Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 39, Num. 4, 2007, pp. 201-205

Indian Journal of Pharmacology, Vol. 39, No. 4, July-August, 2007, pp. 201-205

Research Paper

Protective effect of Kalanchoe pinnata pers. (Crassulaceae) on gentamicin-induced nephrotoxicity in rats

Gaurav Vijay Harlalka, Chandragauda Raosaheb Patil, Mahesh Ramu Patil

Correspondence Address: R. C. Patel College of Pharmacy, Near Karwand Naka, Shirpur -425405, Dhule, Maharashatra
gauravh12@yahoo.co.in

Date of Submission: 19-Jul-2006
Date of Decision: 01-Sep-2007
Date of Acceptance: 27-Sep-2007

Code Number: ph07049

Abstract

Keywords: Antioxidant, Bryophyllum pinnata , gentamicin, nephrotoxicity

Extensive use of plants belonging to the Bryophyllum species in complementary and alternative therapy has been widely reported. These plants are also used in the treatment of certain diseases like urolithiasis, hypertension and diabetes involving altered kidney function. ^[1],[2] However, systematic and scientific reports on the investigation of K. pinnata for its effects on renal function are scarce. In the present study, an effort has been made to evaluate the effects of the aqueous leaf extract of this plant on gentamicin-induced nephrotoxicity in rats.

Gentamicin-induced nephrotoxicity is a model of acute renal failure caused by oxidative stress generated through the induction of superoxide anions. ^[3] Hence, in vitro antioxidant activity of this extract has been further investigated.

Materials and Methods

Plant material

Fresh leaves were collected from the herbal garden of our institute and authenticated as K. pinnata (Lam.) Pers. ( Crassulaceae ) by the Botanical Survey of India, Pune, India. The voucher specimen (number - 120445) was submitted for future reference. The aqueous extract of the plant was prepared by using the cold maceration process. The extract was dried in a vacuum evaporator below 40^° C and stored in air-tight, amber-colored containers at room temperature.

Animals

Healthy, male albino Wistar rats each weighing 150-200 g were used for this study. The rats were housed in polypropylene cages and maintained under standard conditions (12 h light and dark cycles, at 25±3^° C and 35-60% humidity). Standard pelletized feed and tap water were provided ad libitum . The study was approved by the Institutional Animal Ethical Committee of R. C. Patel College of Pharmacy, Shirpur, India, registered under CPCSEA, India (Registration No. 651/02/C/CPCSEA).

Dose and route of administration

According to earlier reports, the LD ₅₀ value for the aqueous extract of leaves of K. pinnata is 560 mg/kg i.p. in rats. ^[4] The extract has been found to be ineffective orally in mice and rats as it did not produce any gross observable effect even at doses as high as 2000 mg/kg p.o. Lack of any observable biological effect when administered by the oral route was attributed to inadequate absorption of the active phytoconstituents of this plant. ^[5],[6] In a preliminary study conducted in our laboratory, the leaf extract of K. pinnata did not exert any observable effects in rats when given by the oral route. However, intraperitoneal administration of the same extract was found to cause diuresis. Based on this observation and earlier reports, the dose of 125 mg/kg given by intraperitoneal route, which caused significant diuresis without any observable toxicity, was used throughout this study.

Nephroprotective activity

Eighteen male Wistar albino rats were assigned to three groups: control group, Gentamicin-treated group and Gentamicin- as well as K. pinnata -treated group, each group containing six rats. The gentamicin-treated group received 100 mg/kg/day gentamicin (Hi Media Laboratories, India) by the intraperitoneal (i.p.) route. ^[7] The K. pinnata -treated group received 100 mg/kg/day gentamicin i.p. and 125 mg/kg/day of the aqueous extract of K. pinnata i.p. for eight days. Rats in the control group were given sterile saline solution i.p. for the same number of days. After dosing on the 8 ^th day, individual rats were placed in separate metabolic cages for 24 hours for urine collection to determine urine output and urine creatinine content. ^[8] Blood samples were collected via retro-orbital puncture at the end of these 24 hours. The serum was rapidly separated and processed for determination of blood urea nitrogen (BUN) and serum creatinine using commercially available kits of Span Diagnostics Ltd, India. ^[4] Changes in body weight were recorded. Three rats per group were sacrificed and both kidneys were isolated from each rat. ^[9] The kidneys were weighed and processed for histopathological examination. ^[10]

Histopathological examination

The kidneys were sectioned longitudinally in two halves and were kept in 10% neutral formalin solution. ^[11] Both kidneys were processed and embedded in paraffin wax and sections were taken using a microtome. The sections were stained with hematoxylin and eosin and were observed under a computerized light microscope (Motic images 2000, version 1.3, China).

