Therapeutic Effects of Aqueous Extracts of Cerasus Avium Stem on Ethylene Glycol- Induced Kidney Calculi in Rats Ehsaneh Azaryan1*, Mohammad Malekaneh2, Maryam Shemshadi nejad3, Fatemeh Haghighi4 Purpose: To investigate the therapeutic effects of the aqueous extract of Cerasus Avium stem on kidney calculi. Materials and Methods: In this experimental study, forty-eight (48) male Wistar rats were randomly allocated into six (6) groups and were studied during a 30 day period. Group A served as normal control and Group B re- ceived 1% ethylene glycol in drinking water (EG group). C, D, E, and F Groups, received 1% ethylene glycol from day 1 and were used as prevention and treatment subjects. Rats in prevention groups of low dose (C) and high dose (D) extract, were gavaged with 200 and 400 mg/kg extract respectively from first day of the experiment and treat- ment groups of low dose (E) and high dose (F) extract, were gavaged with 200 and 400 mg/kg extract respectively from the 15th day of the experiment. Results: On the 30th days of the experiment, serum level of magnesium and potassium decreased significantly in EG group compared with A,C,D,E and F groups (P < .05), while serum level of calcium, creatinine, uric acid, so- dium and urine level of calcium, creatinine, uric acid, increased significantly in EG group compared with A,C,D,E and F groups (P < .05). In the prevention and treatment groups, the number of deposits decreased significantly compared with EG group on the 30th day (P < .05). Conclusion: Cerasus Avium stem has a therapeutic effect on calcium oxalate stones in rats with nephrolithiasis and reduces the number of calcium oxalate deposits. Keywords: Cerasus Avium stem; ethylene glycol; kidney calculi; calcium oxalate INTRODUCTION Urinary tract stones are the third important cause of urinary tract diseases(1). The spread of urinary tract stones is increasing due to changes in peoples’ diet and life style(2). The annual incidence of urolithiasis in Iran in 2005 was 147.2 for men and 129.6 for women per 100,000 population. In the same year in Iran, the aver- age cumulative recurrence rate was 16% after 1 year, 32% after 5 years, and 53% after 10 years(3). Kidney stone formation is a complex process that results from a succession of several physicochemical events includ- ing supersaturation, nucleation, growth aggregation and retention within renal tubules(4). Oxalate, struvite, urate, brushite, cystine were the most commonly reported urolithiases in man and animal species. However, ep- idemiological studies have shown that majority (70%) of stones commonly contain calcium oxalate(5,6). In the present study, we successfully induced CaOx forma- tion in the rat’s kidney by adding EG to drinking water, which is in line with other studies(7-9). Calcium oxalate stone formation is a multi-factorial process involving various etiological factors. Hyperoxaluric rat model is the most potent experimental model for preclinical evaluation of antiurolithiatic efficacy of medicinal herbs because the physiolog- ical process mimics the etiology of kidney stone formation in human and an- imal(10). The hepatic enzymes metabolize EG to oxal- ic acid by glyoxalate mechanism, which is combined with calcium ion in the renal tubular epithelium to form calcium oxalate crystals(11). Extracorporeal shock wave lithotripsy and percutaneous nephrolithotomy tech- niques mainly include the