella foenum graecum (Fabaceae)

were purchased from the market. The plant materials were

identified by Dr.S.N.Mishra, Sr.scientist, Horticulture

College, and Mandsaur.

2.2. Extraction of drugs The dried roots of Coleus barbatus and Trigonella

foenum graecum were washed thoroughly to clean soil

matter, before crushing in size reduction mill and reduced to

moderately coarse powder. The size reduced drug was filled

in soxhlet apparatus for solvent extraction. First the powdered

drug was defatted with petroleum ether. Defatted drug was

then dried and again filled in soxhlet apparatus for solvent extraction with solvent ethanol. Solvent was removed under

reduced pressure at low temperature. Defatted drug was then

dried and again filled in soxhlet apparatus for solvent

extraction with solvent ethanol. (Pulok, M., 2002)

2.3. Rat treatment and urine sample collection

Six groups and each group having five albino rats of

either sex weighing 120-180 gm. were selected. The animal

were housed in cages under standard laboratory condition(

in a temperature and humidity controlled room 25± 1o C ,

50% relative humidity with a 12 hr on-off life cycle) for a period of 14 days prior to the experiment. The animal

received chow and tap water ad libitum. Group I was used as

normal control group. Group II which was given ethylene

glycol (EG) 0.75% only was used as untreated nephrolithiasis

rats group. Group III was given EG 0.75% and 500mg/kg

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International Journal of Toxicology and Applied Pharmacology

Universal Research Publications. All rights reserved

2 International Journal of Toxicology and Applied Pharmacology 2011, 1 (1) 1-5

body weight ethanolic extract of Coleus barbatus. Group IV

was given EG 0.75% and ethanolic extract of Trigonella

foenum graecum 200mg/kg body weight. Group V was given

EG 0.75% and combination of both extract. Group VI was

given EG 0.75% and standard drug (cystine) 500mg/kg body weight. EG was added in their drinking water to induce a

chronic low grade hyperoxalluria and generate CaOx

deposition into kidneys. Twenty four hour urine sample from

each rat were collected after four weak in presence of sodium

azide as antibacterial agent during four weeks to analyze

some urinary components, to measure urinary volume and

pH. At the end of the experimental study all animal were

sacrificed after ether anesthesia. Kidneys harvested from

animals were analyzed histologically after they were fixed in

formalin and embedded in paraffin. (Hadizadeh, M 2007,

Atmani, F., 2004, Khan, S.R., 1985)

2.4. Analytical procedure

Urinary oxalate was estimated according to the method described by Hodgkinson et al (Hodgkinson

1970). 1 ml of urine was acidified by concentrated HNO3 to

solubelize crystals and then adjusted to pH 7 by NaOH in the

presence of color indicator, the bromothymol blue. About 2

ml of saturated CaSO4 and 14 ml of pure ethanol were added

to precipitate oxalate overnight. The sample were centrifuged

at 450 X g for 10 min and then filtered on filter paper. The

precipitate obtained was solubilized in 10 ml of water

acidified by 2 ml concentrated sulfuric acid. The samples

were titrated by a solution of KMnO4.

Table 1: Biochemical parameter of Urine

Sl.

no. Group Urine parameter (mg/ dl)

Oxalate Calcium Creatinine

1. Normal control 3.97± 0.05 2.79±0.14 4.50± 0.07

2. Model control 11.82± 0.06 7.93± 0.24 6.90± 0.14

3. Coleus barbatus (500mg/kg) 3.54±0.03** 3.81±0.10** 4.19±

0.04**

4. Trigonella foenum graecum

(200mg/kg) 3.68± 0.03** 3.47±0.09*

4.41±

0.06**

5. Combination 3.00± 0.23*** 3.04±0.02** 4.01±

0.02**

6. Standard (Cystone tablet

500mg/kg) 2.90± 0.04 *** 2.58±0.16*** 4.00±.04***

P<0.0001, the treated groups are compared with groups I and II. Values are expressed in Mean±SEM, Statistics: one way ANOVA followed

by Dunnet’s test, *** highly significant, ** significant and * less significant

Table 2: Total urinary volume

Sl.No. Groups

Total urinary volume (ml)

Mean± SE

1. Normal control

2.38 ±. 086

2. Model control

1.66 ± 0.13

3. Coleus barbatus extract (500 mg/kg)

4.80 ± 0.07**

4.

