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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2012  |  Volume : 23  |  Issue : 4  |  Page : 773-781
Nephroprotective effect of Kabab chini (Piper cubeba) in gentamycin-induced nephrotoxicity

1 Department of Ilmul Advia, National Institute of Unani Medicine, Bangalore, India
2 Department of Ilmul Advia, A.K. Tibbiya College, Aligarh Muslim University, Aligarh, India

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Date of Web Publication9-Jul-2012


Kabab chini (KC) (Piper cubeba) is an important drug in Unani Medicine, widely described to be effective in renal diseases, and physicians are using it as a protective and curative agent in various renal disorders from ancient times. The present study was designed to evaluate the nephroprotective effect of KC against gentamycin-induced nephrotoxicity in Wistar rats. This was studied in two different sets of tests, in which both the protective as well as the curative effects were evaluated in groups of albino rats. The powder of the test drug was administered orally in a dose of 810 mg/kg and 1220 mg/kg, in suspension form, in the pre- and post-treated models. The nephroprotective effect was assessed on the basis of biochemical estimation of serum urea and creatinine levels and histopathological examination of the treated kidney. The effect observed in the pre-treated and post-treated groups was compared with plain as well as negative control groups using one-way ANOVA with Dunnett's multiple pair comparison test. The findings of the two tests demonstrated that KC produced a significant nephroprotective effect in both pre-treated and post-treated groups. The results of our study indicate that KC possesses significant benefit against gentamycin-induced nephrotoxicity.

How to cite this article:
Ahmad QZ, Jahan N, Ahmad G, Tajuddin. Nephroprotective effect of Kabab chini (Piper cubeba) in gentamycin-induced nephrotoxicity. Saudi J Kidney Dis Transpl 2012;23:773-81

How to cite this URL:
Ahmad QZ, Jahan N, Ahmad G, Tajuddin. Nephroprotective effect of Kabab chini (Piper cubeba) in gentamycin-induced nephrotoxicity. Saudi J Kidney Dis Transpl [serial online] 2012 [cited 2022 Jan 24];23:773-81. Available from: https://www.sjkdt.org/text.asp?2012/23/4/773/98159

   Introduction Top

The berries of Kabab chini (KC) (Piper cubeba), commonly known as Cubeb, are used extensively as a condiment, particularly in the tropics. [1],[2],[3],[4] Old Arabian and Persian physicians are said to have used the fruit in urogenital diseases. [5] In Indonesia, Cubeb is valued as a medicinal plant and is used in traditional me­dicine to treat gonorrhea, syphilis, abdominal pain, diarrhea, enteritis and asthma. [6] It has been described in the classical Unani literature in detail and various actions such as Mudirre baul (diuretic), Mufattit wa Mukhrije Hasat (lithotriptic), Dafeye taffun (antiseptic), Muqa-wwiye Kulyah (kidney tonic), Mohafize Kulyah (nephroprotective), etc. have been ascribed to it. Although a number of other effects have also been described to be possessed by this drug, it is more often used in urogenital disorders. [7],[8],[9],[10] Although it has not been studied as such for its nephroprotective and related effects, some of its constituents such as yatein, hinokinin, cubebin and dihydrocubebin have been reported to possess anti-inflammatory, analgesic, anti-oxidant and anti-cancer activities. [11],[12],[13],[14],[15] A number of polyhydroxy cyclohexanes have been isolated from Piper cubeba and have shown to display tumor-inhibitory, anti-leukemic and antibiotic activities. [16] In view of the above, therefore, the present study was designed to evaluate scientifically the nephroprotective activity of KC in albino rats. The effect of the test drug was studied in animals subjected to nephrotoxicity by the administration of gentamycin. The study was conducted in groups of animals given KC before (pre-treated) or after (post-treated) ad­ministering gentamycin in order to evaluate its protective as well as curative effects.

   Materials and Methods Top

The present study was undertaken in the Department of Ilmul Advia, National Institute of Unani Medicine (NIUM), Bangalore, India. Before starting the experiment, the research protocol was submitted for ethical clearance. The Institutional Animal Ethics Committee (IAEC) of the NIUM, Bangalore, approved the protocol for the study. (Registration number: 953/C/06/ CPCSEA, dated 07/12/2007.)