surgical removal of stones. But, these techniques cause undesirable side effects such as tubular necrosis, hypertension, hemorrhage and subsequent fibrosis of the kidney leading to cell injury and recurrence of renal stone formation(12) Nowadays, most contemporary researchers are using and focusing on homemade and natural remedies as well as their ef- fects on the treatment of kidney stone(13). In addition, other parts of some plants such as the stem and root are frequently used in alternative medicine(14). Cerasus Avium is a tree from the family of the genus Prunus Rosacea and one of rare and valuable species in north- ern forests of Iran(15). In recent years, Cerasus Avium stems have been widely used in folk medicine. After having been dried and boiled Cerasus Avium stem is used for treatment(14). Its fruit stalks are sold by herbal druggists in Iran and are used as decoction to relief re- nal stones, edema and hypertension.(16) However, there is no evidence for the therapeutic usage of this tradi- tional medicine. Therefore, we aimed to evaluate the effects of aqueous extract of Cerasus Avium stem on the treatment of CaOx calculi in a rat model. 1Department of Biochemistry, payamenoor university of Mashhad, Mashhad,Iran. 2Department of clinical Biochemistry, Birjand University of Medical sciences, Birjand, Iran. 3Department of biology, Faculty Of science, university of Sistan and Baluchestan, Zahedan,Iran. 4Department of Pathology, Birjand Uniniversity of Medical sciences, Birjand, Iran. *Correspondence: Department of Biochemistry, payamenoor university of Mashhad, Mashhad,Iran. Email :Ehsaneh.Azaryan@gmail.com. Received November 2016 & Accepted May 2017 ENDOUROLOGY AND STONE DISEASE Vol 14 No 04 July-August 2017 4024 MATERIALS AND METHODS Preparation of aqueous extract of Cerasus. Avium stem The Cerasus Avium stems were purchased from a lo- cal herb store in Birjand, Iran. They were powdered and dried. Then, 500 g of powdered herb was mixed, through the soaking method, with distilled water. Af- ter 24 hours, the extract solution was separated by fil- ter paper. Then, the resulting solution was incubated at 40°C, until it completely dried, Then the residues were weighed (30g) and kept. Before prescribing to the an- imal, the desired concentration of the extract was pre- pared in distilled water. Treatment of animals The experiment was conducted in accordance with the guide for the care and use of laboratory animals and the study was approved by the Ethics committee of Birjand University of Medical Science. Forty-eight Wistar rats with an average weight of 200 ± 20 g were procured from Pasteur Institute of Iran. The animals were acclimatized to standard laboratory Effect of Cerasus Avium on nephrolithiasis-Azaryan et al. Table 1. Effect of Cerasus avium stem extracts on urinary and serum parameters in control and experimental animals. All values are expressed as mean ± SEM for 8 animals in each group. parametres days Agroup B group C group D group E group F group Serum (mg/dl) Calcium 15 8.8 ± 0.17 10.25 ± 0.33 a,* 10.5 ± 0.4 a,* 10.18 ± 0.25 a,* 10.28 ± 0.25 a,* 11.03 ± 0.41a,* 30 8.92 ± 0.49 11.22 ± 0.31a,* 9.28 ± 0.18 b,* 9.32±0.25 b,* 9.41± 0.17b,* 9.63±0.42 b,* Creatinine 15 0.62 ± 0.06 0.7 ± 0.07 a 0.59 ± 0.09 b 0.7 ± 0.07 a 0.68 ± 0.03 ab 0.75 ± 0.03 a 30 0.55 ± 0.02 0.78 ± 0.05 a,* 0.5 ± 0.05 b,* 0.58 ± 0.03 b,* 0.65 ± 0.02 ab,* 0.64 ± 0.04 ab,* Potassium 15 