Protective effect of Pueraria tuberosa Linn. in arsenic induced nephrotoxicity in rats.

 

V. Uma Rani¹*, Dr. M. Sudhakar¹, Dr. A. Ramesh²

¹Malla Reddy College of Pharmacy, Maisammaguda, Dhulapally, Secunderabad, Affiliated to Osmania University, Telangana, India.

²Vishnu Institute of Pharmaceutical Education and Research, Narsapur, Telangana, India.

 

ABSTRACT:

Pueraria tuberose Linn. is an  inexpensive  and  efficient  source  to  provide  all the required nutrients and medicinal  benefits  for  a healthy and rejuvenating body. The present investigation has been undertaken to evaluate the role of Pueraria tuberosa Linn. in modifying the sodium arsenate induced biochemical alterations in albino rats. Administration of sodium arsenate in rats induces oxidative stress, which leads to the generation of free radicals in the body. These free radicals interact with tissue leading to tissue damage i.e, kidney damage. Animals were divided into 6 groups, Group I (control group) rats were treated with normal saline (1mg/kg), Group II ( Positive Control) rats were treated with sodium arsenate (1mg/kg).Group III rats were treated with sodium arsenate (1mg/kg) and Vitamin E (100 mg/kg).Group IV rats were treated with sodium arsenate (1mg/kg) and Pueraria tuberosa linn. extract (50 mg/kg), Group V rats were treated with sodium arsenate (1mg/kg) and Pueraria tuberosa linn. extract (100 mg/kg) , Group VI rats were treated with sodium arsenate (1mg/kg) and hydroalcoholic extract of Pueraria tuberose linn (200mg/kg). After 21 days treatment, blood samples were collected and analyzed for the serum parameters viz BUN test, serum calcium test, serum iron test, Acid phosphatase test, Creatinine test. Antioxidant parameters like Malondialdehyde, catalase, Reduced glutathione and glutathione reductase were estimated. The kidney is removed and sectioned for histopathological examination. The hydroalcoholic extract of Pueraria tuberosa linn (tuber) inhibits the oxidative stress hypothesis mechanism. It was concluded that the extract of Pueraria tuberose linn (tuber) exerts nephroprotective activity could be attributed by presence of natural antioxidants, free radical scavenging.

 

 

 

INTRODUCTION:

Many metals play important roles in the functioning of enzymes, cell-signaling processes, and gene regulation. Arsenic is not known to have any biological role. Increasing concern has been expressed about the rapidly rising level of chemicals in the environment, particularly Arsenic, which has well-known hazardous effects. This ubiquitous environmental pollutant enters the atmosphere from production of ground water and soil. Arsenic can disrupt biological systems by altering the molecular interactions, cell signaling, and ultimately cellular function. The toxic effect of arsenic is well documented in mammals, in which it leads to a broad range of physiological, biochemical, and behavioral dysfunctions (Courtois E et al., 2003).Arsenic exposure occurs mainly through the respiratory and gastrointestinal systems. Kidney is a frequent target for many toxicants (Meyer SA et al., 2001). Autopsy studies of arsenic-exposed humans indicate that among soft tissue, kidney is the largest repository (33%) of arsenic. Arsenic-induced renal damage is mostly rooted in lipid per oxidation (LPO) and disturbance of the prooxidant–antioxidant balance by generation of reactive oxygen species (ROS) (Gurer H et al., 2000; Bechara EJH et al., 2004

The nephrotoxicity is due to poisonous effect of some toxic chemicals and certain type ofdrugs on the kidneys. Nephrotoxins are the agents displaying nephrotoxicity. The nephrotoxiceffect develops more in patients who are already suffering from the renal impairment. Thechronic administration of certain drugs causes nephrotoxicity. If it occurs, then the kidneysfails to remove excess urine and waste. The kidneys are important organs as they involved inthe excretion of waste products, toxic substances in the body. In kidney failure, the toxic metabolites retain in the body leading severe complications like edema, ascites, hepatomegaly, etc.Nephrotoxicity occurs when the renal blood is exposed to a nephrotoxic drug or toxin that causes damage to the kidneys. When kidney damage occurs, the kidney fails to remove excess urine, and waste. The kidney filters the metabolic toxic waste substances from the blood and helps in excreting these substances from the body in the form of urine.

 

MATERIALS AND METHODS:

Plant:

The tubers of Pueraria tuberosa Linn. were collected and authenticated. The tubers were shade dried, grounded coarsely and stored in air tight containers.