Method of preparation, dosage and mode of administration of test drug

The dried berries of KC were provided by the pharmacy of NIUM, Bangalore. The test drug was properly identified and authenticated by Dr. Siddamallayya N, of the Regional Research Institute (Ay.) Bangalore, vide Ref. No. RRI/ BNG/SMP/Drug Authentication/2008-09/356, and was powdered finely with the help of an electric grinder.

The dose of the test drug for the rats was calculated by multiplying the human therapeu­tic dose (7 g) described in the Unani literature by the conversion factor of seven. [17] The dose thus calculated for experimental study was found to be 810 mg. However, to study the dose-dependent effect of the test drug, another dose was also employed by increasing the calculated dose by 50%, which was found to be 1220 mg. Five grams of gum acacia was taken in 100 mL of distilled water. It was shaken vigo­rously for some time to make a homogenous suspension. This suspension was divided into two equal parts. One part was mixed with 8.5 g of KC powder while the other, with 12.5 gm of powder. Both the samples were mixed and shaken well to get a suspension of uniform distribution. The test drugs were prepared fresh every time before administration to the animals.

Experimental animals

Healthy adult albino rats of Wistar strain weighing 150-200 g were used in the study. Animals were maintained on standard diet and water ad libitum unless stated otherwise, and housed in clean polypropylene cages at room temperature (25 ± 2°C) with a 12-h light:12-h dark cycle.

Test for gentamycin-induced nephrotoxicity

This test was carried out by the method of Shirwaikar et al. [18] Male Wistar rats weighing 150-200 g were divided into six groups con­sisting of 10 animals each and treated as fol­lows [Table 1]:
Table 1: Treatment schedules used in the different groups of animals studied.

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  1. Plain control: Normal saline 3 mL orally for 14 days.
  2. Negative control: Gentamycin 80 mg/kg body weight, intramuscular (i.m.), for seven days.
  3. Pre-treated test group - A: Animals in this group were treated with the powder of KC in a dose of 810 mg/kg body weight, orally, once daily for 14 days and, from the eighth day, they were administered gentamycin injection i.m. at a dose of 80 mg/kg body weight once daily, in addition to KC.
  4. Post-treated test group - A: Animals were administered gentamycin injection i.m. as in the previous test at a dose of 80 mg/kg body weight daily for seven days to induce nephrotoxicity and, from the eighth day, KC was administered at a dose of 810 mg/kg body weight orally, once daily to all the animals for the next 14 days.
  5. Pre-treated test group - B: The animals in this group were treated with KC at a higher dose of 1220 mg/kg body weight orally once daily for 14 days and, from the eighth day, they were injected gentamycin i.m. at a dose of 80 mg/kg body weight once daily for seven days.
  6. Post-treated test group - B: The animals in this group were administered gentamycin i.m. in a dose of 80 mg/kg body weight for seven days to induce toxicity. From the eighth day onwards, they were treated with KC given orally in a dose of 1220 mg/kg body weight daily for 14 days.
At the end of the experiment, the rats were sequentially anesthetized with inhaled diethyl ether for about 30-40 s. About 4-5 mL of whole blood was collected by cardiac punc­ture in a 10-mL capacity plain sample bottle. The blood samples were allowed to clot com­pletely for about 3-5 h, after which they were centrifuged at 2000 revolutions per minute for 15 min. This was aimed at separating the sera from the clotted blood cells. The sera were carefully separated into new, well-labeled, cor­responding plain sample bottles at room tem­perature (23-26°C). The sera were assayed for urea and creatinine using diagnostic kits.

Histopathological studies of rat kidneys

After the animals were sacrificed, postmortem examination was performed. Their kidneys were identified and carefully dissected out en bloc for histopathological examination. After rinsing in normal saline, sections were taken from each harvested kidney. The tissue was fixed in 10% formosaline, dehydrated with 100% ethanol solution and embedded in paraf­fin. It was then processed into 4-5-μm-thick sections stained with hematoxylin and eosin and examined under a photomicroscope.

