Indian Journal of Natural Products and Resources

Vol. 2(1), March 2011, pp. 28-33

Antiurolithiatic activity of Crataeva magna Lour. bark

Suman Kumar Mekap1, Satyaranjan Mishra

2, Sabuj Sahoo

3*and Prasana Kumar Panda

3

1Sri Satya Sai College of Pharmacy, Bhopal-Indore Road, Pachama, Sehore-466 001, Madhya Pradesh, India 2K.V. Virani Institute of Pharmacy & Research Centre, Badhada, Savarkundla-364 515, Gujarat, India

3 Pharmacognosy and Phytochemistry Division, University Department of Pharmaceutical Sciences, Utkal University,

Bhubaneswar -751 004, Orissa, India

Received 25 March 2010; Accepted 18 January 2011

Crataeva magna Lour. bark, commonly known as Baruna, belonging to family Capparaceae, has been investigated for its

antiurolithiatic activity in two conventional models (in vivo) of Urolithiasis in rats. The two methods chosen were lactose (30%)

+ ethylene glycol (1%) and ammonium chloride (2%) + ethylene glycol (0.75%) induced urolithiasis, respectively. The ethanol

extract (400 mg/kg bw) reduced the elevated levels of serum calcium (3.25 ± 0.30) and urine calcium (2.33 ± 0.18) significantly

(P<0.05), employing lactose (30%) + ethylene glycol (1%) induced urolithiasis model. The ethanol extract (400 mg/kg bw)

reduced the urine uric acid level significantly employing both models, viz. lactose (30%) + ethylene glycol (1%) (0.82 ± 0.07;

P<0.001) and ammonium chloride (2%) + ethylene glycol (0.75%) (0.85 ± 0.12; P<0.001) when compared to toxic group. The

ethanol extract (400 mg/kg bw) employing both models resulted in reduced serum creatinine and calcium, urine oxalate and

kidney weight significantly with a marked increase in final body weight and urine volume output when compared to toxic

group. The results shown by the ethanol extract (400 mg/kg bw) group was compared to standard polyherbal drug (Cystone;

5 ml/kg bw) treated group and thus exhibited potent antiurolithiatic activity.

Keywords: Ammonium chloride, Bark, Baruna, Crataeva magna, Ethylene glycol, Lactose, Urolithiasis.

IPC code; Int. cl. (2011.01) A61K 36/18, A61K 129/00, A61P 13/04

Introduction Urolithiasis in its different forms is frequently

encountered during urological complications. Some

common causes are inadequate urinary drainage,

foreign bodies in the urinary tract, microbial infections,

diet with excess oxalates and calcium, vitamin

abnormalities, viz. Vitamin A deficiencies, Vitamin D

excess, metabolic diseases like hyperparathyroidism,

cystinuria, gout and intestinal dysfunction1. Generally

stones of two types i.e., non calcium and calcium

stones are formed2. Calcium, albumin, creatinine, urate

and oxalate are some necessary analytical markers in

serum and urine for clinical diagnosis of this type of

urological disorders3-5

.

In recent times, focus on plant research has

increased all over the world and a large body of

evidence has collected to show immense potential of

medicinal plants used in various traditional systems.

Crataeva magna Lour. bark belonging to family

Capparaceae, has many therapeutic benefits such as

its use in tribal area as antispasmodic, anti-

hypertensive, anti-inflammatory, to treat kidney

stones, anti-hyperglycemic, anti-protozoal, anti-

bacterial, anthelmintic and as an analgesic agent6.

The present study is focused on the investigation of

the antiurolithiatic activity of ethanol and aqueous

extracts of C. magna bark.

Materials and Methods

Collection of plant materials

The bark of C. magna (Plate 1) was collected from

young matured plant from Utkal University campus during the month of May and identified by the Botanist of Department of Botany, Utkal University, Bhubaneswar by comparing with the specimen

present in the Departmental herbarium. After authentification fresh plant materials were collected in bulk, washed under running tap water to remove adhering dust, shade dried and pulverized in a mechanical grinder. The coarse powder was used for further studies.