 

Animal selection:

Thirty six Wistar Albino rats of either sex weighing 200gms-250gms were obtained from departmental animal facility where they were housed under standard husbandry conditions (25 ± 2 °C temp., 60–70% relative humidity and 12 h photoperiod) with standard rat feed and water ad libitum. Experiments were conducted in accordance with the guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India and experimental protocols were approved by the Institutional Animal Ethics Committee (CPCSEA/1217/2016/2).

 

Methodology:

Preparation of  the hydroalcoholic extract:

The tubers of Pueraria tuberosa Linn. were collected, shade dried and powdered.The powdered plant material was successively dried.  This dried powder was used for soxhlet extraction.  Extraction was done by using the soxhlet apparatus at a temperature below 60^oC for 24 hours. Powder was extracted with 70% water and 30% alcohol. The solvent thus obtained was evaporated under vacuum to get a semi-solid form of the extract. Thus, extract obtained was used for evaluating the activity.

 

Preliminary phytochemical screening:

Preliminary phytochemical screening was done for the presence of carbohydrates, amino acids, proteins, saponins,  alkaloids,  flavonoids, glycosides, sterols,  tannins, tri-terpenoids and phenolic compounds according to the procedures described in “Text book of Practical Pharmacognosy” by C.K. Kokate.

 

Experimental Design

Thirty six Wistar Albino both male and female rats of weight 200g-250g were selected for this study. Animals were divided into six groups of six animals each. Group 1: Control group (received distilled water 1ml), Group 2: Sodium arsenate (1 mg/kg, p.o.), Group 3:  Sodium arsenate + vitamin E (1 mg/kg, p.o. + 100 mg/kg, p.o.), Group 4: Sodium arsenate + HEPT (1 mg/kg, p.o. + 50 mg/kg, p.o.), Group 5: Sodium arsenate + HEPT(1 mg/kg, p.o. + 100 mg/kg, p.o.),Group 6: Sodium arsenate + HEPT(1 mg/kg, p.o. + 200 mg/kg, p.o.). All the groups were treated once daily for a period of 30 days. The animals were weighed and behavioral observations were recorded before and at the end of the experiment. After the administration of last dose, the animals were given rest overnight and then on the next day, they were sacrificed under light ether anesthesia. The organs were removed, cleaned, washed with phosphate buffer saline (pH 7.4) for various studies.

 

Serum sample preparation:

Blood samples were allowed to stand at room temperature for 30 min and serum was isolated by centrifugation at 4000rpm for 15 min and used for estimation of Creatinine (Bowers LD., 1980), Blood urea nitrogen (BUN),Serum iron,Serum calcium and Acid phospatase (Ohkawa H et al., 1979).

 

Biochemical assays

Kidneys were minced separately and homogenized (10% w/v) in ice-cold 0.1 M sodium phosphate buffer (pH 7.4). The homogenate was centrifuged at 8000 rpm for 30 min at 4°C twice to get the enzyme fraction. The supernatant was used for biochemical assays.

 

Lipid peroxidation (LPO)

LPO was estimated colorimetrically by measuring malondialdehyde (MDA) formation as described by Nwanjo and Ojiako, 2005. In brief, 0.1 ml of homogenate was treated with 2 ml of a 1:1:1 ratio of TBA–TCA–HCl (TBA 0.37%, TCA 15%, HCl 0.25 N) and placed in water bath at 65°C for 15 min, cooled, and centrifuged at 5,000 rpm for 10 min at room temperature. The optical density of the clear supernatant was measured at 535 nm against a reference blank. The MDA formed was calculated by using the molar extinction coefficient of thiobarbituric acid reactants (TBARS; 1.56×10⁵l/mole cm⁻¹). The product of LPO was expressed as nmol of MDA formed per g of tissue.

 

Catalase (CAT)

Catalase (CAT) activity was estimated following the method of Aebi, 1993. The homogenate (100 µl) was treated with ethanol (10 µl) and placed on an ice bath for 30 min. To this, 10 µl of 25% triton X-100 was added and again kept for 30 min on ice. To 200 µl phosphate buffer (0.1 M), 50 µl of treated liver and kidney homogenate and 250 µl of 0.066 M H₂O₂ (prepared in 0.1 M phosphate buffer, pH 7.0) were added in a cuvette. The decrease in optical density was measured at 240 nm for 60 s. The molar extinction coefficient of 43.6 cm⁻¹ was used to determine CAT activity. One unit of activity is equal to the moles of H₂O₂degraded/min/mg protein.