   Statistical Analysis Top

The parameters mentioned above were asses­sed in all the groups and the findings were expressed as mean ± SEM. The different va­lues determined were compared with each other and comparison was made using one­way ANOVA with Dunnett's multiple pair comparison test. The difference of mean was considered significant at a P-value of 0.05 or less.

   Results Top

Effect of test drug on gentamycin-induced nephrotoxicity

Serum urea

Serum urea in the plain control group was estimated to be 30 ± 2.302 mg/dL. In the nega­tive control group treated with gentamycin only, it was 156 ± 39.56 mg/dL. In the pre-treated group A, where animals were admi­nistered KC orally at a dose of 810 mg/kg along with gentamycin, the serum urea was found to be 40.6 ± 5.469 mg/dL. There was a significant decrease as compared with the negative control group (P < 0.01). Similarly, the post-treated group A, in which animals received gentamycin along with KC at a dose of 810 mg/kg, also demonstrated a decrease in serum urea from 156 ± 39.56 mg/dL to 47.14 ± 8.096 mg/dL as compared with the negative control group (P <0.01). In the pre-treated group B, where the animals were administered KC for seven days before gentamycin administration, a significant decrease was found in serum urea, which was 43.37 ± 3.035 mg/dL. In the post-treated group B, where the animals were admi­nistered gentamycin before the administration of test drug, the level of serum urea decreased significantly to 57.66 ± 8.417 mg/dL (P <0.01) as compared with negative controls. Results are summarized in [Table 2], [Table 3] and graphically presented in [Figure 1].
Figure 1: Graphic representation of the effect of Kabab Chini on blood urea in gentamycin-treated rats.

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Table 2: Effect of KC (Piper cubeba) on serum urea and creatinine in gentamycin-induced toxicity.

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Table 3: Histopathological features as seen in the kidney of gentamycin-treated animals.

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Serum creatinine

The serum creatinine in the plain control group was 0.7571 ± 0.1008 mg/dL, whereas in the negative control group, it increased to 3.0125 ± 0.2741 mg/dL. In the pre-treated group A, the serum creatinine level was 1.4275 ± 0.1145 mg/dL (P <0.01). Similarly, the post-treated group A also showed a significant de­crease in serum creatinine from 3.0125 ± 0.2741 mg/dL to 1.0225 ± 0.09246 mg/dL as compared with the negative control group (P <0.01). In the pre-treated group B, the serum creatinine level was 1.03375 ± 0.1274 mg/dL, which showed a significant decrease (P <0.01), while in the post-treated group B, the serum creatinine was 1.07 ± 0.085 mg/dL (P <0.01). The results are summarized in [Table 2] and presented graphically in [Figure 2].
Figure 2: Graphic representation of the effect of Kabab chini on serum creatinine in gentamycin-treated rats.

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Histopathological examination

In the plain control group, microscopic exa­mination of the kidney showed a good number of glomeruli. Numerous blood vessels were seen in both cortex and medulla. No histopathological abnormalities were observed [Figure 3].
Figure 3: Renal histology in the plain control group showing normal structure of kidney.

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In the negative control group, histopathological findings showed kidney structure distorted by severe necrosis of tubules. The stroma was edematous. The tissue was infiltrated by numerous chronic inflammatory cells. Engorged blood vessels and areas of hemorrhage were seen. Features suggested severe tubular nec­rosis [Figure 4].
Figure 4: Renal histology in the negative control group showing severe tubular necrosis.

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In the pre-treated group A, histopathological findings showed the stroma with a mild degree of edema. There was a mild degree of glomerular congestion. The tissue was sparsely infil­trated by inflammatory cells. Features sugges­ted moderate tubular damage [Figure 5].
Figure 5: Renal histology in the pre-treated A group showing mild tubular damage.