In vitro antioxidant activity

For all the in vitro antioxidant models mentioned below, ascorbic acid was used as a reference standard. The concentrations of ascorbic acid were 10, 20, 30, 40, 50 µg/ml and that of extract were 50, 100, 150, 200, 250 µg/ml.

DPPH free radical-scavenging activity

To determine the antioxidant activity of the leaf extract, a method based on the reduction of a methanolic solution of the colored free radical 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) was used. ^[12] The methanolic solution of DPPH (0.1 mM, 1 ml) was incubated with 3 ml of different concentrations of the leaf extract ranging from 50 to 250 μg/ml. Incubation was carried out at room temperature (25°C) for 30 min. For each concentration, the assay was run in triplicate. At the end of the incubation period, the optical density of each sample was determined at 517 nm. ^[13] Ascorbic acid (Loba Chemie, India) solution was used as a standard. EC ₅₀ values (concentration required to scavenge 50% of the free radicals) for both Ascorbic acid and the leaf extract were determined. The radical scavenging activity of the tested sample was expressed as an inhibition percentage (IP).

DPPH Scavenged (%) = (A _DPPH - A _test / A _DPPH ) x 100

where A _DPPH is the absorbance of the 0.1 mM of DPPH solution and A _test is the absorbance in the presence of the extract or Ascorbic acid.

Nitric oxide radical-scavenging activity using the Griess Illosvay reaction ^[12],[14]

Free radical-scavenging activity was evaluated by studying the inhibition of the generation of Nitric Oxide from Sodium Nitroprusside. An aqueous solution of sodium nitroprusside at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions. The nitrite ions thus produced can be quantified using their reaction with Griess reagent that leads to formation of a chromophore, the concentration of which is proportional to that of the generated nitrite ions. Scavengers of nitric oxide compete with oxygen leading to a reduced production of nitric oxide.

In this assay, 1.0 ml of Sodium nitroprusside (5 mM) in phosphate-buffered saline (PBS) was mixed with 3.0 ml of different concentrations (50-250 µg/ml) of the extract dissolved in the distilled water. The assay mixture was then incubated at 25°C for 150 minutes. These solutions were treated with Griess′ reagent and the optical density of the resultant chromophore determined spectrophotometrically at 546 nm and compared with the absorbance of standard solutions of ascorbic acid simultaneously run in identical assay units. The experiment was run in triplicate. As a blank, the assay mixture similarly run in the absence of the extract or ascorbic acid was used.

Reducing power assay ^[15]

As a measure of antioxidant activity, the reducing power of the extract was also determined as follows: 1 ml of different concentrations of the K. pinnata extract solutions were added to 2.5 ml of 1% potassium ferricyanide in different test tubes and the resultant mixture incubated at 50^° C for 20 min. Then, 2.5 ml of 10% trichloroacetic acid was added to each tube. The tubes were centrifuged for 10 min at 3000 rpm. The supernatant from each tube (2.5 ml) was taken in a separate test tube and 2.5 ml of distilled water and 0.5 ml (0.1%) ferric chloride solution were added to each tube. The absorbance of these assay mixtures was measured at 700 nm. Increase in the absorbance of the reaction mixture was considered to be the reducing power of the extract.

In parallel to this, the reducing power of ascorbic acid was also determined for comparison.

Anti-lipid peroxidation

Decomposition of the lipid membrane of cells leads to the formation of Malondialdehyde (MDA) along with other aldehydes and enols as end-products. Malondialdehyde (MDA) formed during lipid peroxidation then reacts with thiobarbituric acid (TBA) to form a colored complex which can be spectrophotometrically measured at 532 nm. ^[16]

Anti-lipid peroxidation in liver homogenate ^[17],[18]

Preparation of liver homogenate

Rat liver was perfused with ice-cold 0.15 M KCl through the portal vein. The perfused liver was isolated and 10% (w/v) homogenate was prepared in PBS using a tissue homogenizer below 4^° C. This homogenate was used to study in vitro lipid peroxidation.