 

Glutathione (GSH)

Reduced glutathione (GSH) was determined by the method of Ellman, 1959. In brief, 1 ml of supernatant was taken after precipitating 0.5 ml of liver and kidney homogenate with 2 ml of 5% TCA. To this, 0.5 ml of Ellman's reagent (0.0198% DTNB in 1% sodium citrate) and 3 ml of phosphate buffer (1 M, pH 8.0) was added. The color developed was read at 412 nm. Reduced GSH concentration is measured by using a drawn standard curve and was expressed as mg/g of tissue.

 

Gluthathione Reductase

To 350 mcl of Potassium Phosphate buffer (100Mm,pH 7.5), 50mcl of  NADPH, 500 mcl of oxidised glutathione and 100 mcl of heart homogenate were added and made the volume upto 1mml with distilled water. The reaction is started by the addition of the NADPH solution. Mix by inversion, place the cuvette in spectrophotometer and start the kinetic program.

 

Histopathalogical estimation:

At the end of the experiment (day 31), all rats were anesthetized by light ether, kidney were excised out and fixed in formalin (10%). Five micron thin section were prepared by using eosin and these section were stained with haematoxyline and eosin. For histological alterations these slides were observed under light microscope.

 

 

Statistical Analysis:

The experimental results were expressed as the Mean ± SEM with six rats in each group. The intergroup variation between various groups were analyzed statistically using one-way analysis of variance (ANOVA) using the Graph Pad Prism version 5.0, followed by Dunnett’s multiple comparison test (DMCT). Results were considered statistically significant when P < 0.05.

 

RESULTS:

Preliminary phytochemical screening of Pueraria tuberosa Linn.

The main chemical constituents that are found in the hydroalcoholic extract of Pueraria tuberose flavonoids are abundantly found. Carbohydrates, Tannins, alkaloids, carbohydrates, saponins, phenolic compounds, gums and mucilage.

 

Effect of Hydroalcoholic extract of Pueraria tuberose on Serum Parameters in Sod. arsenate induced Nephrotoxicity in rats (Table 1)

Pueraria tuberosa Linn alone does not have any significant change on renal enzymes but treatment of Pueraria tuberosa Linn. extract along with arsenic trioxide treatment induced changes like it caused a significant decrease in BUN test, Serum calcium test, serum iron test, acid phosphatase test, creatinine test.

 

Effect of Hydroalcoholic extract of Pueraria tuberose on Antioxidant Parameters in Sod. arsenate induced Nephrotoxicity in rats(Table 2)

Arsenic exposure produced significant decrease P<0.0001 in catalase (12.45±0.1), in GSH (18.23±0.23),in glutathione reductase (10.56±0.2), and significant increase P<0.0001 in MDA (98.06±2.5) in Arsenic control group when compared to normal control group. Hydroalcoholic extract of Pueraria tuberose pretreatment before Arsenic exposure showed significant increase P<0.0001 in catalase (25.8±0.3), GSH (23.85±0.8) , glutathione reductase (18.91±0.69) and significant decrease P<0.0001 in MDA (77.93±1.0) in Arsenic extract 3 group when compared to Arsenic treated group.

 

 

 

 

 

 

 

 

 

 

 

 

Table 1:Effect of Hydroalcoholic extract of Pueraria tuberose on Serum Parameters in Sod. arsenate induced Nephrotoxicity  in rats.

Groups	Blood Urea Nitrogen (mg/ml)	Creatinine (mg/ml)	Serum Uric acid (mg/ml)	Serum calcium(mg/ml)	Acid phosphatase
Normal control	70±3.8	8.3±0.039	10.2±2.0	11.5±3.8	6.05±0.32
Na.Arsenate (1mg/kg)	150±28.51^	20.3±0.290^	25.01±1.1^	27.2±6.2^	10.90±1.21^
Na.Arsenate(1mg/kg) + Vitamin E(100mg/kg)	85±5.68#	12.8±0.08#	15.05±3.9#	14.2±5.6#	7.81±0.85#
Na.Arsenate(1mg/kg) +Pueraria tuberosa Linn.(50mg/kg)	130±11.14*	17.2±0.069*	22.9±1.15*	25.9±4.5*	6.51±0.05*
Na.Arsenate(1mg/kg) +Pueraria tuberosa Linn.(100mg/kg)	110±9.91**	15.1±0.068**	19.0±2.2**	22.71±4.51**	4.0±0.05**
Na.Arsenate(1mg/kg) +Pueraria tuberosa Linn.(200mg/kg)	90±6.66***	13.9±0.05***	16.3±3.33***	15.0±4.10***	3.5±0.04***

