Indian Journal of Experimental Biology Vol. 42. July 2004. pp. 686-690

Effect of lupeol isolated from Crataeva nurvala Buch.-. Ham. stem bark extract against free radical induced nephrotoxicity in rats

Annie Shirwaikar. Manjunath Setty M & Praveen Bommu

Department of Pharmacognosy. Manipal College of Pharmaceutical Sciences. Manipal 576 104. India. and

Krishnanand B.

Department of Pathology. Kasturba Medical College. Manipal 576 104, India.

Received 27 August 2003; Revised 6 May 2004

Lupeol. isolated from Crataeva nurvala stem bark in doses 40 and 80 mg/kg body weight. po. for 10 days, decreased the concentration of blood urea nitrogen. creatinine and lipid peroxidation and increased glutathione and catalase activities in cisplatin (5 mg/kg body weight. ip) induced nephrotoxicity in rats. The increased glutathione and catalase activities are indicative of antioxidant properties of lupeol.

Keywords: Lupeol. Crataeva nurvala, Nephrotoxicity. Rats, Free radical

IPC Code: Int. Cl7 A61P

The plant Crataeva nurvala Buch.-Ham. belonging to the family Capparidaceae is known as three-leaved caper in English, neermatalam. nirval in Malayalam, Varuna in Sanskrit and barun as well as barna in Hindi. The medicinal value of C. nurvala has been described against a wide variety of urinary disorders including urolithiasis, in the ancient text "Sushruta Samhita"l. Among the compounds isolated from the stem bark, lupeol was identified as a major component in association with u- and ~-amyrin 2 •

The cytoprotective action of lupeol isolated from C. nuvala stem bark against free radical toxicity has been investigated in experimental urolithiasis. Lupeol adminstration induced a remarkable decrease in kidney oxalate level and also was effective in counteracting the free radical toxicity by bringing about a significant decrease in peroxidative levels and an increase in antioxidant status. These observations highlight the antioxidant property of lupeol and its cytoprotection against free radical toxicity 3. Various studies have reported that the dysfunction of renal proximal tubule cells, associated as a limiting side effect of the antineoplastic cisplatin is due to the involvement of oxidative stress via free radical formation 4-6 •

Fax: (+) 91-820-571998. Tel.: (+) 91-820-571201 ext. 22482; E-mail: annieshirwaikar@yahoo.com

The triterpene celestrol, has been shown to have a potent inhibitory effect against lipid peroxidation in liver mitochondria7

• In vitro studies have shown that ursolic acid and oleanolic acid isolated from Ocimum sanctum and Eugenia jambolana offered remarkable protection against free radical damage8

. The structural similarity of lupeol with these compounds promoted us to investigate its activity against free radical damge manifested during cisplatin nephrotoxicity in Wistar rats.

Materials and Methods Plant material-The botanically identified plant

material of Crataeva nurvala was collected in the month of September 2002 from Udupi district, Karnataka, India. A voucher specimen no. PP 516 has been preserved in the herbarium of the College of Pharmaceutical Sciences, Manipal, India.

Isolation of lupeol-C. nurvala stem bark powder was extracted exhaustively with petroleum ether by cold percolation and the resulting extract was eluted successively with hexane, hexane:benzene and benzene. The benzene eluate on concentration deposited a white component lupeol (yield 0.6%) which was filtered and recrystallised from methanol: acetone (60:40) and identified by comparison with standard spectral details9

•

White crystalline substance; m.p. 215°C; [u]o -+27.2 0; IR bands (KBr) em .1: 3321, 2945, 2864,

SHIRW AIKAR et at.: EFFECT OF LUPEOL AGAINST NEPHROTOXICITY IN RATS 687

1639, 1458, 1379, 1035 cm· l. IHNMR showed seven

methyl groups at 8 1.70 (CH3-22), 80.77 (CH3-19), 8 0.84 (CH3-28.6), 8 0.88 (CH3-19.5), 80.99 (CH3-28), 8 0.968 (CH3-22) and 8 1.08 (CH3-26.5), one proton doublet at 8 3.11 (H-3) and two signals at 8 4.5 (~- H-24) and 8 4.6 (a - H - 24) attributed to methylene groups at C - 24. Mass spectrum' shows molecular ion peak at mlz 425, and other significant peaks at mlz 409,393, 318, 307, 289,258, 203, 189, 154, 136, and 121.