Trigonella foenum graecum extract

(200mg/kg)

4.50 ± 0.10**

5. Combination

5.00 ± 0.05 ***

6. Standard drug (500 mg/kg) 5.30 ± 0.09 ***

P<0.001, the treated groups are compared with groups I and II. Values are expressed in Mean±SEM, Statistics: one way ANOVA followed

by Dunnet’s test, *** highly significant, ** significant and * less significant

3 International Journal of Toxicology and Applied Pharmacology 2011, 1 (1) 1-5

Calcium analysis was performed by using a Merck

thermo spectronic u.v.1 beam spectrophotometer equipped

with a Varian hollow cathode and a deuterium background

corrector. Creatinine was estimated based on principle, the production of an orange colour by the interaction of

Creatinine with alkaline sodium pirate. The colour produced

was compared in a colorimeter, and the Creatinine content of

the urine estimated by comparison with a Creatinine solution

of known concentration. (Atmani, F., 2004, Barbas, C.,

2002)

Table 3: Determination of urinary pH

Sl.No. Groups

pH of urine

1. Normal control 7.21 ± 0.08

2. Model control 9.83 ± 0.24

3. Coleus barbatus extract (500 mg/kg) 7.55 ± 0.12**

4.

Trigonella foenum graecum extract

(200 mg/kg)

7.87 ± 0.14*

5. Combination 7.28 ± 0.11***

6. Standard drug (500 mg/kg) 7.36 ± 0.10**

P<0.001, the treated groups are compared with groups I and II. Values are

expressed in Mean±SEM, Statistics: one way ANOVA followed by Dunnet’s

test, *** highly significant, ** significant and * less significant

2.5. Data analysis

Standard evaluation was done using one-way

analysis of variance (ANOVA) Statistical significance was

set at P< 0.0001. Results are presented as mean ± standard errors (S .E.). (Hadizadeh, M 2007)

3. Results

The changes in the urine parameters in the

experiment animals during the study are presented (Table 1,

2, 3).The urine concentration of oxalate; calcium and

Creatinine was increased significantly in animals

administered with 0.75 percentage ethylene glycol. Four

weeks treatment with ethanolic extract of Coleus barbatus

significantly decreased urine concentration of oxalate (3.13

±0.03), calcium (2.81±0.10) and Creatinine (4.16 ± 0.04) as

compared to model control (oxalate – 11.32 ± 0.07, calcium – 7.82± 0.24, Creatinine – 6.97 ± 0.14) (Table 1 ).The

concentration of oxalate (3.47± 0.03), calcium (3.28±0.09)

and Creatinine (4.42 ± 0.06) of group IV treated with

ethanolic extract of Trigonella foenum graecum were found

to be significant as compared to model control (Group II ).

The concentration of oxalate (3.03 ± 0.23), calcium

(3.03±0.02) and Creatinine (4.09 ± 0.02) of group V treated

with combination of ethanolic extract Coleus barbatus &

Trigonella foenum graecum, were found to be more

significant as compared to group II,group II,group IV. (Table

1). Moreover the group treated with combination of the two

drugs (500 mg Coleus barbatus + 200 mg Trigonella foenum

graecum) was found to be most significant from the entire

group V. The percentage reductions of all parameters of urine

were found more in group V and in group VI (standard). Urinary volume significantly decreased in the animals treated

with the 0.75 % of ethylene glycol. Urinary volume were

increased by 221(Group III), 208 (GroupIV), 240(Group V),

261(group VI) percentage compared to model control group.

(Table 2) Urinary pH significantly increased in the animals

treated with the 0.75 % of ethylene glycol. Urinary pH were

decreased by 23 (Group III), 20 (GroupIV), 27(Group V), 25

(group VI) percentage with compared to model control group

(Table 3).

In histopathological observations gross examination

of rats kidney from control group showed a normal cortical

structure of the kidney including glomeruli and distended tubules, sclerotic glomeruli, and increased cellularity between

tubules. In ethylene glycol treated group (model control

group) showed cortical structure of the kidney including

glomeruli and affected distended tubules, sclerotic glomeruli,

and cellularity between the tubules. Group III, (500 mg

ethanolic extract of Coleus barbatus) showed the recovered

crypts or small sacs. Group IV, (200 mg/kg ethanolic extract

of Trigonella foenum graecum) widespread focal interstitial

lymphoplasmacytic infiltration with mild tubulitis, normal

glomeruli and arteriolar vessels. There is sign of sclerotic

glomeruli and increased cellularity between tubules. Group V (combination of both extract.) Shows minor interstitial

lymphoplasmacytic infiltration in glomeruli with occasional

penetration of tubular epithelium. In addition, increased

cellularity between tubules is clearly visible. Group VI

Standard (Cystine500 mg/kg) recovered distended tubules,

sclerotic glomeruli, and increased cellularity between tubules.

