Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 68 No. 5 pp. 711ñ715, 2011 ISSN 0001-6837 Polish Pharmaceutical Society BERGENIA CILIATA EXTRACT PREVENTS ETHYLENE GLYCOL INDUCED HISTOPATHOLOGICAL 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 of ethylene glycol induced urolithiasis in adult female Wistar rats. The hydro-alcoholic extract of Bergenia ciliata/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 and absolute organ weight of ethylene glycol treated rats. Also histopathological results showed disrupted renal parenchyma, degenerative changes in glomeruli and focal calcification in glomerulo-tubular structures in ethylene glycol treated animals. Administration of Bergenia ciliata extract/cystone along with ethylene glycol showed significant protective effect in body weight and organ weight with few stray areas of calcifications in glomeruli. Moreover, Bergenia ciliata extract shows higher renoprotective index than cystone at the same dose level. Keywords: Bergenia ciliata, ethylene glycol, antiurolithiasis, calcium oxalate, histopathology Urolithiasis is one of the most painful ailments of the urinary tract disorder. With a prevalence of > 10% and an expected recurrence rate of ~ 50%, stone disease has an important effect on the healthcare system (1, 2). Calcium oxalate (CaOx) is the primary constituent of the majority of stones formed in the urinary system of patients with urolithiasis. The medical management of lithiasis, today, includes lithotripsy and surgical procedures which show some significant side effects such as renal damage, hypertension or renal impairment (3). Plants are known to provide a source of inspiration for novel drug compounds and this is a sequel to the fact that medicines derived from plants have made large contributions to human health and well being (4). A number of plant drugs have been used in India and elsewhere which claim efficient cure of urinary stones (5). Bergenia is a genus of 10 species of flowering plants in the family Saxifragaceae. It is native to central Asia and found in Afghanistan, China and in Himalaya. Bergenia ciliata Wall., (B. ciliata) commonly known as Paashaanbhed in the Indian systems of medicine, is used as a tonic for the treatment of fevers, diarrhoea, and pulmonary infections (6). Traditionally, its rhizomes are used in Ayurvedic formulations. The antilithic property of the crude extract has been investigated in vitro and it was concluded that B. ciliata extract had no effect in preventing stone formation in rats but was significantly beneficial in dissolving preformed stones (7). Alcoholic extract of B. ciliata exhibits significant anti-inflammatory, analgesic, and diuretic properties (8). In the present study the antilithiatic activity of Bergenia ciliata has been investigated to validate its folkloric use. EXPERIMENTAL The plant material was collected from local market of Ahmedabad, India in the month of May, 2010 and was identified as rhizomes of B. ciliata by Department of Botany, Gujarat University, India. The rhizomes of B. ciliata were coarsely powdered and extract was prepared according to the WHO protocol CG-06. Five grams of powder and 100 ml of 70% methanol were mixed and stirred on a magnetic stirrer and then filtered twice with Whatman filter paper no. 41. The extract obtained was dried and stored in air tight container at 4 O C. The dried extract (BCE) was dissolved in distilled water and used for further study. Experimental animals Healthy female rats of Wistar strain weighing between 220ñ250 g of equivalent age groups were * Corresponding author: e-mail: ramtejverma2000@yahoo.com; saha30@rediffmail.com 711
712 SARMISTHA SAHA and RAMTEJ JAYRAM VERMA obtained from Torrent Research Centre, Ahmedabad, India. They were acclimatized for 15 days in polypropylene cages under controlled conditions (temperature 25 ± 2 O C; relative humidity 50ñ55%; 12 h light/ dark cycle) in the Animal House of Zoology Department, Gujarat University, Ahmedabad, India. Animals were maintained on certified pelleted rodent feed supplied by Amrut Feeds, Pranav Agro Industries Ltd., Pune, India and water ad libitum. The experimental procedures were approved by the Committee for the Purpose of Control 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 in albino rats. Urolithiasis was induced by oral administration of 0.75% v/v ethylene glycol (Merck Ltd., Mumbai, India) in drinking water till 28 th day. Cystone (Batch No. A029156 B; manufactured November 2009; Mfg. Licence No. L AUS-127) was obtained from Himalaya Drug Company, Bangalore, India. All the chemicals used in the present experiment were of analytical reagent grade. Experimental design and treatment schedule In the experiment, a total of 40 rats were randomly divided into