Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 68 No. 5 pp. 711ñ715, 2011 ISSN 0001-6837Polish Pharmaceutical Society

Urolithiasis is one of the most painful ailmentsof the urinary tract disorder. With a prevalence of >10% and an expected recurrence rate of ~ 50%,stone disease has an important effect on the health-care system (1, 2). Calcium oxalate (CaOx) is theprimary constituent of the majority of stones formedin the urinary system of patients with urolithiasis.The medical management of lithiasis, today,includes lithotripsy and surgical procedures whichshow some significant side effects such as renaldamage, hypertension or renal impairment (3).

Plants are known to provide a source of inspira-tion for novel drug compounds and this is a sequel tothe fact that medicines derived from plants have madelarge contributions to human health and well being(4). A number of plant drugs have been used in Indiaand elsewhere which claim efficient cure of urinarystones (5). Bergenia is a genus of 10 species of flow-ering plants in the family Saxifragaceae. It is native tocentral Asia and found in Afghanistan, China and inHimalaya. Bergenia ciliata Wall., (B. ciliata) com-monly known as Paashaanbhed in the Indian systemsof medicine, is used as a tonic for the treatment offevers, diarrhoea, and pulmonary infections (6).Traditionally, its rhizomes are used in Ayurvedic for-mulations. The antilithic property of the crude extracthas been investigated in vitro and it was concluded

that B. ciliata extract had no effect in preventing stoneformation in rats but was significantly beneficial indissolving preformed stones (7). Alcoholic extract ofB. ciliata exhibits significant anti-inflammatory, anal-gesic, and diuretic properties (8). In the present studythe antilithiatic activity of Bergenia ciliata has beeninvestigated to validate its folkloric use.

EXPERIMENTAL

The plant material was collected from localmarket of Ahmedabad, India in the month of May,2010 and was identified as rhizomes of B. ciliata byDepartment of Botany, Gujarat University, India.The rhizomes of B. ciliata were coarsely powderedand extract was prepared according to the WHOprotocol CG-06. Five grams of powder and 100 mLof 70% methanol were mixed and stirred on a mag-netic stirrer and then filtered twice with Whatmanfilter paper no. 41. The extract obtained was driedand stored in air tight container at 4OC. The driedextract (BCE) was dissolved in distilled water andused for further study.

Experimental animals

Healthy female rats of Wistar strain weighingbetween 220ñ250 g of equivalent age groups were

BERGENIA CILIATA EXTRACT PREVENTS ETHYLENE GLYCOL INDUCEDHISTOPATHOLOGICAL CHANGES IN THE KIDNEY

SARMISTHA SAHA and RAMTEJ JAYRAM VERMA*

Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad ñ 380009, India

Abstract: The present investigation is an attempt to evaluate the effect of Bergenia ciliata extract on kidney ofethylene glycol induced urolithiasis in adult female Wistar rats. The hydro-alcoholic extract of Bergenia cilia-ta/standard drug cystone were administrated simultaneously at a dose of 150 and 300 mg/kg body weight/day,p.o. along with ethylene glycol (0.75% v/v) for 28 days. Significant changes were observed in body weight andabsolute organ weight of ethylene glycol treated rats. Also histopathological results showed disrupted renalparenchyma, degenerative changes in glomeruli and focal calcification in glomerulo-tubular structures in eth-ylene glycol treated animals. Administration of Bergenia ciliata extract/cystone along with ethylene glycolshowed significant protective effect in body weight and organ weight with few stray areas of calcifications inglomeruli. Moreover, Bergenia ciliata extract shows higher renoprotective index than cystone at the same doselevel.

Keywords: Bergenia ciliata, ethylene glycol, antiurolithiasis, calcium oxalate, histopathology

711

* Corresponding author: e-mail: ramtejverma2000@yahoo.com; saha30@rediffmail.com

712 SARMISTHA SAHA and RAMTEJ JAYRAM VERMA

obtained from Torrent Research Centre,Ahmedabad, India. They were acclimatized for 15days in polypropylene cages under controlled condi-tions (temperature 25 ± 2OC; relative humidity50ñ55%; 12 h light/ dark cycle) in the AnimalHouse of Zoology Department, Gujarat University,Ahmedabad, India. Animals were maintained oncertified pelleted rodent feed supplied by AmrutFeeds, Pranav Agro Industries Ltd., Pune, India andwater ad libitum. The experimental procedures wereapproved by the Committee for the Purpose ofControl and Supervision of Experiment on Animals(Reg. ñ 167/ 1999/ CPCSEA), New Delhi, India.

Ethylene glycol induced urolithiasis model

Ethylene glycol-induced hyperoxaluria model(9) was used to assess the antiurolithiatic activity inalbino rats. Urolithiasis was induced by oral admin-istration of 0.75% v/v ethylene glycol (Merck Ltd.,Mumbai, India) in drinking water till 28th day.Cystone (Batch No. A029156 B; manufacturedNovember 2009; Mfg. Licence No. L AUS-127)was obtained from Himalaya Drug Company,Bangalore, India. All the chemicals used in the pres-ent experiment were of analytical reagent grade.

