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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 21  |  Issue : 6  |  Page : 1058-1065
The reno-protective effect of aqueous extract of Carum carvi (black zeera) seeds in streptozotocin induced diabetic nephropathy in rodents

1 Department of Pharmacology, University of Health Sciences, Khayaban-e-Jamia Punjab, Lahore, Pakistan
2 Department of Pathology, University of Health Sciences, Khayaban-e-Jamia Punjab, Lahore, Pakistan
3 Department of Pharmacology, Rawalpindi Medical College, Rawalpindi, Pakistan

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Date of Web Publication4-Nov-2010


To assess the effect of aqueous extract of Carum carvi seeds in experimentally induced diabetic nephropathy (DN) in rodents, we studied 48 adult male Wistar rats divided into 4 groups: normal controls (group A), diabetes positive control (group B), and experimental (groups C and D). They received Carum carvi extract as a renoprotective agent. Rats having fasting blood glucose levels over 280 mg/dL were included in this study. Group C rats received STZ (60 mg/kg) and aqueous extract of Carum carvi at 30 mg/kg of body weights. On the other hand group D rats received STZ (60 mg/kg) and aqueous extract of Carum carvi at 60 mg/kg of body weight. Blood samples were collected on the 60 th day, and kidneys were also extracted for examination. The diabetic group rats showed a variable increase in the serum levels of glucose, urea, creatinine, total urinary protein and microalbuminuric levels. Body weight decreased and urine volume increased in the diabetic groups. 30 mg/kg body weight of Carum carvi dose decreased the levels of these parameters in rats. On the other hand, 60 mg/kg body weight of Carum carvi dose significantly decreased the levels of the biochemical parameters. The morphological examination of group C rats showed no changes whereas the rats in group D showed moderate changes. Carum carvi constituents, especially flavonoids and carvone have strong anti-oxidant activity, which provides reno-protection against diabetes and its complications. In conclusion, high dose of Carum carvi aqueous seeds extract (60 mg/kg) showed reno-protection against STZ induced dia­betic nephropathy in rats.

How to cite this article:
Sadiq S, Nagi AH, Shahzad M, Zia A. The reno-protective effect of aqueous extract of Carum carvi (black zeera) seeds in streptozotocin induced diabetic nephropathy in rodents. Saudi J Kidney Dis Transpl 2010;21:1058-65

How to cite this URL:
Sadiq S, Nagi AH, Shahzad M, Zia A. The reno-protective effect of aqueous extract of Carum carvi (black zeera) seeds in streptozotocin induced diabetic nephropathy in rodents. Saudi J Kidney Dis Transpl [serial online] 2010 [cited 2021 Sep 27];21:1058-65. Available from: https://www.sjkdt.org/text.asp?2010/21/6/1058/72292

   Introduction Top

Nephropathy is one of the important micro­angiopathic complications of diabetes mellitus. Recent studies indicate that reactive oxygen species play a key intermediate role in the pa­thophysiology of diabetic nephropathy. [1],[2] Hyper­glycemia, not only generates more reactive oxygen metabolites, but also attenuates anti­oxidative mechanisms through non enzymatic glycosylation of anti-oxidant enzymes. [3],[4]

The mechanism of production of free radical generation by high glucose is complex. High glucose causes glycosylation of circulating and cellular protein and may initiate a series of auto-oxidative reactions that results in the for­mation and accumulation of advanced glyco­sylation end-products (AGE) in tissue pro­teins. [5] The AGE has oxidizing potential and can promote tissue damage by free radicals. [1],[5] The activation of hyperglycemia-induced se­condary mediators, such as protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), and cytokine production are also res­ponsible for oxidative stress induced renal in­jury in the diabetic conditions. [6]

Various experimental animals have been uti­lized to investigate diabetic nephropathy, how­ever, streptozotocin (STZ) induced hypergly­cemic rats have been used in this model of dia­betic nephropathy.