Drugs and chemicals-Cisplatin was obtained from Biochem Pharmaceutical Industries, Mumbai, India. Biochemicals used were of analytical reagent grade and obtained from Sigma Chemical Co. (St. Louis, MO, USA). Chemicals of high purity were obtained from E.Merck (India) Pvt. Ltd, Mumbai, India.

Animals -Male Wistar rats (30), weighing 170-210 g, bred in the Central Animal House, M.A.H.E, Manipal, were used. The animals fed with pellet diet (Hindustan Lever Ltd., India) and water ad libitum were maintained at 21 0 -230 C under a constant 12h light and dark cycle. The study was conducted after obtaining institutional animal ethical committee clearence.

Treatment schedule-Three days after acc1imat­isation to the laboratory conditions, the rats were randomly assigned to five experimental groups of 6 each as follows:

• Group 1: rats were administered single ip dose of 0.9% NaCl (2rnl1kg) and after five days were treated orally with olive oil (2ml/kg/day) for 10 days.

• Group 2: rats were administered single ip dose of cisplatin (5mg/kg) for five days.

• Group 3 : rats were administered single ip dose of cisplatin (5mg/kg) and after five days were treated orally with olive oil (2ml/kg/day) for 10 days.

• Group 4 : rats were administered single ip dose of cisplatin (5mg/kg) and after five days were treated orally with lupeol (40mg/kg/day) in olive oil for 10 days.

• Group 5 : rats were administered single ip dose of cisplatin (80mg/kg) after five days were treated orally with lupeol (80mg/kg/day) in olive oil for 10 days.

Animals were anaesthetised with diethyl ether, and rapidly decapitated. Blood was collected and the

serum was separated by centrifugation on 16th day in groups 1,3,4 and 5 and after 5 days in gruop 2. The kidneys were excised, blotted on a filter paper and weighed. The cortex was dissected out and a part thereof placed in formalin saline for subsequent histological processing while the other part was used for biochemical estimations.

Assessment of renal function-The weight (g) of the animals were noted on the first and last day of treatment and the percentage change in body weight was calculated.

Urea concentration in the blood was estimated by the modified Berthelot's method 10, using a kit obtained from Agappe Diagnostics, Maharastra, India. Creatinine level in serum was estimated by alkaline picrate method 11.12, using a kit obtained from Dr.Reddy's Laboratories, Hyderabad, India.

Thiobarbituric acid reactive substances (TBARS)I3, glutathionel4

, catalase I 5 and protein l6 in tissue homogenate were estimated.

Histological methods-Small pieces of the cortex of the left kidney of each animal were fixed in 10% neutral buffered formalin, dehydrated in graded alcohol and embedded in paraffin wax. Sections (5/lm thick) were stained with haematoxyline and eosin (H&E) and subjected to microscopic examination for the presence of glomerular congestion, tubular casts, peritubular congestion, epithelial desquamation, blood vessel congestion, interstitial edema and inflammatory cells.

Statistical analysis-Results given as mean ± SE were analysed using one way ANOV A followed by post hoc Sheffe's Test using SPSS computer software version 7.5. The statistical significance of difference was taken as P<0.05.

Results Results of biochemical examination are presented

in Tables 1 and 2. Histological examination-On the 16th day (Table

2) the cisplatin treated rat kidney sections of group 3 showed marked congestion of the glomeruli with numerous tubular casts associated with epithelial desquamation (Fig. 1 b) as compared to the normal rat kidney (Fig. la) Marked peritubular congestion and edema were also observed. The interstitium showed infiltration with inflammatory cells and congestion. These features suggest that cisplatin induces acute tubular necrosis.

While group 4 (Fig.lc) continued to show mild glomerular and peritubular congestion, group 5 showed

688 INDIAN J EXP BIOL, JULY 2004

complete normalization of kidney section (Fig.ld). However mild glomerular, peri tubular congestion and inflammatory cells were still observed in group 5.

Discussion Cisplatin induced renal injury was evidenced by the

elevated biochemical markers such as blood urea and serum creatinine and by the histopathological features of acute tubular necrosis. Lupeol isolated from C. nurvala stem bark when administered at two dose levels i.e. 40 and 80 mg/kg body weight, significantly decreased the raised blood urea and serum creatinine and brought about a marked recovery in kidneys as evidenced microscopically.

