Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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The Inhibitory Ability of Orobanche Extract on Calcium Oxalate Lithiasis in Male Albino Rats

A.M. Kamal1, M.S. Taha1,*
1Biological Applications Department, Nuclear Research Center, Atomic Energy Authority, Egypt.
The present study aimed to evaluate the inhibitory effect of Orobanche extract in ethylene glycol induced nephrolithiasis. Thirty male albino rats were divided into five groups each group contains 6 animals, group (1) control group, group (2) animals were supplied with 0.75% ethylene glycol in drinking water, group (3) animals were administrated Orobanche extract 3g/kg orally, group (4) animals were administrated Cystone 500 mg/kg in addition to 0.75% ethylene glycol, group (5) animals were administrated Orobanche extract 3g/kg orally in addition to 0.75% ethylene glycol the experiment continued for 28 days. Serum and the kidney homogenates were analyzed for various biochemical parameters and urine was examined microscopically for crystals. Orobanche treatment group and Cystone treatment group significantly decreased phosphorus, Calcium and Oxalate in kidney tissue of nephrolithiasis rats and significantly decreased kidney and liver marker in serum of nephrolithiasis rats. Conclusion this result revealed that Orobanche extract could be a potential candidate for phytotherapy against nephrolithiasis. 
Kidney stone formation is a common disorder. Estimated to occur in approximately 12% of the population and it occurs more frequently in men than in women within the age of 20–49 years (Edvardsson et al., 2013), the majority of stones up to 80% are composed mainly of calcium oxalate (Patel et al., 2014), current treatments for patients with CaOx urolithiasis are limited and do not always lead to a sufficient reduction in urine oxalate (UOx) excretion and the standard drugs used to prevent urolithiasis (UL) are not effective in all patients, however, most of which have adverse effects due to their long term use. Many remedies taken from plants have been reported to be effective treatments for urolithiasis with little side effects (Butterweck and khan (2009) However, several research works considered the screening of bioactive compounds from plants in different parts of the world, other studies indicated the presence of these compounds in the parasitic plant such as Orobanche (Nagaraja et al., 2010). Up to date, little is known about the possible active compounds from the parasitic plants, so far no scientific study has been reported regarding antiurolithiatic property of the parasitic plant. Orobanche is a parasitic herbaceous plant in the family of Orobanchaceae McNeal et al., (2013). A previous study of Qu et al., (2016) revealed that Orobanche contains phenol propanoid glycosides (PhGs). Abbes et al., (2014) also investigated the antioxidant activity of methanolic and aqueous extract of Orobanche and revealed that Orobanche might be a potential resource of antioxidant and antibacterial activity. Moreover, Scharenberg et al., (2018) described bioactivities for Orobanche extracts and natural products isolated from Orobanche species and showed it has antioxidative and anti-inflammatory effects analgesic, antifungal and antibacterial activities. Therefore, in the present study, an effort has been made to establish the scientific validity for the ameliorating effect of Orobanche extract in ethylene glycol (EG)-induced hyperoxaluric oxidative stress and renal cell injury in rat kidney.                                                      
A total of thirty male albino rats weighing between 150-200g were used for this study. The experimental protocol was approved by the Research Ethics Committee, serial number 13A/19 of the National Center for Radiation Research and Technology, Egypt. The animals were obtained from the animal house of the Nuclear Research Center, Atomic Energy Authority, Egypt. The animals were housed in 6 rats per cage, provided with food and tap water adlibitum and acclimated for one week before the experiment.                                             
Preparation of samples
The plant specimens were collected from Faba bean cultivars in in “Field Crop Research Institute” at the Agricultural Research Center, Giza, Egypt, During March, the plants were washed carefully with running tap water then, rinsed with distilled water and shade dried for a period of time then ground into powder. Extraction was freshly prepared by adding 3g of Orobanche powder in 60ml hot boiled water and stand for 24 hours then filter the extract.
Acute Toxicity Studies
Acute toxicity studies were performed in mice and rats. Single doses of extract and the water as the vehicle at similar volumes were given orally by stomach tube to adult albino Swiss mice or rats. Clinical signs, symptoms and mortality were recorded through a 14-day observation period. TheLD50 values were tested acutely toxicity study results: Rats and mice may tolerate the extract comparable to 5 g/kg with no mortalities (EL-Sawy et al., 2015).
Classification of Orobanche
The plants’ specimen was collected during March month and examined taxonomy (Table 1).