Caraway, locally known as Black Zeera is a member of the group of aromatic umbelliferous plants. Caraway is one of the oldest spices cul­tivated in Europe. It is naturally found in Nor­thern and Central Europe, Siberia, Turkey, Iran, India, and North Africa. The fruit of caraway is a schizocarp, which at harvest splits into two halves called seeds. [7] Caraway has been used since ancient times, especially for the treat­ment of digestive disorders. Caraway is known to have antihyperglycemic effect. Eddouks and Lemhadri showed that aqueous extract of cara­way has antihyperglycemic effect in STZ in­duced diabetic rats. [8] The plant extract and vo­latile oils from Carum carvi have also been used as antiulcerogenic agents and anti-flatulent colic in infants. [9],[10] Caraway has been commonly used in phytomedicine as antibacterial, anti­proliferative, and laxative agent. [11],[12],[13] The major constituents of its seeds are carvone, flavonoids and limonene. In addition, myrcene, beta car­yophyllene, thujone, anethole, and pinene are present as minor components. [7] The flavonoid constituents of caraway have been separated by means of chromatography on cellulose co­lumns and constituents such as quercetin-3-glu­curonides, isoquercitrin, quercetin 3-0 caffeyl­glucoside, and kaempferol 3-glucoside were obtained. [14]

The purpose of the present experimental stu­dy is to observe the effects of Carum carvi on STZ induced diabetic nephropathy in Wistar rats.

   Materials and Methods Top


Forty eight adult male Wistar rats weighing 200-250 g were procured for this study. They were kept in the experimental research labo­ratory of the University of Health Sciences, Lahore, Pakistan under day and night conditions. Prior to the commencement of the experiments, all the animals were kept for one week under the same laboratory conditions, at a tempera­ture of 22 ± 2 ºC, relative humidity of 70 ± 4% and 12 hour light/day cycle. They received nutritionally standard diet and tap water. The care and handling of rats were in accordance with the internationally accepted standard guide­lines for use of experimental animals.

Plant materials and preparation of the extract

Seeds of Carum carvi were collected from the local market of Lahore and were authen­ticated from a botanist (Dr. Ghazala, Professor of Botany, Punjab University, Lahore, Pakistan). Carum carvi seeds were coarsely powdered using a grinder. Dried powdered Carum carvi seeds (50 g) were mixed with 100 mL of water and the mixture was blended. After blending, the mixture was stirred with the help of a mag­netic stirrer for 3 days. After the decant had settled down, the supernatant was separated and centrifuged; then it was incubated after centri­fugation in order to obtain the powdered form of the extract. The seeds (voucher no. 0786) and extract (voucher no. 0787) were deposited in the Pharmacology laboratory, University of Health Sciences, Lahore. This supernatant got standardized from the Pakistan Council for Scientific and Industrial Research (PCSIR) la­boratories in Lahore.

Experimental Procedure

After acclimatization, 12 rats were labeled as controls. All the other rats were starved for 16 hours and diabetes was induced using a single intraperitoneal injection of freshly dissolved STZ (60 mg/kg) in 0.01M citrate buffer (pH 4.5). Following the STZ injection, the rats were given drinking water supplemented with suc­rose (15 g/L) for 48 hours to limit the early mortality as stores of insulin were released due to the damage induced by the STZ in the pan­creatic islets. One week after the STZ injec­tion, the rats were assessed for diabetes and those with fasting blood glucose over 280 mg/ dL were included in this study. To prevent sub­sequent development of ketonuria, the diabetic rats were given subcutaneous injections of in­sulin (2-4 U/rat) to maintain blood glucose le­vels in the desirable range (above 280 mg/dL). [15]

Thereafter, all rats were divided into four groups each having 12 animals. The control rats (Group A) were fed on standard diet with tap water and received no drugs. The group B i.e. diabetic control rats received 60 mg/kg of STZ as a single intraperitoneal injection and were fed on standard diet and tap water. The group C i.e. experimental group rats received 60 mg/kg of STZ as a single intraperitoneal injection and aqueous extract of Carum carvi seeds in a daily oral dose of 30 mg/kg for a period of sixty days. Group D i.e. experimental group rats received 60 mg/kg of STZ as a single intraperitoneal injection and aqueous ex­tract of Carum carvi seeds using a dose of 60 mg/kg body weight daily (orally) for a period of 60 days. [16]

Sample collection

Four hours after administration of the last dose of the extract i.e. on 60 th day and after overnight fasting, the animals were weighed and anesthe­tized under ether vapor. A sample of 2 mL blood was drawn from the tail vein in all animals. Blood was transferred to sterile vacuotainers with gel and allowed to clot at room tempera­ture for one hour. It was then centrifuged for 10 minutes at a speed of 3000 rpm. Serum was se­parated and stored in sterile eppendorf tubes at -20ºC for analysis of biochemical parameters. [17]

Twenty four hour urine samples were collec­ted using metabolic cages and analyzed. The animals were kept individually in metabolic cages, and they were given only water. Body weight was also measured initially and at the end of the experiment.