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In the post-treated group A, histopatological examination showed that there was mild inters­titial edema. Mild degree of congestion was also seen in the glomeruli. Numerous engorged blood vessels were seen. Mild tubular changes were noted. The tissue was free from inflam­matory cells [Figure 6].
Figure 6: Renal histology in the post-treated A group showing moderate tubular necrosis with significant reversal of inflammatory changes.

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In the pre-treated group B, histopathological findings showed mild interstitial edema, mild degree of peritubular and glomerular conges­tion and numerous engorged blood vessels. The tissue was free from inflammatory cells. Features suggested mild tubular changes [Figure 7]. In the post-treated group B, histopathology showed mild interstitial edema, mild degree of glomerular congestion and few congested blood vessels. Mild tubular damage was observed. The tissue was sparsely infiltrated by chronic inflammatory cells [Figure 8].
Figure 7: Renal histology in the pre-treated B group showing mild tubular changes with significant reversal of inflammatory changes.

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Figure 8: Renal histology in the post-treated B group showing mild tubular damage with significant reversal of inflammatory changes.

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   Discussion Top

Gentamycin is a known nephrotoxic agent re­ported to induce a significant degree of nephro-toxicity at different dose levels. Its nephro-toxic potential was established at a dose level of 80 mg/kg in albino rats. [19],[20] In the present study, this was evidenced by significant (P <0.01) elevations in the serum urea and creatinine concentrations when compared with the plain control group. The nephrotoxic effect was further corroborated by the histological findings in which many of the glomeruli showed diffuse eosinophilic sclerosis, engorged blood vessels and areas of hemorrhage, indicating severe tubular necrosis [Figure 5] and [Figure 8], while in the plain control group, normal histopathological features were seen [Figure 4]. This func­tional and structural derangement caused by the toxic agent is in agreement with other re­ports showing its nephrotoxic effects. [21],[22],[23] The markers of kidney function and structure in this study were grossly seen to be within the normal limits in the groups of animals treated with KC thus demonstrating nephroprotective effect. In the pre-treated group A, biochemical mar­kers of kidney function were found to be sig­nificantly lower than in the control group, while histological examination showed features suggestive of mild tubular damage [Figure 6]. Similarly, the animals in the post-treated group A demonstrated a significant decrease in se­rum urea and creatinine levels as compared with the negative control group (P <0.01). Mic­roscopic examination demonstrated moderate degree of tubular necrosis [Figure 7]. Thus, the pre-treated and post-treated groups showed that KC produced a significant degree of nephroprotection based on the biochemical markers of kidney function and the histopathological features. In inter-group compa­rison, however, the effect in the pre-treated group was more significant (P <0.05).

An almost similar pattern was observed in groups treated with a higher dose of KC as both the indices of kidney function and its matrix were found within the normal limits and dose-dependant response was observed as com­pared with that of low-dose therapy (P <0.01). The test drug, by not allowing the biochemical markers of kidney function as well as structural integrity to change beyond the normal limits even on administration of a high dose of gentamycin, clearly indicated that it possessed a striking nephroprotective effect. The findings further suggested that KC at a higher dose was more effective, but there was no difference in the post-treatment and pre-treatment effects. It suggested that at a higher dose, KC is equally effective both as protective as well as curative agent.

It has been reported that gentamycin accu­mulates in renal tubules, particularly in the S 1 and the S 2 segment of the proximal tubules in lysosomal and endosomal vacuoles and elicits an array of morphological and functional alte­rations of increasing severity through phospolipidosis. Therefore, renal dysfunction in this case represents renal tubular damage involving the cortical region, which is manifested by the increased level of urea and creatinine in serum and structural changes on histopathiological studies. [20],[21],[22] The findings of the study also poin­ted toward the site of action, i.e. the cortical region of the kidney, and also the likely mecha­nism of action, i.e. phospholipase-like activity. In another study, KC has been reported to produce an anti-inflammatory effect. [23] The role of anti-inflammatory effect apparently looks counter-productive to an injured organ, as anti-inflammatory agents usually delay the healing process. [24] However, the test drug pro­duced a healing effect, as was seen in the curative groups where the injury induced by the toxins improved significantly. This is pro­bably because a number of inflammatory me­diators such as endothelin-1, monocyte chemo-attractant protein-1, etc. are excessively pro­duced because of the toxic effect. Thus, an anti-inflammatory property in this condition may help improve the kidney function by neu­tralizing the pro-inflammatory mediators. Fur­ther, the anti-inflammatory drugs inhibit the production of vasodilator prostaglandin PGE2 thereby causing vasoconstriction, which is the primary cause of decrease in glomerular filtra­tion. This further aggravates the toxicity of aminoglycosides [25],[26] but, the test drug itself, despite having anti-inflammatory effects as re­ported by some authors, [23] produced significant nephroprotective effect in gentamycin-induced toxicity. This is indeed a testament of the drug having a striking nephroprotective effect.