Experimental design and treatment schedule

In the experiment, a total of 40 rats were ran-domly divided into eight groups containing five ani-mals in each group. Group I served as control andreceived regular rat feed and drinking water ad libi-tum. Ethylene glycol (0.75%) (EG) in drinkingwater was administered to animals of GroupsIVñVIII for induction of renal calculi till 28th day.Group II and III served as standard and plant controland received only standard antiurolithiatic drug,

cystone and BCE (300 mg/kg body weight), respec-tively. Group V and VI received standard drug, cys-tone at two dose levels (150 and 300 mg/kg b.w.)simultaneously from the 1st till 28th day along withEG. Similarly, Group VII and Group VIII receivedBCE at two the same dose levels (150 and 300mg/kg b.w.) simultaneously along with EG from the1st till 28th day and served as preventive regimen. Allextracts were given once daily by oral route. At theend of 28th day of treatment, the abdomen was cutopen to remove both kidneys from each animal.Isolated kidneys were cleaned off extraneous tissueand weighed on a balance. The right kidney wasfixed in 10% formalin, processed in series of gradedalcohol and xylene, embedded in paraffin wax, sec-tioned at 5 µm and stained with hematoxylin andeosin for histopathological examination. The his-toarchitecture was obtained under microscope.

The kidney body weight ratio was calculatedusing this formula:

The kidney protecting activity of the B.ciliataextract was expressed as renoprotective index whichwas calculated using the formula:

B ñ CR = (1 ñ ññññññ) × 100

T ñ Cwhere B is the mean value of B. ciliata or cystonealong with the ethylene glycol (Groups VñVIII), T isthe mean value of ethylene glycol alone (Group IV)and C is the mean value of control animals (Group I).

RESULTS

There was no significant difference in initialbody weight among the groups but at the end of the

Absolute weight of kidneyKidney ñ Body Weight Ratio = ññññññññññññññññññññññññ

Weight of whole animal

Table 1. Effect of oral administration of B. ciliata on body weight (g) in normal and ethylene glycol induced urolithiatic Wistar rats.

Experimental groupsDay of treatment

First 28th

I. Untreated control 241.20 ± 0.97 245.40 ± 0.68

II. Cystone control (SD) 241.60 ± 2.14 245.60 ± 0.60

III. B. ciliata (BCE) 241.40 ± 1.86 244.60 ± 2.04

IV. Ethylene glycol (EG) 242.00 ± 1.22 211.80 ± 1.39a*

V. EG + SD150 242.20 ± 1.88 224.20 ± 1.69a*b*c* (37)

VI. EG + SD300 240.40 ± 1.63 234.80 ± 2.06a*b* (69)

VII. EG + BCE150 242.00 ± 1.22 232.80 ± 0.97a*b* (63)

VIII. EG + BCE300 242.40 ± 1.12 245.60 ± 0.60b*c* (100)

Results are expressed as the mean ± SEM; n = 5. Values shown in parenthesis indicate renoprotective percentage.a as compared withuntreated group; b as compared with toxin-treated (group IV); cas compared with toxin + standard-treated (group VI); No significant dif-ference was noted between groups IñIII. Level of significance *p < 0.001.

Bergenia ciliata extract prevents ethylene glycol induced histopathological... 713

Table 2. Effect of oral administration of B. ciliata on absolute organ weight and kidney to body weight ratio in normal and ethylene gly-col induced urolithiatic Wistar rats.

Experimental groups Kidney weight

Absolute organ weight (g) Kidney to body weight ratio

I. Untreated Control 1.44 ± 0.04 0.60 ± 0.01

II. Cystone Control (SD) 1.45 ± 0.06 0.63 ± 0.02

III. B. ciliata (BCE) 1.46 ± 0.04 0.61 ± 0.01

IV. Ethylene glycol (EG) 1.90 ± 0.07a* 0.90 ± 0.02 a*

V. EG + SD150 1.77 ± 0.08aÜ (28) 0.83 ± 0.02 aÜ

VI. EG + SD300 1.55 ± 0.12bÜ (76) 0.66 ± 0.05 bÜ

VII. EG + BCE150 1.75 ± 0.04aÜ (33) 0.75 ± 0.01 aÜ

VIII. EG + BCE300 1.54 ± 0.05bÜ (79) 0.63 ± 0.01 bÜ

Results are expressed as the mean ± SEM; n = 5. Values shown in parenthesis indicate renoprotective percentage. a as compared withuntreated group; b as compared with toxin-treated (group IV); c as compared with toxin + standard-treated (group VI); No significant dif-ference was noted between groups IñIII. Level of significance Üp < 0.05; *p < 0.001.