The assay mixtures contained 0.5 ml of homogenate, 1 ml of 0.15 M KCl and 0.5 ml of different concentrations of the extract. Lipid peroxidation was initiated by adding 100 µl of 1 mM ferric chloride. The reaction mixture was incubated for 30 minutes at 37^° C. After incubation, the reaction was stopped by adding 2 ml of ice-cold 0.25 N HCl containing 15% trichloroacetic acid and 0.38% TBA as well as 0.2 ml of 0.05% butylated hydroxyl toluene. The reaction mixture was heated for 60 min at 80^° C, cooled to room temperature and centrifuged at 5000 rpm for 15 minutes. Optical density (O.D.) of the supernatant from each tube was measured at 532 nm against a blank which contained all reagents except liver homogenate and plant extract. Identical experiments were performed to determine the normal (without drug and ferric chloride) and induced (without drug) lipid peroxidation. The percentage of anti-lipid peroxidation effect (% ALP) was calculated by following formula:

% ALP = (Ferric chloride O.D. - Sample O.D./ Ferric chloride O.D. - Normal O.D.) x 100

Statistical analysis

The data obtained was analyzed using one-way ANOVA followed by Dunnette′s multiple comparison test. P < 0.05 was considered significant.

Results

Nephroprotective Activity: Urine creatinine, serum creatinine, blood urea, blood urea nitrogen and the weights of the kidneys were found to be significantly increased in rats treated with only gentamicin; whereas treatment with the aqueous extract of the leaves of K. pinnata was found to protect the rats from such effects of gentamicin. As shown in [Table - 1], urine volume was found to be significantly increased in the rats treated with K. pinnata leaf extract.

The body weights of the rats treated with gentamicin were also found to be significantly reduced as compared to control group and K. pinnata-treated rats.

Histopathological examination

Control rats showed normal glomerular and tubular histology whereas gentamicin was found to cause glomerular, peritubular and blood vessel congestion and result in the presence of inflammatory cells in kidney sections from the gentamicin-treated group. Concurrent treatment with the extract was found to reduce such changes in kidney histology induced by gentamicin [Figure - 1] and [Table - 2].

In vitro antioxidant activity

DPPH method

EC ₅₀ value for the Standard Ascorbic Acid Solution was found to be 11.25 µg/ml whereas EC ₅₀ for the aqueous extract of leaves of K. pinnata was found to be 116.25 µg/ml [Table - 3].

Nitric oxide radical-scavenging activity

EC ₅₀ of the Standard Ascorbic acid solution was found to be 15.5 µg/ml while it was 90 µg/ml for the extract [Table - 3].

Reducing power assay

Proportional increases in reducing power of both Ascorbic acid and the extract of K. pinnata were observed with increasing concentrations.

Anti-lipid peroxidation in liver homogenate

EC ₅₀ of the standard Ascorbic acid solution was found to be 14.0 µg/ml while EC ₅₀ for the aqueous extract of the leaves of K. pinnata was found to be 125 µg/ml [Table - 3].

Discussion

Our study results show that the aqueous extract of leaves of Kalanchoe pinnata possesses potent nephroprotective and in vitro antioxidant activity. This plant contains different classes of phytochemicals such as flavonol glycosides like quercetin-3-L-rhamnosido-L-arabinofuranoside, quercetin-3-diarabinoside and kaempferol-3-glucoside, many alkanes C25-C35 (n-hentriacontane, n-triacontane predominating) and alkanols C ₂₆ -C ₃₄ . Pentacyclic triterpenoids like α-amyrin, b-amyrin and sterols like sitosterol have also been isolated from the non-saponifiable fraction. Furthermore, the presence of other phenolic constituents like p -coumaric, ferulic, syringic, caffeic and p -hydroxybenzoic acids ^[19] and organic acids like isocitric ^[20] and citric acids has been reported. ^[21]

Quercetin and kaempferol are detected in the leaves. An earlier report ^[22] suggests that quercetin has a marked protective effect on cadmium-induced nephrotoxicity that results from an increase in Metallothionein, a small cysteine-rich protein and eNOS (endothelial nitric oxide synthase) expression and the inhibition of COX-2 (cyclooxygenase-2) and iNOS (inducible nitric oxide synthase) expression.

The results of our study suggest that K. pinnata contains constituents having nephroprotective and antioxidant activities, which are comparable to that of ascorbic acid. Further investigations using specific fractions of this extract can help to isolate and identify potential nephroprotective and antioxidant constituents.

Acknowledgment

References

Copyright 2007 - Indian Journal of Pharmacology

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