Extraction

About 200 g of coarse dried powder of bark was

taken in the Soxhlet apparatus and extracted

——————— *Correspondent author: E-mail: sabujbiotech@rediffmail.com;

Phone: +91-9437344766 (Mob.)

MEKAP et al: ANTIUROLITHIATIC ACTIVITY OF CRATAEVA MAGNA BARK

29

successively using ethanol (95%) and water7,8

. The

extraction for each solvent was carried out for 18 to

20 hours. The extract was collected by evaporating

the solvents and percentage yield was calculated.

Experimental design

Wistar albino rats weighing 140-200 g of either sex were used. The experimental protocol was approved by the Institutional Animal Ethics Committee and animals were maintained under standard conditions for an acclimatization period of 15 days before performing the experiment. All rats were housed individually in metabolic cages and temperature maintained at 22±2°C. The condition in the animal house was approved by Committee for the purpose of Control and Supervision on Experiments on Animals (Regd. No. 990/c/05/CPCSEA). The acute oral toxicity study was done according to OECD guideline at dose range 100 to 4000 mg/kg. No mortality of animals was observed at the dose range and hence two different doses 200 and 400 mg/kg was taken for the study

9.

Lactose (30%) and Ethylene glycol (1%) induced urolithiasis10

Forty two healthy adult Wistar albino strain rats of either sex weighing 140-200 g were randomly divided into seven groups. Each group consists of 6 animals. Group 1: Normal rats were fed with standard rat chow diet and tap water ad libitum for 4 weeks. Group 2: EG intoxicated rats with diet for inducing urolithiasis were fed with a lactose rich Lab diet [the lactose rich lab diet contains: 3.68% sucrose, 30% lactose, 23.4% protein, 10% fat, 5.3% crude fiber, 6.9% ash minerals (calcium 0.95%, phosphorus 0.67%, magnesium 0.21%), vitamin A 22 IU/g, vitamin D3 4.5 IU/g, vitamin E 49 IU/g] with 1% EG (in drinking water)

for 4 weeks. Group 3: (Standard drug treated rats) were fed with lactose rich lab diet + 1% EG + Cystone (5 ml/kg) (Himalaya Lab., India) for 4 weeks. Group 4: test group treated with 200 mg/kg of aqueous extract were fed with lactose rich lab diet + 1% EG + aqueous extract 200 mg/kg for 4 weeks. Group 5: test group treated with 400 mg/kg of aqueous extract were fed with lactose rich lab diet + 1% EG + aqueous extract 400 mg/kg for 4 weeks. Group 6: test group treated with 200 mg/kg of ethanol extract were fed with lactose rich diet + 1% EG + ethanol extract, 200 mg/kg for 4 weeks. Group 7: test group treated with 400 mg/kg of ethanol extract were fed with lactose rich diet + 1% EG + ethanol extract, 400 mg/kg for 4 weeks.

Ammonium chloride (2%) and Ethylene glycol (0.75%) induced

urolithiasis11

Forty two healthy adult Wistar albino strain rats of either sex weighing 140-200 g were randomly divided into seven groups. Each group consisted of 6 animals. The treatment period was considered for 10 days. Group 1: Normal rats were fed with standard rat chow diet and tap water ad libitum for 10 days. Group 2: EG and ammonium chloride intoxicated rats were given normal lab diet + drinking water containing 0.75% [v/v] ethylene glycol (EG) and 2% [w/v] ammonium chloride (AC) for 10 days to induce urolithiasis. Group 3: Standard group were fed with normal lab diet + drinking water containing 0.75% [v/v] EG and 2% [w/v] AC + Cystone (5 ml/kg) for 10 days. Groups 4 and 5: the test groups treated with aqueous extract 200 mg/kg and 400 mg/kg of body weight were fed with normal lab diet + drinking water containing 0.75% [v/v] EG and 2% [w/v] AC with aqueous extract 200 and 400 mg/kg body weight, respectively for 10 days. Groups 6 and 7: the test groups, treated with ethanol extract 200 mg/kg and 400 mg/kg of body weight were fed with normal lab diet + drinking water containing 0.75% [v/v] EG and 2% [w/v] AC with and ethanol extract of C. magna at dose of 200 and 400 mg/kg body weight, respectively for 10 days.