In vivo and in vitro studies have demonstrated, that reactive oxygen metabolites viz. free radical species, superoxide, hydroxyl radical anion and hydrogen peroxide are important mediators of tissue injury 17-20.

Oxygen free radicals have been implicated in several biological processes, potentially important in I I d· 21 22 R h . I . g omeru ar Iseases . . eports suggest t at CISP atm

induces nephrotoxicity by InItlatlOn of lipid peroxidation and depletion of cellular thiols23

.24

.

Cisplatin inhibits the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathoine peroxidase) in rat kidneys25, thereby suggesting that cisplatin cytotoxicity results from generation of reactive oxygen species. The results obtained in the

Fig. I-Photomicrograph of (a) normal rat kidney showing normal glomeruli and tubules, (b) Cisplatin intoxicated showing glomerular congestion, (c) Lupeol (40mgikg) treated rat kidney showing mild glomerular and peritubular congestion, and (d) Lupeol (80mg/kg) treated rat kidney showing marked reduction in extent of tubular damage [H & E x 200].

SHIRW AIKAR et al.: EFFECT OF LUPEOL AGAINST NEPHROTOXICITY IN RATS 689

Table I-Effect of lupeol isolated from C. Ilurvala stem bark on various biochemical parameters in cisplatin induced renal damage [Values are mean ± SE from 6 rats in each group]

Groups % change in Blood urea Serum creatinine Glutathione TBARS Catalases body weight nitrogen (mg/dL) (Ilg/mg protein) (mMIlOO g tissue) (Ilg of H20 2 Imin

(mg/dL) Img protein)

1 13.17 ± 3.43 36.06± 3.00 0.57 ±0.07 4.40 ± 0.20 1.42 ± 0.15 380.14±21.96 2 -7.86±2.14' 98.21 ±2.31' 1.72±0.13" 2.21 ± 0.23" 2.36 ± 0.08a 254.35 ± 14.34 " 3 -13 .18 ± 1.72 a 73.32 ± 4.15 a 1.35 ± 0.08 a 1.34 ± 0.31 " 2.94 ± 0.11 a 210.16 ± 19.86" 4 -9.64 ± 2.35 59.03 ± 2.09 be 0.93 ± 0.02 be 2.54 ± 0.2 b 1.88 ± 0.05 b 361.30 ± 26.18 b 5 -6.71 ± 1.97 41.65 ± 1.91 be 0.78 ± om be 3.l8±0.27b 1.47 ± 0.07 be 376.Q2 ± 30.34 be

P values: a <0.05 Vs Control (Group 1); b <0.05 Vs cisplatin 16th day (Group 3); c <0.05 Vs cisplatin 6th day (Group 2). One way ANOVA followed by Post hoc Sheffe's test.

Table 2-Effect of lupeol on histological features of cisplatin induced renal damage

Histological features

Glomerular congestion Tubular casts

Group 1 Group 2 Group 3 Group 4

Peri tubular congestion Epithelial desquamation Blood vessel congestion Interstitial edema Inflammatory cells

- normal; + mild; ++ severe.

present study correlate with previous reports that lipid peroxidation contributes to cisplatin induced nephrotoxicity. Effects of several antioxidants like N,N! diphenyl-p-diphenyJenediamine and butylated hydroxy anisole and thiols like dithiothreitol and glutathione on cisplatin induced lipid peroxidation and glutathione depletion26 have been reported.

In the present study lupeoJ, was found to increase the glutathione level and catalase activity and to decrease the concentration of TBARS in the kidney cortex. Hence the possible mechanism of nephroprotection by lupeol may be attributed to its antioxidant and free radical scavenging properties.

To conclude, the results of the present study show that the lupeol isolated from the stem bark of C. nurvala possesses marked nephroprotective activity and could have a promising role to play in the treatment of acute renal injury induced by nephrotoxins, like cisplatin. Further work envisages evaluating the nephroprotective activity of this plant in chronic renal failure models.

Acknowledgement This study was supported by Manipal College of

Pharmaceutical Sciences, MAHE, Manipal, India.

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