eight groups containing five animals in each group. Group I served as control and received regular rat feed and drinking water ad libitum. Ethylene glycol (0.75%) (EG) in drinking water was administered to animals of Groups IVñVIII for induction of renal calculi till 28 th day. Group II and III served as standard and plant control and received only standard antiurolithiatic drug, cystone and BCE (300 mg/kg body weight), respectively. Group V and VI received standard drug, cystone at two dose levels (150 and 300 mg/kg b.w.) simultaneously from the 1 st till 28 th day along with EG. Similarly, Group VII and Group VIII received BCE at two the same dose levels (150 and 300 mg/kg b.w.) simultaneously along with EG from the 1 st till 28 th day and served as preventive regimen. All extracts were given once daily by oral route. At the end of 28 th day of treatment, the abdomen was cut open to remove both kidneys from each animal. Isolated kidneys were cleaned off extraneous tissue and weighed on a balance. The right kidney was fixed in 10% formalin, processed in series of graded alcohol and xylene, embedded in paraffin wax, sectioned at 5 µm and stained with hematoxylin and eosin for histopathological examination. The histoarchitecture was obtained under microscope. The kidney body weight ratio was calculated using this formula: Absolute weight of kidney Kidney ñ Body Weight Ratio = ññññññññññññññññññññññññ Weight of whole animal The kidney protecting activity of the B.ciliata extract was expressed as renoprotective index which was calculated using the formula: B ñ C R = (1 ñ ññññññ) 100 Tñ C where B is the mean value of B. ciliata or cystone along with the ethylene glycol (Groups VñVIII), T is the 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 initial body weight among the groups but at the end of the Table 1. Effect of oral administration of B. ciliata on body weight (g) in normal and ethylene glycol induced urolithiatic Wistar rats. Day of treatment Experimental groups First 28 th 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 ± 1.39 a* V. EG + SD150 242.20 ± 1.88 224.20 ± 1.69 a*b*c* (37) VI. EG + SD300 240.40 ± 1.63 234.80 ± 2.06 a*b* (69) VII. EG + BCE150 242.00 ± 1.22 232.80 ± 0.97 a*b* (63) VIII. EG + BCE300 242.40 ± 1.12 245.60 ± 0.60 b*c* (100) Results are expressed as the mean ± SEM; n = 5. Values shown in parenthesis indicate renoprotective percentage. a as compared with untreated group; b as compared with toxin-treated (group IV); c as compared with toxin + standard-treated (group VI); No significant difference 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 glycol induced urolithiatic Wistar rats. Kidney weight Experimental groups 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 1.90 ± 0.07a* 0.61 ± 0.01 0.90 ± 0.02 a* 1.77 ± 0.08aÜ (28) 1.55 ± 0.12bÜ (76) 0.83 ± 0.02 aü 0.66 ± 0.05 bü 1.75 ± 0.04aÜ (33) 1.54 ± 0.05bÜ (79) 0.75 ± 0.01 aü 0.63 ± 0.01 bü IV. Ethylene glycol (EG) V. EG + SD150 VI. EG + SD300 VII. EG + BCE150 VIII. EG + BCE300 Results are expressed as the mean ± SEM; n = 5. Values shown in parenthesis indicate renoprotective percentage. a as compared with untreated group; b as compared with toxin-treated (group IV); c as compared with toxin + standard-treated (group VI); No significant difference 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), prophylactic treatment with cystone at the dose of 300 mg/kg, (Fig. 4)
714 SARMISTHA SAHA and RAMTEJ JAYRAM VERMA alone treated group at both the doses tested (Table 2). However EG along with BCE 300 treatment group showed significant protective effect in organ weight compared to that of animals in EG along with SD 300 group. Administration of the extract at both the doses used (150 and 300 mg/kg b.w.) did not produce any significant change in the kidneybody weight ratio when compared with the control (Table 2). The architectural appearance of the kidneys from the rats in the control group, presented a normal histological appearance with no calcium oxalate depositions with normal glomeruli, tubules surrounded by the Bowmanís capsule, proximal and distal convoluted tubules without any inflammatory changes and normal blood vessels (Fig. 1). On the other hand, disrupted renal parenchyma showing loss of structural arrangement of renal tubules, early degenerative changes in glomeruli and focal calcification in glomerulo-tubular structures and congested blood vessels were observed in the renal tissue of urolithiatic rats (Fig. 2). The renal tissue of EG along with BCE shows only few stray areas of calcification in glomeruli and normal tubular structures with no congestion in blood vessels (Figs. 5 and 6). The renal tissue of standard drug treatment still shows moderate calcification in many tubules and few glomeruli (Figs. 3 and 4). DISCUSSION AND CONCLUSION Figures 5, 6. Light microscopic architecture and calcification in the kidney section. Paraffin section of kidney, stain: hematoxylin and eosin, magnification 100. Prophylactic treatment with B. ciliata extract at the dose of 150 mg/kg (Fig. 5), prophylactic treatment with B. ciliata extract at the dose of 300 mg/kg (Fig. 6). treatment the ethylene glycol treatment caused a significant loss in the body weight. The simultaneous administration of BCE/cystone along with EG showed significant effect by restoring the body weight of treated rats (p < 0.001); BCE at a dose of 300 mg/kg was most effective as showing 100% renoprotective index (Table 1). The isolated kidneys were weighed and compared between the groups. The weight of kidney in EG treated group was significantly higher than the control groups (p < 0.001). The kidneys in the EG along with BCE/cystone treated groups had significantly lowered weight (p < 0.05) as compared to EG Renal CaOx deposition induced by ethylene glycol is frequently used to mimic the urinary stone formation in humans (9, 10). Pathologic studies (11) have shown that the renal failure from EG is associated with proximal tubule cell necrosis leading to production of several metabolites (glycol aldehyde, glycolate, glyoxylate and oxalate, in that order) and accumulation of large calcium oxalate monohydrate crystals in tubular lumen. Therefore, we evaluated the medicinal effect of BCE on CaOx urolithiasis using this model. An Ayurvedic compound preparation (Cystone) was found to contain water soluble substances, which inhibited the initial precipitation of calcium and phosphate ions in the form of a mineral phase bound to the organic matrix and the subsequent growth of the preformed mineral phase (12). In the present study, concurrent administration of EG with cystone/bce causes significant protective effect in EG induced changes. The effect is dosedependent. The effectiveness of plant extract is comparatively higher compared to cystone treatment, as is depicted by renoprotective index (Tables 1 and 2).
Bergenia ciliata extract prevents ethylene glycol induced histopathological... 715 The kidneys excised from ethylene glycol treated group were larger and heavier than from the control animals. When observed under light microscope, many birefringent crystalline deposits in the histological preparations were seen in tubules of all regions of kidney. In BCE along with EG treated rats, such deposits were small and less abundant. Microscopic examination of kidney sections derived from EG induced urolithiatic rats showed calcification inside the tubules which causes dilation of the proximal tubules. Co-treatment with BCE decreased the calcification in different parts of the renal tubules and also prevented damages to the tubules and calyxes. Organ-body weight ratio is a marker of cell constriction and inflammation (13). The non-significant effect on the rat kidney-body weight ratio following the administration of various doses of the plant extract (Table 2) suggests that the extract did not induce inflammation or constriction of the kidney cells (13). The present study reports that the hydro-alcoholic extract of rhizomes of B. ciliata administered to rats with ethylene glycol induced lithiasis, reduced and prevented the growth of urinary stones more effectively as compared to cystone treatment. However, this is a preliminary work and needs further investigation. REFERENCES 1. Tisselius H.G.: Kidney Stones: Medical and Surgical Management. p. 33. Lippincott Raven, Philadelphia 1996. 2. Knoll T.: Eur. Urol. Suppl., 6, 717 (2007). 3. Tombolini P., Ruoppolo M., Bellorofonte C., Zaatar C., Follini M.: J. Nephrol. 13, 71 (2000). 4. Iwu M.W., Duncan A.R., Okunji C.O.: New antimicrobials of plant origin. pp. 457-462. ASHS Publication, Alexandria 1999. 5. Mukharjee T., Bhalla N., Aulakh G.S., Jain H.C.: Indian Drugs 21, 224 (1984). 6. Nadkarni, A.K.: Indian Materia Medica. pp. 1075ñ1077. Popular Prakashan, Bombay 2000. 7. Garimella T.S., Jolly C.I., Narayanan S.: Phytother. Res. 15, 351 (2001). 8. Gehlot N.K., Sharma V.N., Vyas D.S.: Ind. J. Pharmacol. 8, 92 (1976). 9. Atmani F., Slimani Y., Mimouni M., Hacht B.: BJU Int. 92, 137 (2003). 10. Tsai C.H., Chen Y.C., Chen L.D., Pan T.C., Ho C.Y., Lai M.T., Tsai F.J., Chen W.C.: Urol. Res. 36, 17 (2008). 11. Cruzan G., Corley R.A., Hard G.C., Mertens J.J.W.M., McMartin K.E., Snellings W.M., Gingell R., Deyo J.A.: Toxicol. Sci. 81, 502 (2004). 12. Jethi R.K., Duggal B., Sahota R.S., Gupta M., Sofat I.B.: Ind. J. Med. Res. 78, 422 (1983). 13. Moore K.L., Dalley A.F.: Structure of the penis. In: Clinical Oriented Anatomy. pp. 287-299 4th edn. Lippincot Williams and Williams, Philadelphia 1999. Received: 21. 09. 2010