Urine and blood sampling

The crystalluria and stone formation was verified by different biochemical marker analysis of urine and serum. The urine samples of the test animals in different groups were collected in their respective end day of the experiment [on 28

th day in lactose rich diet +

(1%) EG model and on 10th day in (0.75%) EG + (2%)

AC model]. The collected urine sample volume were measured followed by centrifugation at 3000 rpm for

Plate 1―Crataeva magna bark

INDIAN J NAT PROD RESOUR, MARCH 2011

30

10 minutes. After centrifugation the urine samples were examined under light microscope (LAICA, DME Germany 400X) to ensure the presence of oxalate microcrystal followed by biochemical analysis (urine oxalate, calcium and uric acid). The blood samples were collected from the animals under anaesthesia (ether) before sacrificing. The collected blood samples were then centrifuged to obtain serum for the analysis of serum creatinine and serum calcium.

Kidney analysis

The kidney was fixed in bouin liquid12

soaked in paraffin, cut at 3-4 µm intervals and the slices stained using hematoxylene and eosin

13. Tissue

slices were photographed using optical microscopy under polarized light (LAICA, DME Germany 400 X)

Statistical analysis

Results were indicated in terms of mean ± SEM.

Statistical significance of data were assessed by

analysis of variance (One way-ANOVA), followed by

comparison between different groups using ‘Tukey-

Kramer’ multiple comparison test. The significance

was considered at the level of P<0.05.

Results and Discussion

The results of serum and urine biochemistry is

indicated in Tables 1 and 2. Table 1 comprises: the

urine and serum biochemistry data of Lactose (30%)

+ Ethylene glycol (1%) induced urolithiasis model

along with the body weight, urine volume and kidney

weight data. Table 2 comprises: the urine and serum

biochemistry, body weight, urine volume and kidney

weight data of Ammonium chloride (2%) + Ethylene

glycol (0.75%) induced urolithiasis model.

The acute urolithiasis in both the conventional

models was evidenced by the significant elevation in

urine and serum biochemical parameters along with

the reduced urine output as compared to the normal

rats.

The ethanol extract (400 mg/kg bw) employing

Lactose (30%) + Ethylene glycol (1%) induced

urolithiasis resulted a significant reduction (P< 0.001) in

urine uric acid (0.82 ± 0.07) and oxalate (1.54 ± 0.26)

level as compared to toxic group. Serum calcium

(3.25 ± 0.30) and urine calcium (2.33 ± 0.18) level

was significantly (P<0.05) lowered when compared

with the toxic group along with significant elevation

in urine volume (13.38 ± 1.12; P<0.05) output,

However, the extract showed a reduction in serum

creatinine level employing both models but the

reduction is not significant when compared with

urolithic control group.

The ethanol extract (400 mg/kg bw) employing

ammonium chloride (2%) + Ethylene glycol (0.75%)

induced Urolithiasis model exhibited significant

Table 1―Urine and serum biochemistry of 30% lactose and 1% EG induced urolithiasis on 28th day of experiment

Body wt in g Group

I F

Urine volume

in ml

Kidney wt in g Urine

Calcium

mg/dl

Oxalate mg/dl Uric acid Serum

Creatinine

mg/dl

Serum

Calcium

mg/dl

Group 1 172±9.46 191±10.02 18.4±0.97 1.503±0.025 2.25±0.19 0.50±0.03 0.69±0.04 7.45±0.36 4.10±0.21

Group 2 180±6.26 188±6.28 9.96±0.69*** 1.851± 0.035*** 3.27± 0.21* 4.07±0.13*** 1.9±0.10 8.65±0.31 2.21±0.20***