Figures 1ñ4. Light microscopic architecture and calcification in the kidney section. Paraffin section of kidney, hematoxylin and eosin(H&E): ◊ 100 of control (Fig. 1), urolithic (Fig. 2), prophylactic treatment with cystone at the dose of 150 mg/kg (Fig. 3), prophylactictreatment with cystone at the dose of 300 mg/kg, (Fig. 4)

714 SARMISTHA SAHA and RAMTEJ JAYRAM VERMA

treatment the ethylene glycol treatment caused a sig-nificant loss in the body weight. The simultaneousadministration of BCE/cystone along with EGshowed significant effect by restoring the bodyweight of treated rats (p < 0.001); BCE at a dose of300 mg/kg was most effective as showing 100%renoprotective index (Table 1).

The isolated kidneys were weighed and com-pared between the groups. The weight of kidney inEG treated group was significantly higher than thecontrol groups (p < 0.001). The kidneys in the EGalong with BCE/cystone treated groups had signifi-cantly lowered weight (p < 0.05) as compared to EG

alone treated group at both the doses tested (Table2). However EG along with BCE 300 treatmentgroup showed significant protective effect in organweight compared to that of animals in EG alongwith SD 300 group. Administration of the extract atboth the doses used (150 and 300 mg/kg b.w.) didnot produce any significant change in the kidney-body weight ratio when compared with the control(Table 2).

The architectural appearance of the kidneysfrom the rats in the control group, presented a nor-mal histological appearance with no calcium oxalatedepositions with normal glomeruli, tubules sur-rounded by the Bowmanís capsule, proximal anddistal convoluted tubules without any inflammatorychanges and normal blood vessels (Fig. 1). On theother hand, disrupted renal parenchyma showingloss of structural arrangement of renal tubules, earlydegenerative changes in glomeruli and focal calcifi-cation in glomerulo-tubular structures and congest-ed blood vessels were observed in the renal tissue ofurolithiatic rats (Fig. 2). The renal tissue of EGalong with BCE shows only few stray areas of cal-cification in glomeruli and normal tubular structureswith no congestion in blood vessels (Figs. 5 and 6).The renal tissue of standard drug treatment stillshows moderate calcification in many tubules andfew glomeruli (Figs. 3 and 4).

DISCUSSION AND CONCLUSION

Renal CaOx deposition induced by ethyleneglycol is frequently used to mimic the urinary stoneformation in humans (9, 10). Pathologic studies (11)have shown that the renal failure from EG is associ-ated with proximal tubule cell necrosis leading toproduction of several metabolites (glycol aldehyde,glycolate, glyoxylate and oxalate, in that order) andaccumulation of large calcium oxalate monohydratecrystals in tubular lumen. Therefore, we evaluatedthe medicinal effect of BCE on CaOx urolithiasisusing this model. An Ayurvedic compound prepara-tion (Cystone) was found to contain water solublesubstances, which inhibited the initial precipitationof calcium and phosphate ions in the form of a min-eral phase bound to the organic matrix and the sub-sequent growth of the preformed mineral phase (12).In the present study, concurrent administration ofEG with cystone/BCE causes significant protectiveeffect in EG induced changes. The effect is dose-dependent. The effectiveness of plant extract iscomparatively higher compared to cystone treat-ment, as is depicted by renoprotective index (Tables1 and 2).

Figures 5, 6. Light microscopic architecture and calcification inthe kidney section. Paraffin section of kidney, stain: hematoxylinand eosin, magnification 100◊. Prophylactic treatment with B. cil-iata extract at the dose of 150 mg/kg (Fig. 5), prophylactic treat-ment with B. ciliata extract at the dose of 300 mg/kg (Fig. 6).

Bergenia ciliata extract prevents ethylene glycol induced histopathological... 715

The kidneys excised from ethylene glycoltreated group were larger and heavier than from thecontrol animals. When observed under light micro-scope, many birefringent crystalline deposits in thehistological preparations were seen in tubules of allregions of kidney. In BCE along with EG treatedrats, such deposits were small and less abundant.

Microscopic examination of kidney sectionsderived from EG induced urolithiatic rats showedcalcification inside the tubules which causes dilationof the proximal tubules. Co-treatment with BCEdecreased the calcification in different parts of therenal tubules and also prevented damages to thetubules and calyxes. Organ-body weight ratio is amarker of cell constriction and inflammation (13).The non-significant effect on the rat kidney-bodyweight ratio following the administration of variousdoses of the plant extract (Table 2) suggests that theextract did not induce inflammation or constrictionof the kidney cells (13). The present study reportsthat the hydro-alcoholic extract of rhizomes of B.ciliata administered to rats with ethylene glycolinduced lithiasis, reduced and prevented the growthof urinary stones more effectively as compared tocystone treatment. However, this is a preliminarywork and needs further investigation.

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Received: 21. 09. 2010