Archives of Anatomy and Physiology Protective and curative effects of Boerhaavia di ﬀ usa L. against ﬂ uoride induced renal oxidative stress and antioxidant enzymes in rats

This work elucidated the protective effect of leaf extract of the Boerhaavia diffusa L. (punarnava) on kidney damage following ﬂ uoride administration in rats. Forty eight rats were randomly divided into eight group’s six rats in each. Group I was administered deionized water orally served as control. Group II and III were administered with 300 and 600 ppm NaF/kg bw/day for 40 days. Group IV were orally administrated with 500mg/kg b.w/day of leaf extract of Boerhaavia diffusa L. for 20 days Group V and VI were pre-treated with 500 mg/kg bw/day of leaf extract of the Boerhaavia diffusa L. for 20 days and then exposed to 300 and 600 ppm NaF/kg bw/day for 40 days. Group VII and VIII were exposed ﬁ rstly to 300 and 600 ppm NaF/kg bw/day and then post-treated with leaf extract of the Boerhaavia diffusa L. for 20 days. The level of MDA exhibited signi ﬁ cantly (p<0.001) increase while GSH and activities of SOD, CAT, and GPx revealed signi ﬁ cant (p<0.001) decline in kidney of rats treated with 300 and 600 ppm of NaF. The results indicate that pre and post-treatment signi ﬁ cantly decrements (p<0.001) in the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and glutathione peroxidise (Gpx) alongwith signi ﬁ cant increase (p<0.001) in the level of malondialdehyde (MDA).The current work suggests that the leaf extract of Boerhaavia diffusa L conferred therapeutic bene ﬁ ts on NaF-induced nephrotoxicity, particularly when administered before more than after the insult.


Introduction
Drug-induced nephrotoxicity is an important cause of renal failure. Kidney is one of the target organs attacked by excessive amounts of fl uoride. Fluoride has a role in cellular respiratory process like in free radical reactions. Fluoride reacts with polyunsaturated fatty acids and initiates lipid peroxidation leading to necrosis and apoptosis [1,2]. As the primary organ concerned with excretion and retention of fl uoride, kidney is quite sensitive to the toxicity of fl uoride [3]. The situation of serious imbalance between oxidant and antioxidant is referred to as oxidative damage. In many diseases, tissue damage is accompanied by an imbalance in the oxidant and antioxidant status. Exposure to fl uoride results in generation of anion superoxide, increased oxygen concentration and its downstream consequences such as hydrogen peroxide, hydroxyl radicals, which are important in mediating the toxic effects of fl uoride. Intake of high levels of fl uoride is known to cause structural changes, altered activities of enzymes, and infl uence the metabolism of lipid. Acute poisoning can terminate in death due to blocking of cell metabolism since fl uoride inhibits enzymatic processes, mainly metalloenzymes responsible for important vital processes [4].
Boerhaavia diff usa L. has many medicinal properties and enjoys an important place among medicinal herbs in India since ancient times [5]. Punarnava leaves are consumed by the people as food supplements with broad spectrum disease defending properties and with no reported side-effects, the results of the present studies may have future therapeutic relevance in the areas where humans are exposed to fl uoride either occupationally or environmentally [6]. The aim of the present study is to investigate the oxidative damage caused in renal tissue and the protective effects of leaf extract of Boerhaavia diff usa L.

Preparation of leaf extract of Boerhaavia diffusa L.
Fresh leaves of Boerhaavia diff usa L. were washed in running tap water to remove adhering dust and wiped to dryness. The leaves were then dried under shade. The shade dried leaves were fi nely grind using a mechanical blender. The powder obtained was used for ethanol extraction in a soxhlet extractor. The excessive solvent from the extract was recovered with rotary vacuum evaporator and then the concentrated extract was dried to constant weight in a hot air oven at 40°C.The leaf extract of Boerhaavia diff usa L. was prepared by the method given by Narendhirakannan [7].

Ethical aspects
Experimental protocols and procedures used in this study were approved by the animal ethical committee of Punjabi University, Patiala (Animal Maintenance and Registration No. 107/99/ CPCSEA /2014-23).
Young Wistar albino rats, weighing between 100-200gm were housed in polypropylene cages with stainless grill tops and fed with standard rat pellet diet (Hindustan lever limited, India) and water was given ad libitum. Animals were maintained at a constant room temperature of 20-22°C and 60% humidity. Rats were allowed a 2-week acclimatization period and then they were randomly divided into eight groups. Rats of group I received 1ml deionized water /kg b.w. / day orally daily by a gastric tube for 40 days, and served as control. Rats of group II and III were orally administered with 300 and 600 ppm NaF /kg bw /day for the same duration. Group IV antidote control group was orally administrated with 500mg/kg b.w/day of leaf extract of Boerhaavia diff usa L. for 20 days. Animals of Group II and III were pre and post-treated with 500mg/kg bw/day of leaf extract of Boerhaavia diff usa L. for 20 days .At the end of the experimental period, rats were fasted overnight and sacrifi ced under ether anesthesia.

Preparation of tissue homogenate
The renal tissue was washed with ice-cold 0.9% saline and homogenized quickly with ice cold 0.1M phosphate buffer (pH 7.4) using glass tefl on homogenizer to give a 10% homogenate. The homogenate was centrifuged at 10, 000 rpm for 20 min and the supernatant were used for estimation of malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx).