Table 1: Scientific Classification.

Preliminary phytochemical analysis
Gas chromatography- mass spectrometry (GC / MS) analysis of Orobanche aegyptiaca was performed and revealed the presence of many bioactive compounds (Table 2).

Table 2: Analysis of the chemical composition of Orobanche.

Chemicals and reagents
Ethylene glycol and antiurolithiatic drug, Cystone were purchased from El-Goumhouria Company for trading medicines chemicals and medical appliances, Cairo Egypt. All diagnostic kits used for estimating the biochemical parameters were obtained from Biodignostic Company, Dokki, Giza, Egypt.
Experimental Design
A total of thirty male albino rats were divided equally into 5 groups each contain 6 rats per cage, group (1) served as a control, group (2), animals have received 0.75%v/v in drinking water for 28days for induction nephrolithiasis. Group (3), animals have received only Orobanche extract 3g/kg of body weight orally, group (4) animals has received ethylene glycol in drinking water followed by standard antiurolithiatic drug, Cystone 500mg/kg of body weight orally, group (5) animals has received ethylene glycol in drinking water followed by Orobanche extract 3g/kg of body weight orally. At the end of the experiment, after 28days blood was collected from retro-orbital under anesthetic conditions and serum was separated and analyzed for Creatinine, urea, AST, ALT, bilirubin.
Preparation of kidney homogenate
The kidneys were homogenized in cold potassium phosphate buffer (0.05 M, pH 7.4) for assaying tissue calcium (Ohnishi 1977), oxalate (Hodgkinson, 1970), phosphorus (Maher et al., 1988), Malondialdehyde (MDA) (Wong et al., 1987) and glutathione peroxidase (GPx) (Rotruck et al., 1973).
Serum biochemical analysis
The activities of serum AST and ALT aminotransferase were assayed by the method of Reitman and Frankel (1957). Bilirubin, urea and creatinine were determined according to the method described by (Wahlefeld et al., 1972), Neumann and Ziegenhorn (1977) and (Henry et al., 1974) respectively.
Microscopic Examination

After 28 days of the experiment (at the end of the experiment). Each rat was placed independently in a metabolic cage and urine of 24hr was collected from rats and examined microscopically for crystals.
Statistical analysis
Data were expressed as mean±SE. The statistical significance between subgroups was analyzed with one-way ANOVA using SPSS, 20 software. Followed by Duncan’s multiple range test.  Results were considered significant if the P-value<0.05. 
As shown in Table (3): In this investigation administration of lithogenic substances such as ethylene glycol (EG) in rats showed impaired renal function with increase P, Ca and ox. Stone formation in ethylene glycol is caused by hyperoxaluria, which causes increased renal retention and excretion of oxalate (Mandavia et al., 2013). Two types of kidney stones, calcium oxalate crystal forms when calcium combines with oxalic acid, Calcium phosphate stone crystals form when calcium atoms combine with phosphoric acid instead of oxalic acid and produce the calcium phosphate kidney stone. Calcium phosphate kidney stone tends to occur in people who produce more alkaline urine than those who produce calcium oxalate kidney stones (Callaghan and Bandyopadhyay 2012). Additionally, a high-phosphorus diet caused kidney stone formation (Bushinsky et al., 2000). Alterations in phosphorus homeostasis also may contribute to calcium kidney stones. Studies to date of calcium and phosphorus regulatory hormones and nephrolithiasis have been cross-sectional and focused on urine factors rather than actual kidney stone formation (Taylor et al., 2015).

Table 3: Effect of Orobanche extract on phosphorus, calcium and oxalate in kidney tissue of nephrolithiasis male albino rats.

Calcium oxalate crystal deposition is associated with severe oxidative stress to renal tissue. It leads to lipid peroxidation of membranes by the generation of reactive oxygen species (Khan 2004). Our result is coincidence with the previous study of (Velpandian et al., 2012) and (Liu et al., 2018) they reported that hyperoxaluria and crystal deposition induced the production of ROS and renal epithelial cells injury in a rat model of calcium oxalate nephrolithiasis. This finding was confirmed by urine examination as shown in Fig 1 (b) show a high density of CaOx crystals. Administration of Orobanche (EG+OR) significantly reduced (P<0.05) P, Ca and OX, Suggesting, the antiurolithiasis activity of Orobanche has been attributed to the high phenolic contents and particularly flavonoids have antiurolithiasis activity responsible for the reduction of CaOx crystal aggregation Zeng and Jiang (2019). Similarly, EG+CYT treated group significantly reduces P, Ca and OX. These results support the use of Orobanche extract as an effective alternative in treating ethylene glycol induced urolithiasis. This result was confirmed by urine examination as shown in Fig 1 (D and E) treatment with Cystone or Orobanche reduce the risk of stone formation.