Values are expressed as mean ± SEM, n=6. ^#    P<0.001 vs normal control,  ^a     P<0.01 vs normal control,   **   P<0.01 vs lead control. The intergroup variation between various groups were analyzed statistically using Dunnett’s multiple comparison test. 

 

 

Table 2. Effect of Pueraria tuberosaon Antioxidant Parameters in Sod.arsenate induced Nephrotoxicity in rats.

Groups	MDA (nm/gtissue)	Catalase (K/min)	GSH(ug/ml)	GR(u/ml)
Normal control	25.67±0.714	45.42±0.171	40.93±0.418	20.48±0.056
Na.Arsenate (1mg/kg)	50.15±1.16^	25.35±0.0714^	20.30±0.195^	10.00±0.008^
Na.Arsenate(1mg/kg) + Vitamin E(100mg/kg)	28.85±0.596#	40.82±0.114#	35.333±0.118#	15.5±0.079#
Na.Arsenate(1mg/kg) +Pueraria tuberosa Linn.(50mg/kg)	45.15±0.477*	28.12±0.094*	25.24±1.454*	7 .35±0.122*
Na.Arsenate(1mg/kg) +Pueraria tuberosa Linn.(100mg/kg)	35.23±1.249**	33.55±0.094**	28.66±0.193**	10.77±0.079**
Na.Arsenate(1mg/kg) +Pueraria tuberosa Linn.(200mg/kg)	30.15±0.477***	37.67±0.215***	32.12±0.461***	12.23±0.099***

Values are expressed as mean ± SEM. n=6; ^p<0.0001 compared normal group, #p<0.0001compared to lead group,*p<0.0001 compared to lead group, **p<0.0001 compared to lead group, ***p<0.0001 compared to lead group.

 

 

Histopthological studies:

 

Figure I: Control Kidney showed normal Glomerulus and tubular structures.   No reactive changes like degeneration, Inflammation in the kidney were observed. Tissue is stained with Haematoxylin and Eosin at magnification 100X.

 

Figure - II. Na. Arsenate treated Kidney:. Tissue showed Focal interstitial nephritis, glomerulus region appeared normal and inflammation observed. Stained with Haematoxylin and Eosin at magnification 100X.

 

Figure- III:. Na. Arsenate+ Vit E(100mg/kg) treated Kidney: Glomerulus region appeared normal. But mild cystic degeneration was observed in the tubular region accumulated with fluids and is stained with Haematoxylin and Eosin   at magnification 100X.

 

Figure- IV: Na. Arsenate + Pueraria tuberosa linn (50mg/kg) treated–Kidney: Showed Focal interstitial nephritis, glomerulus and tubular structure appeared normal. Degenerative, inflammatory changes were observed in the kidney and is stained with Haematoxylin and Eosin at magnification 100X.

 

Figure- V:  Na. Arsenate +Pueraria tuberosa linn (100mg/kg) treated–Kidney: No degenerative, necrotic and inflammatory changes were observed and is stained with Haematoxylin and Eosin at magnification 100X.

 

Figure – VI:  Na. Arsenate + Pueraria tuberosa linn (200mg/kg) treated–Kidney: Tissue appeared normal, glomerulus and tubular structures appeared normal. No reactive changes in the kidney observed and is stained with Haematoxylin and Eosin at magnification 100X.

 

DISCUSSION:

The present study was focused to evaluate the protective effect of Pueraria tuberose Linn.  Extract against Sodium arsenate induced kidney damage via the scavenging of Reactive Oxygen Species. The results of the present study revealed that arsenic intoxication causes significant decrease in BUN(blood urea nitrogen), serum calcium, acid phosphatase, creatinine levels, and significant increase in catalase and blood glutathione level.