Assesment of biochemical parameters
The level of MDA in the kidney tissue of rats was determined by the method of Ohkawa [8]. The GSH content in kidney tissue was measured by the method of Dringen [9]. The activities of SOD [10]. CAT [11], and GPx was determined [12], in kidney tissue of rats.

Statistical analysis
Results were expressed as mean ± standard deviation (SD). Statistical signifi cance of difference between the experimental groups was evaluated by one way ANOVA followed by Bonferroni and Post hoc Dunnetts multiple comparison test. The correlation between two variables was analyzed by STATISTICA 7 software. A two tailed p value < 0.05 was considered statistically signifi cant. All computations were performed using SPSS 17.0 statistical software (IBM).

Malondialdehyde (MDA)
The level of MDA in kidney tissue of test rat showed a signifi cant (F=567.9, p<0.001) increase after 40 days of fl uoride treatment. More prominent increase (156.2 %) was registered in highest dose group (600 ppm NaF/kg b.w/day Figure 1  2.119; mean difference 0.851 to 2.744) with 500 mg / kg bw /day of leaf extract of Boerhaavia diff usa L. as compared to respective NaF treated groups (Figure 4).

Superoxide dismutase (SOD)
The activity of SOD in kidney tissue of test rat revealed a signifi cant (F=13.0491, p<0.001) decrement after 40 days of fl uoride treatment. More prominent decreased (-70.40 %) was reported treated with highest dose group (600 ppm NaF/kg b.w/day) (Figure.

Catalase (CAT)
The activity of CAT in kidney tissue of test rat showed a

Discussion
This study was undertaken to estimate the prophylactic and curative effect of Boerhaavia diff usa L. against sodium fl uorideinduced oxidative stress in kidney tissue of rat.
The present study demonstrate an elevation in level of renal MDA in rats treated with 300 and 600 ppm of NaF /kg bw/day.
The present study revealed that there was close relationship between fl uoride-induced nephrotoxicity and oxidative stress.
This fi nding is consistent with those of previous studies [13][14][15][16]. In our experiments, rats exposed to 300 and 600 ppm of NaF /kg bw/day for 40 days showed decrease in content of reduced glutathione. The present results are in accordance with previous     fl uorosis reports [3,13,19]. Decrease in the level of fl uoride after mitigation with leaf extract also resulted in signifi cant increase in content of reduced glutathione as compared with 40 days of fl uoride treatment. Reduced glutathione neutralizes the hydroxyl radical and plays a major role against infl ammatory responses and oxidative stress. Reduced glutathione is an important naturally occurring antioxidant, which prevents free radical damage and helps detoxifi cation by conjugating with chemicals. Under oxidative stress, reduced glutathione is consumed by reduced glutathione related enzymes to detoxify the agents that increase lipid peroxidation [20]. Oxidative stress is induced by increasing production of reactive oxygen species, such as superoxide anion, hydrogen peroxide and hydroxyl radicals. Reactive oxygen species can induce lipid peroxidation, inactive cellular enzymes, depolymerize polysaccharides, and induce deoxyribonucleic acid breaks and chromosome breakage [17]. Experimental evidences have indicated that exposure to fl uoride results in oxidative stress in both vitro and in vivo in soft tissues [21].
The investigation indicates the inhibition of oxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase in kidney tissue of rats during 300 and 600 ppm NaF /kg bw/day intoxication. It was observed that sodium fl uoride exposure in rats caused a signifi cant (p<0.001) decrement in total activity of superoxide dismutase, catalase, and glutathione peroxidase. This fi ndings are in accordance with [13,15,16,22]. Boerhaavia diff usa L. leaf extract administration signifi cantly (p<0.001) accelerated the renal activities of superoxide dismutase, catalase, and glutathione peroxidase in fl uoridated rats kidney tissue. Superoxide dismutase is a naturally occurring intracellular enzyme that catalyzes the breakdown of superoxide radicals. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen. Catalase is a common enzyme found in nearly all living organisms that are exposed to oxygen, where it catalyzes the decomposition of hydrogen peroxide to water and oxygen.
Our studies revealed that leaf extract of Boerhaavia diff usa L .has the capability to provide protection against fl uorideinduced renal injury mediated, by reactive oxygen species and the other related toxicants. Thus, extract of Boerhaavia diff usa L. seems to have the potential to be considered as a benefi cial antioxidant. This extract of Boerhaavia diff usa L. may function simply by quenching free radicals and the other related toxic intermediates generated during oxidative stress due to fl uoride or may improve the antioxidant enzyme status of the tissue in the face of the oxidative stress. Toxicity of superoxide anion free radical and hydrogen peroxide could involve the formation of much more reactive hydroxyl radical (•OH) [23]. The results of the present study may be of future therapeutic relevance particularly in the areas where humans are chronically exposed to fl uoride either occupationally or environmentally. B oerhaavia diff usa L. can also serve as pharmacological intervention and, the bio-active fractions obtained therefrom may be used also as a future antioxidant supplement to combat oxidative stressinduced renal damage due to fl uoride. The present study refl ects the antioxidant and free radical scavenging activity of leaf extract of Boerhaavia diff usa L. (punarnava).

Conclusion
Our result describes the protective effect of leaf extract Boerhaavia diff usa L against fl uoride-induced kidney tissue damage in experimental rats. However, the nephroprotective effect of leaf extract of Boerhaavia diff usa L. was observed to be signifi cantly higher when it was administered before NaF treatment than after NaF treatment.