Fig 1: CaOX crystals viewed in light microscope in urine.(A) control group (B) EG group, (C) OR group (3g/kg/), (D)( EG+CYT) group receiving (500mg/kg) (E) treated (EG+OR)group (3g/kg).

Microscopic Examination
As shown in Table (4) there was a significant increase (P<0.05) in urea and creatinine in ethylene glycol group compared to the control group the elevated urea and creatinine levels in serum indicated a diminished renal function in hyperoxaluric conditions. Our results are in line with previous studies (Aggrawal et al., 2012 and kumari et al., 2016). They showed that urea and creatinine levels in serum were elevated due to EG. However administrated of Orobanche extract (EG +OR) significantly reduced (P<0.05) level of urea and creatinine by improving glomerular filtration which indicates that Orobanche exerts nephroprotective effect this effect may be due to presence phytoconstituents such as flavonoid, phenolic acid Vanillic acid, Glucopyranose terpenes some earlier reports show that flavonoid-rich plants extracts possess antiurolithiatic activity (Ahmed et al., 2013). These results were coincidence with an earlier study of (Sindu et al., 2015) who reported that rats treated with vanillic acid (50mg/kg and 100mg/kg) restored the elevated levels of renal function markers and reduced antioxidant status to near normalcy. Severe hepatic damages were observed by significant elevation of hepatic enzymes ALT, AST and bilirubin activity in the serum of ethylene glycol group as shown in Table (4) this finding were in accordance with other studies of (El Sawy et al., 2014 and kumari et al., 2016) they recorded elevation in ALT and AST enzymes, the elevated levels of hepatic enzymes activities may indicate liver tissue damage probably by altered cell membrane permeability leading to the leakage of enzymes from tissue to the serum. Administration of Orobanche significantly  reduce the elevations in the liver enzyme, this result was coincidence with an earlier study of (Itoh et al., 2010) who showed that phenolic acid such as vanillic acid suppressed the transaminase enzyme.

Table 4: Effect of Orobanche Extract on liver and kidney function in the serum of nephrolithiasis male albino rats.

As shown in Table (5) there was a significant elevation (P<0.05) in the MDA level in kidney tissue associated with a significant decrease in glutathione peroxidase compared to the control group. The elevated concentration of MDA in the EG group is indicative of oxidative tissue damage, as lipid peroxides. It seems clear that renal epithelial cell injury plays a vital role in calculus development and the lithogenic effect of EG must be mainly attributed to the oxidative damage induced by high levels of oxalate generated by ethylene glycol, decreased tissue antioxidant enzymes may be followed by elevated free radical production in the early stages and in the later stages of nephrolithiasis, which may put the renal tissue under oxidative stress (Thamilselvan et al., 2003). These results are coincidence with the previous study of (Karadi et al., 2006; Khan 2014); (Golshan et al., 2017), they reported that administration of ethylene glycol induced oxidative stress. Increase glutathione peroxidase (GPx) in Orobanche treated group further confirms the anti-oxidative potential of Orobanche in renal tissue against EG-induced oxidative damage. These results support the protective effect of Orobanche against oxidative damage suggesting, that the protective effect attributed to flavonoids and phenolic compounds. Flavonoids compounds can scavenge the free radicals and attenuate lipid peroxide. It reactivates the antioxidant defense system resulting in increasing the capacity of detoxification (Ahmed et al., 2013).

Table 5: Effect of Orobanche extracts on lipid peroxide and glutathione peroxidase in kidney tissue of nephrolithiasis male albino rats.

From the result obtained it could be concluded that the administration of Orobanche extract is useful as a preventive and therapeutic against the formation of oxalate kidney stones. The underlying mechanism could be due to its diuretic effect, antioxidant, nephroprotective property, Further studies are necessary to clarify the mechanism underlying this effect.
The author thanks Dr. Samir Salam Talab Lecturer in taxonomy and Flora department, faculty of Science Zagazig University and Dr. Yasmin Hassan, Lecturer of Botany Department. Faculty of Science, Suez Canal University, Egypt for helping in taxonomy examination.

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