From the above results it was noted that the combination

(ethanolic extracts of Coleus barbatus & Trigonella foenum

graecum) was most significant.

4. Discussion Urinary stone disease is mainly the result of super

saturation of urine with certain urinary salts such as CaOx,

the most common constituent of kidney stones. (Daudon et

al 1993) Urorolithiasis is caused by several biochemical

mechanisms. It is a generalized increase in the calcium

content of the kidneys. (Grases, F., 1995) The major causes

include those associated with an increase in the urinary levels

of calcium crystals precipitation. Kidney stones usually arise

because of an imbalance between the kidney’s need to

conserve fluid and the need to extrude waste products of low

solubility. This imbalance often is precipitated by alternations

in diet, fluid intake, climate and extent of physical activity. The majority of patients with calcium containing stones

excretes excessive amounts of urinary calcium and often has

urine that is supersaturated solution of calcium and oxalate

salts ( Sarkisian M.R 2001) Many in vivo models have been

developed to understand the mechanisms involved in the

formation of urinary stones and to ascertain the effects of

4 International Journal of Toxicology and Applied Pharmacology 2011, 1 (1) 1-5

Fig 1 Group I Normal control shows the cortical structure of the kidney

including glomeruli and distended tubules, sclerotic glomeruli, and increased

cellularity between tubules.

Fig 2 Group II, Kidney stone Model control Shows cortical structure of

the kidney including glomeruli and affected distended tubules, sclerotic

glomeruli, and cellularity between the tubules. This demonstrated a thin and

attenuated filtration barrier with an absence of definitive pedicels and crypts

appears. Hematuria, glycosuria and renal failure are also present.

Fig 3 Group III, (500 mg ethanolic extract of Bergenia ligulata) Shows

extensive interstitial lymphoplasmacytic infiltration with definite tubulitis

and intimal cell prominence with subintimal vacuolation in arterial vessels of

kidney. This also shows the recovered crypts or small sacs.

Fig 4 Group IV, (ethanolic extract of Trigonella foenum graecum 200

mg/kg) widespread focal interstitial lymphoplasmacytic infiltration with

mild tubulitis, normal glomeruli and arteriolar vessels. There is sign of

sclerotic glomeruli and increased cellularity between tubules.

Fig 5 Group V (combination of both extract.) Shows minor interstitial

lymphoplasmacytic infiltration in glomeruli with occasional penetration of

tubular epithelium. In addition, increased cellularity between tubules is

clearly visible. The glomerular lesions are poorly characterized.

Fig 6 Group VI, Standard (Cystone 500 mg/kg): Shows the cortical

structure of the kidney including glomeruli and recovered distended tubules,

sclerotic glomeruli, and increased cellularity between tubul

5 International Journal of Toxicology and Applied Pharmacology 2011, 1 (1) 1-5

various therapeutic agents on development and progression of

the disease. Rat is the most frequent used animal to induce

CaOx deposition into kidneys and mimic the etiology of the formation of stones in humans. Accordingly, it evaluated the

effectiveness of medicinal plant, Coleus barbatus &

Trigonella foenum graecum & their combination on rats

rendered nephrolithiasis by administration of EG. In fact, the

plant is widely used in India in the treatment of kidney stone

formation as an antilithiasic and diuretic agent. It was noticed

urinary volume increase in the rats those receiving EG and

plant extracts compared to those receiving EG only. It was

making a general look over data it can underline two

important points. First, oxalate level remain constant in the

urine of nephrolithiatic rats treated with the ethanolic extract

of Coleus barbatus & Trigonella foenum graecum and their combination.

While it increased in untreated rats groups. Secondly, urinary

concentration of calcium and Creatinine of treated groups

remained at a level comparable to control group rats. In the

present study of kidney stone, after the administration of

ethanolic extract of Coleus barbatus & Trigonella foenum

graecum and their combination to the group of rats urine

analysis shows that the occurrence of stone was decreased

when compared to the kidney stone control group and

combination (ethanolic extract of Coleus barbatus &

Trigonella foenum graecum as effective as the standard group. Further studies are necessary to clarify the mechanism,

which is still not clear so far.

5.References

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Source of support: Nil; Conflict of interest: None declared