Group 3 182±6.47 195±6.23 14.93±0.57*** 1.618± 0.030*** 2.26± 0.25* 1.21± 0.19*** 0.71±0.04 7.72±0.28 3.48±0.28**

Group 4 186±3.71 195±3.87 10.41±0.45 1.816±0.040 3.10±0.12 3.77±0.28 1.66±0.17 8.50±0.71 2.27±0.21

Group 5 167±4.47 177±4.30 10.9±0.64 1.774±0.030 2.59±0.21 3.24±0.30 1.56± 0.18** 8.04±0.21 2.35±0.17

Group 6 172±3.42 184±2.51 11.5±0.58 1.673± 0.030** 2.75±0.21 2.62± 0.32** 1.36± 0.13*** 8.13±0.58 2.58±0.19

Group 7 189±2.70 204±2.95 13.38±1.12* 1.661± 0.030** 2.33± 0.18* 1.54± 0.26*** 0.82± 0.07*** 7.91±0.71 3.25±0.30*

Values are given in mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. For n=6;

Group 2(Toxic group) was compared with Group 1 (Normal group) and all the other groups were compared with Group 2 (toxic group).

Group 1: Normal control group (Normal lab diet with normal water ad libitum);

Group 2: Urolithic control group (Lactose rich diet + 1% EG in drinking water ad libitum);

Group 3: Standard group (treated with Cystone 5 ml/kg);

Group 4: Treated with aqueous extract 200 mg/kg

Group 5: treated with aqueous extract 400 mg/kg;

Group 6: Treated with ethanol extract 200 mg/kg;

Group 7: Treated with ethanol extract of 400 mg/kg.

I: Initial body weight, F: Final body weight.

MEKAP et al: ANTIUROLITHIATIC ACTIVITY OF CRATAEVA MAGNA BARK

31

reduction in urine uric acid (0.85 ± 0.12; P<0.001),

urine oxalate (2.36 ± 0.42; P<0.01), and serum calcium

(3.29 ± 0.25; P<0.05) level along with significant

elevation in urine volume (12.5 ± 1.0; P< 0.01) output

as compared to toxic rat group.

Ethanol extract 200 mg/kg and the aqueous extracts

200 and 400 mg/kg of body weight in both

the experimental models exhibited minimal

antiurolithiatic activity which was considered below

the significant level.

The results of urine and serum biochemistry

showed significant reduction in urine calcium, uric

acid and oxalate level, serum calcium with significant

elevation in urine volume output, the markers

previously reported which affirmed potent

antiurolithiatic activity14

.

The photomicrograph exhibiting anatomical

changes of kidney is shown in Plate 2 (a-g). The

photo-micrograph of normal control rat kidney (Plate

2a) showing normal organization of tubular

epithelial cells and glomeruli (X-400). Plate 2 (b, c)

represents the section of Urolithic control rat kidney

showing glomerular atrophy and deposition of

crystals (X-400) treated with Lactose rich diet +

(1%) EG in drinking water and (0.75%) EG + (2%)

AC in drinking water, respectively. Plate 2 (d, e)

showing sections of rat kidney treated with standard

cystone (5 ml/kg) showing regenerative changes in

glomeruli and tubules (X-400) employing both

models. The section of rat kidney treated with

ethanol extract 400 mg/kg in 30% lactose + (1%) EG

model and (0.75%) EG + (2%) AC showing

normalcy of tubular epithelial cells and glomeruli

(X-400) is shown in Plate 2 f and g, respectively.

Similar histological observations earlier reported

support the presence of glomerular atrophy and

deposition of crystals and attainment of normalcy of

tubular epithelial cells and glomeruli when treated

with test extract employing both models15

.

The results exhibited by the ethanol extract of

C. magna bark (400 mg/kg bw) in both the

conventional models confirmed its significant

antiurolithiatic potency when compared with the

standard drug (cystone).

Conclusion

The result exhibited by the ethanol extract of

C. magna bark (400 mg/kg bw) showed significant

antiurolithiatic activity. Further studies will be aimed

at extensive investigation, isolation and purification of

active phytoconstituents with potent antiurolithiatic

activity.