 

The treatment with Sodium arsenate significantly altered the serum creatinine, urea and uric acid, which denotes the occurrence of renal toxicity with arsenic. Arsenic concentrates in the kidney during its urinary elimination process that affects the function of proximal convoluted tubules(Burton et al, 1995, Parrish et al, 1999). Acute renal dysfunction due to arsenic exposure is characterized by acute tubular necrosis and cast formation with increase in blood urea nitrogen and creatinine levels (Kimura et al, 2006). The kidney and liver are the primary targets forarsenic-induced toxicity (Nandi et al, 2006). As like in the current observation, arsenic increased the generation of ROS, which enhanced lipid peroxidation and cellular damage in renal tissue, resulted in the release of urea and creatinine (Kokilavani et al, 2005). 

 

In the current investigation, Sodium arsenate treatment increased lipid peroxidation in blood, heart,  liver and kidney tissues. MDA is a marker of endogenous lipid peroxidation. Liu et al (2001) reported that the treatment with arsenic caused a significant increase in the rate of formation of ROSsuch as superoxide anion radical, hydroxyl radical and hydrogen peroxide.The toxic potential exerted by these compounds is through their reactivity with sulphur containing compounds and the generation of ROS (Hughes et al, 2011). According to Wang et al (2006), arsenic induced MDA production could be due to the impairment of cells natural protective system and could be directly related to the GSH depletion. Major arsenic-induced ROS includes superoxide anion (O₂−), hydroxyl radical (OH•), hydrogen peroxide (H₂O₂), singlet oxygen (O₂1) and peroxyl radicals (Halliwell and Whiteman, 2004). Arsenic exposure is also known to stimulate the release of free iron from ferritin (Ahmad et al, 2000) and the resulting free iron is believed to be one of the potent inducer of ROS formation via the Fenton type reaction. Arsenic intoxication decreased the ability of intracellular antioxidant power and hence attenuated the reducing ability of Fe (III) to Fe (II) (Manna et al, 2008). The improper balance between Reactive oxygen species (ROS) metabolites and antioxidant defense results in “oxidative stress”.

 

However, treatment with plant tuber extract in different doses along with arsenic decreased the lipid peroxidation in kidney tissue as compared with arsenic treated animals, thus indicating protective role of this plant extract in arsenic intoxication. Moreover, elevated levels of the antioxidant enzymes (SOD, CAT and GST) and non-enzymatic potential (GSH), further support the antioxidant role of the root extract.

 

In the present investigation the blood lipid peroxidation level showed a highly significant elevation and GSH level, CAT level showed a highly significant depletion following arsenic trioxide exposure. Overproduction of ROS enhances the lipid peroxidation which increases the lipid peroxidation products like malondialdehyde (MDA) and other TBARS levels which lead to degradation of cellular macromolecules.

 

GSH is the major thiol, which binds electrophilic molecular species and free radical intermediates. It plays a central role in the antioxidant defense system, metabolism and detoxification of exogenous and endogenous substances (Muller and Florian, 2000). Arsenic has high affinity for GSH and causes the irreversible excretion of, up to two GSH tripeptides. The metal–GSH conjugation process is desirable in that it results in the excretion of the toxic metal into the bile. However, it depletes the GSH from the cell and thus decreases the antioxidant potential.

 

The protective efficacy of Pueraria tuberose Linn. due to presence of several active components. The active component found in Pueraria tuberose Linn. may provoke the activity of free radical scavenging enzyme systems and renders protection against arsenic-induced damages.

 

Because it is also high in flavonoids, Pueraria tuberose Linn. helps to detoxify the body by increasing the elimination of hardened mucous, crystallized acids and solidified, decaying fecal matter and thus, provides an optimum nutritional environment inside the body system. Pueraria tuberosa Linn. is an extremely effective way of boosting the immune system to fight against diseases. Histopathological changes also support the above results. Histological sections of  kidney  in control and Pueraria tuberose Linn. treated rats showed the normal kidney tissues,  glomerulus and tubular appeared normal. Arsenic intoxication produced various pathological lesions in the kidney tissue such as focal interstitial nephritis, degenerative and inflammatory changes.

 

Hence, the mechanism by which the Pueraria tuberose Linn exerts a nephroprotective effect could be attributed to  presence of natural antioxidants and  its free radical scavenging and antioxidant properties

 

CONCLUSION:

To conclude, the present study demonstrated that deleterious reactive oxygen species or lipid peroxides responsible for arsenic induced toxicity may be alleviated by several active components like flavonoids, found inPueraria tuberose Linn. extract.

 

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Received on 08.11.2016       Accepted on 12.01.2017     

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