Some of the compounds contained in KC, such as cubebin, hinokinin, yatein, dihydrocubebin, etc., have been shown to possess anti-inflammatory, analgesic, antioxidant and anti­cancer activities. [27],[28],[29] This further corroborates the proposition that the test drug produces its effects through diverse mechanisms comple­menting each other and that the collective res­ponse actually translates into nephroprotection.

The findings of the study demonstrated that KC produced a significant effect in both pre-treated and post-treated groups. Further, in-group comparison between pre-treated and post-treated groups showed that the effect of the test drug was more marked in the pre-treated groups as compared with the post-treated groups. Thus, it seems that the protective effect of the test drug is more pronounced than its curative effect.

The protective effect demonstrated by the test drug fortified the Unani concept of tonic drugs that help to maintain the quwa to perform the normal function of the organ even in deviated conditions of structure and function, by streng­thening the affected organ to fight against the toxins. Further, because the test drug possesses different pharmacological effects, it can be said that it produced a protective effect by evolving a holistic paradigm and involving its numerous actions, and addressed almost all aspects of toxic effects on kidney tissue to bring the chemically challenged kidney to a state of functional and structural normalcy. This drug can be successfully used to prevent and treat the kidney disease and also to delay the progression of renal diseases.

On the basis of our findings, it can be con­cluded that the test drug possessed significant nephroprotective effect against gentamycin, a known nephrotoxic agent. Thus, the study vali­dated the claim of the Unani System of Medi­cine that KC is a drug that can be used as a nephroprotective agent in a number of renal diseases.

   References Top

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11.Da Silva R, De Souza GH, Da Silva AA, et al. Synthesis and biological activity evaluation of lignan lactones derived from ()-cubebin. Bioorg Med Chem Lett 2005;15:1033-7.  Back to cited text no. 11
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16.Coimbra Hdos S, Royo Vde A de Souza VA, et al. Analgesic and anti-inflammatory activities of (-)-o benzyl cubebin, a (-)- cubebin deri­vative, obtained by partial synthesis. Boll Chim Farm 2004;143:65.  Back to cited text no. 16
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26.Silva ML, Coímbra HS, Pereira AC, et al. Eva­luation of Piper cubeba extract, (-)- cubebin and its semi-synthetic derivatives against oral pathogens., Phytother Res 2007;172-8.  Back to cited text no. 26
27.Hardik S, Bodiwala G, Singh R, Chinmoy, Sankar D, Shyam SS, et al. Antileish- manial amides and lignans from Piper cubeba and Piper retrofractum. J Natural Med 2007;61: 481-2.  Back to cited text no. 27
28.Aqil F, Ahmed I, Mehmood Z. Antioxidant and free radical scavenging properties of twelve traditionally used Indian medicinal plants. Turk J Biol 2006;30:177-83.  Back to cited text no. 28
29.Karthikeyan J, Rani P. Enzymatic and non-enzymatic antioxidants in selected Piper species. Indian J Exp Biol 2003,41:135-40.  Back to cited text no. 29

Correspondence Address:
Ghufran Ahmad
Department of Ilmul Advia, Faculty of Unani Medicine, Aligarh Muslim University, Aligarh
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DOI: 10.4103/1319-2442.98159

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]

  [Table 1], [Table 2], [Table 3]

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