Table 2―Urine and serum biochemistry of ammonium chloride 2% and EG 0.75% induced urolithiasis on 10th day of experiment

Body wt in g. Group

I F

Urine volume

in ml

Kidney wt in

g

Urine

Calcium

mg/dl

Oxalate mg/dl Uric acid Serum

Creatinine

mg/dl

Serum

Calcium

mg/dl

Group 1 145±4.28 154±5.14 13.9±1.09 1.516±0.065 2.16±0.15 0.54±0.06 0.61±0.05 7.32±0.53 4.16±0.28

Group 2 168±6.38 156±7.13 7.06±0.58*** 1.621±0.054 3.21±0.19* 4.01± 0.26*** 1.77± 0.17*** 8.16±0.60 2.38± 0.23***

Group 3 158±7.76 164±7.12 12.41±0.98** 1.537±0.062 2.39±0.27 1.44± 0.35*** 0.79± 0.07*** 7.74±0.58 3.7±0.37*

Group 4 173±8.91 163±8.22 8.81±0.78 1.591±0.064 3.33±0.22 3.79±0.18 1.65±0.17 8.07±0.45 2.69±0.17

Group 5 162±5.88 156±5.62 10.38±0.94 1.579±0.049 2.97±0.25 3.61±0.28 1.35±0.12 7.89±0.37 2.71±0.19

Group 6 178±7.70 173±8.68 10.55±1.06 1.589±0.039 2.99±0.18 3.67±0.31 1.26±0.21 8.01±0.25 3.02±0.26

Group 7 175±4.83 180±4.86 12.5±1.0** 1.578±0.046 2.35±0.19 2.36± 0.42** 0.85± 0.12*** 7.49±0.32 3.29±0.25*

Values are given in mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. For n=6;

Group 2(Toxic group) was compared with Group 1 (Normal group) and all the other groups were compared with Group 2 (toxic group).

Group 1: Normal control group (Normal lab diet with normal water ad libitum);

Group 2: Urolithic control group (Normal lab diet with 0.75% EG + 2% AC in drinking water ad libitum);

Group 3: Standard group (treated with Cystone 5 ml/kg);

Group 4: Treated with aqueous extract 200 mg/kg

Group 5: Treated with aqueous extract 400 mg/kg;

Group 6: Treated with ethanol extract 200 mg/kg;

Group 7: Treated with ethanol extract of 400 mg/kg.

I: Initial body weight, F: Final body weight.

INDIAN J NAT PROD RESOUR, MARCH 2011

32

Plate 2 (a-g)―Histological sections of rat kidney; a: Section of normal control rat kidney showing normal organization of tubular

epithelial cells and glomeruli (X-400); b: Section of urolithic control rat (treated with 30% lactose rich diet + 1% EG in drinking water)

kidney showing glomerular atrophy and deposition of crystals (X-400); c: Section of urolithic control rat (treated with 0.75% EG + 2%

AC in drinking water) kidney showing glomerular atrophy and deposition of crystals (X-400); d: Section of standard rat (treated with

cystone; 5 ml/kg) kidney in 30% lactose + 1% EG model, showing regenerative changes in glomeruli and tubules (X-400); e: Section of

standard rat (treated with cystone; 5 ml/kg) kidney in 0.75% EG + 2% AC model, showing regenerative changes in glomeruli and tubules

(X-400); f: Section of rat kidney treated with ethanol extract (400 mg/kg) in 30% lactose + 1% EG model, showing normalcy of tubular

epithelial cells and glomeruli (X-400); g: Section of rat kidney treated with ethanol extract (400 mg/kg) in 0.75% EG + 2% AC model,

showing normalcy of tubular epithelial cells and glomeruli (X-400).

MEKAP et al: ANTIUROLITHIATIC ACTIVITY OF CRATAEVA MAGNA BARK

33

Acknowledgement The authors are thankful to the H.O.D., U.D.P.S.,

Utkal University for providing laboratory facilities.

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