Biochemical Analysis

Glucose levels were estimated using commer­cially available kit (Randox, UK) based on glu­cose oxidase method. [18] Serum urea was mea­sured by commercially available kits (Randox, UK), based on enzymatic colorimetric method, while serum creatinine was measured using Jaffe alkaline picrate method (Randox, UK). [19],[20] Total urinary protein was estimated by a colorimetric method and microalbumin level based on an immunoturbimetric assay (Randox,UK). [21],[22]

   Statistical Analysis Top

The data was entered and analyzed using SPSS 17.0 (Statistical Package for Social Sciences). All data are shown as mean ± standard error (SE). One way analysis of variation (ANOVA) was applied to observe group mean differences. Post Hoc Tukey test was applied to determine the mean differences among the groups. A P value of < 0.05 was considered as statistically significant.

   Results Top

The biochemical parameters showed that the injection of STZ caused a significantly (P < 0.01) increased serum glucose, urea and crea­tinine levels in the rats of groups B, C and D as compared to the control group. On the other hand, simultaneous administration of aqueous extract of Carum carvi resulted in a significant (P < 0.01) decrease in the serum glucose, urea and creatinine levels of the rats in groups C and D when compared with group B [Table 1].

The total urinary protein excretion showed that the injection of STZ caused a significantly (P < 0.01) increased total urinary protein ex­cretion in the rats of group B, C and D as com­pared to the controls. On the other hand, the simultaneous administration of aqueous extract of Carum carvi resulted in a significant (P < 0.01) decrease in the total urinary protein le­vels of the rats in group D when compared with group B. When group B was compared with group C, no significant difference was observed in the urinary protein levels (P = 0.092) [Table 1].

The microalbuminuria levels showed that the injection of STZ caused a significantly (P < 0.01) increased microalbuminuria levels in the rats of group B, C and D as compared to the controls. On the other hand, the simultaneous administration of aqueous extract of Carum carvi resulted in a significant (P <0.01) dec­rease in the microalbuminuria in the rats in group D when compared with that of group B. When group B was compared with group C, no significant difference was observed in mic­roalbuminuria (P = 0.173) [Table 1].
Table 1 :Mean ± standard error values of different biochemical parameters in all study groups (A, B, C and D).

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The urine volume showed that the injection of STZ caused a significantly (P < 0.01) in­creased levels of urine volume in the rats of group B, C, and D when compared with the control group. On the other hand, the simul­taneous administration of the aqueous extract of Carum carvi resulted in a significant (P < 0.01) decrease in the levels of urine volume of the rats in group C and D when compared with group B [Table 1].

There was a significant decrease in body weight in the rats of group B, C and D as com­pared with the controls (P < 0.01). On the other hand, the simultaneous administration of aqueous extract of Carum carvi resulted in an insignificant increase in the body weight of the rats in groups C (P = 0.87) and D (P = 0.23) animals when compared with group B [Table 1].

Morphological examination

The morphological studies of kidneys in the control group showed normal glomeruli with normal convoluted tubules. Blood vessels and interstitium were also normal [Figure 1]. Group B (diabetic group) rats injected with STZ, showed a remarkable renal damage in 75% of the glomeruli characterized by diffuse glome­rular sclerosis and hyalinization of glomerular lobules and mild diffuse sclerosis of intercapillary areas encroaching the basement mem­branes [Figure 2]. The interstitium showed chro­nic inflammatory infiltrates of mild to mo­derate severity. Many tubules contained pro­tein casts. Experimental group C animals in­jected with STZ and low dose of Carum carvi extract (30 mg/kg) showed mild changes in 25-­35% of glomeruli and tubules [Figure 3]. These changes were found to be significantly reduced in kidneys of the experimental group D animals. This group of rats showed moderate changes in the tubular and glomerular morphology. How­ever, some glomeruli showed almost normal structure [Figure 4],[Figure 5].
Figure 1 :Photomicrograph of a glomerulus from a normal control rat showing normal structure of the glomerulus and the tubules (H & E ×40).

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Figure 2 :Photomicrograph from a diabetic control rat showing diffuse glomerular sclerosis (H & E ×40).

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Figure 3 :Photomicrograph shows mild changes in one glomerulus, whereas the other glomerulus shows capillary damage in a group C rat (low dose of Carum carvi) (H & E ×40).

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Figure 4 :Photomicrograph of a glomerulus in a group D rat (high dose of Carum carvi) shows a moderate change indicated by patency of capillaries. Some tubules show degenerative changes (H & E ×40).

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Figure 5 :Photomicrograph of a glomerulus in a group D rat shows reversal to nearly normal structure in its tuft (H & E ×40).

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   Discussion Top

Nephropathy is an important microvascular complication of diabetes mellitus. Many in vivo and in vitro studies have indicated that oxida­tive stress is one of the major pathophysio­logical mechanisms involved in the develop­ment of diabetic nephropathy. [23] Hyperglyce­mia enhances the non-enzymatic glycosylation of proteins and form advanced glycosylation end-products (AGE), which are stable and re­sistant to degradation by enzymes and injure cells by the structural rearrangement of pro­teins. Increased serum levels of AGE seem to predict changes in renal morphology such as expansion of mesangial cell matrix and glome­rular basement membrane (GBM) thickening. [24] STZ induces diabetes mellitus (DM) in ro­dents and results in the development of neph­ropathy similar to early stage clinical diabetic nephropathy. Urinary albumin levels are a se­lective marker of glomerular injury and eleva­ted rates of albumin excretion are a harbinger of progressive nephropathy. [25] Urine albumin excretion is considered to be the most sensitive marker of renal injury. [15] An increase in urinary albumin excretion is widely recognized as an early predictor of glomerular damage espe­cially in diabetes mellitus. [26]

Tight control of blood glucose can reduce cli­nical complications in diabetic patients. How­ever, alternative treatment strategies are re­quired to prevent the oxidative stress compli­cations and to optimize recovery. It is well do­cumented that modulations of oxidative stress through treatment with antioxidants can effec­tively reduce the development of diabetes. [27]

The present study shows a significant eleva­tion observed in the levels of serum glucose, urea, creatinine, and total urinary protein and urinary albumin excretion of group B diabetic rats as compared to group A normal rats. Urine volume was also elevated in group B rats, while body weight decreased in group B rats as compared with group A rats. Elevated levels of these parameters in serum and urine are presumptive markers of diabetes associated lesions in kidneys of rats. Co-administration of Carum carvi extract brought the levels of these diagnostic parameters in the serum and urine of group D animals towards normal as com­pared with group B rats. On the other hand, in group C animals, biochemical levels decreased but did not reach the normal level. Our results are in accordance with the reports by others who used chemical antioxidants and diet of natural antioxidant plants. [23],[25],[28],[29],[30]

The morphological examination in group C animals, low dose Carum carvi aqueous seeds extract showed mild changes in some kidneys whereas most of them showed no changes. On the other hand in group D animals, high dose Carum carvi aqueous seed extract resulted in moderate morphological changes towards nor­mal. This shows the reno-protective effect after a high dose of Carum carvi extract in diabetic nephropathy. This also coincides with the other reports where antidiabetic substances were used. [28] The proposed mechanism of Carum carvi in reducing the serum glucose levels in diabetes and its complications could be due to an antioxidant mechanism. Anjaneyulu and Chopra observed that quercetin is a strong antioxidant and it attenuates diabetic nephro­pathy in rats. [23] Oxidative stress can promote the formation of a variety of vasoactive me­diators that can affect renal functions, directly by causing renal vasoconstriction or decrea­sing the glomerular capillary ultrafiltration coefficient, and thus it reduces GFR. [31] It is likely that the scavenging effects of quercetin on free radicals, the inhibition of lipid pero­xidation and the subsequent decrease in the production of vasoactive mediators may play a role in improving renal dysfunction in diabetes. [32]

The main constituents in Carum carvi are carvone, limonene and flavonoids. Elmastas et al reported that carvone had a strong antioxi­dant activity. [33] Therefore, in our study flavo­noids and carvone in Carum carvi might have reno-protective role in STZ induced diabetic nephropathy in rats.

In conclusion, the results of the present study indicate that the co-treatment of Carum carvi aqueous seeds extracts prevent STZ induced diabetic nephropathy in the Wistar rats. The high dose of Carum carvi aqueous seeds ex­tracts showed better results as compared to the low dose, both biochemically and morpholo­gically. The overall reno-protective effect of Carum carvi is probably due to a counter­action of free radicals by its antioxidants i.e, Quercetin (flavonoids) and Carvone.

Further studies are required to observe if higher doses and variable routes of adminis­tration have better protective effect on the kid­neys of diabetic nephropathy.

   References Top

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