5.22 ± 0.5 4.96 ± 0.24 a 5.18 ± 0.25 a 5.63 ± 0.29 a 5.13 ± 0.26 a 5.55 ± 0.35 a 30 5.71 ± 0.3 4.4 ± 0.36 b,** 5.65 ± 0.16a,** 5.08 ± 0.43a,** 5.51 ± 0.19 a,** 5.58 ± 0.37 a,** Sodium 15 162.3 ± 6.27 174.2 ±10.01ab 177 ± 2.98 ab 174 ± 3.3 ab 182 ± 4.04 a 185 ± 5 a 30 164 ± 6.89 189.8 ±1.15 a,* 163.1 ± 3.6 b,* 171.57 ± 4.21b,* 170.33 ± 1.76 b,* 165.66 ± 6.31 b,* Magnesium 15 1.92 ± 0.19 1.78 ± 0.17 c,** 2.32 ± 0.12 ab,** 2.26 ± 0.18abc,** 2.11 ± 0.16abc,** 2.5 ± 0.06a,** 30 2.07 ± 0.12 1.68 ± 0.18 c,* 2.83 ± 0.36 b,* 2.23 ± 0.18 bc,* 4.58 ± 0.21 a,* 4.61 ± 0.23 a,* Acid Uric 15 2.05 ± 0.13 3.4 ± 0.19 a,* 3.25 ± 0.17 a,* 3.10 ± 0.22 a,* 3.63 ± 0.29 a,* 3.53 ± 0.34 a,* 30 2.37 ± 0.30 4.14 ± 0.35 a,* 2.92 ± 0.16 bc,* 2.58 ± 0.28 bc,* 3.32 ± 0.17 b,* 3.01 ± 0.27 bc,* Urin(mg/dl) Calcium 15 1.72 ± 0.21 2.33 ± 0.66 a 2 ± 0.33 a 1.98 ± 0.20 a 2.12 ± 0.31 a 2.06 ± 0.31 a 30 1.18 ± 0.03 1.58 ± 0.09 a,** 1.21 ± 0.15 b,** 1.24 ± 0.07b,** 1.15 ± 0.07 b,** 1.39 ± 0.14 ab,** Creatinine 15 37.37 ± 10.44 66.54 ± 25.70 a 50.52 ± 10.80 a 54.48 ± 8.74 a 60.51 ± 8.60 a 66.13 ± 13.73 a 30 34.42 ± 7.40 81.36 ± 23.64 a** 45.88 ± 9.52 a** 45.18 ± 20.24 a** 50 ± 15.01 a** 55.20 ± 15.05 a** Acid Uric 15 3.15 ± 0.37 6.10 ± 1.50 a 3.84 ± 0.72 a 3.93 ± 1.37 a 5.88 ± 1.70 a 5.92 ± 1.73 a 30 3.41 ±0.55 7.32 ± 2.45 b,** 2.95 ± 0.52 a,** 2.56 ± 0.38 a,** 3.16 ± 0.91 a,** 2.20 ± 0.10 a,** All values are expressed as mean±SEM for 8 animals in each group. *Statistically significant at*P < .01 **Statistically significant at **P < .05 a Comparisons are made with Group A. b Comparisons are made with Group B. c Comparisons are made with Group C. Endourology and Stone Diseases 4025 conditions (temperature: 25 ± 2 ◦C) and maintained on 12-hour light:12-hour dark cycle and they were given standard diet and had free accesses to food and water ad libitum throughout the study. They were random- ly divided into six groups of 8 and treated according to the experimental protocol for 30 days. The control Group (A) only received normal water. The other five groups received 1 % ethylene glycol (Merk,Germany) during the study period. EG group (B) did not receive any other treatment during the study period. Ethylene glycol was added from the first day for thirty(30) days to the water of prevention groups of low-dose (C) and high-dose (D). From the first day of adding ethylene glycol to the water, aqueous extract of Cerasus. Avium stem was added with 200 and 400 mg per kg of body weight. Ethylene glycol was added from the first day for 30 days to the treatment groups of low-dose (E) and high-dose (F). From the fifteenth day of adding ethyl- ene glycol to the water, aqueous extract was added with 200 and 400 mg per kg of body weight. In duration of experiment two rat of A and B groups died. Blood samples The blood samples were collected on days 15 and 30. (in the 15th day using orbital sinus and in the 30th day using cardiac puncture). Blood was collected in non-heparinized tubes and centrifuged at 3500 rpm for 15 min to obtain serum. Serum level of calcium, creati- nine, uric acid, magnesium, potassium, sodium, were measured with an Auto Analyzer.(Prestige) and colori- metric method. Urine samples Twenty-four hour (24 h) urine collection of rats in each group was performed on the 15th and 30th days, indi- vidually in metabolic cages. Food and water was avail- able during experimentation in the cages. For analysis, 1 ml of urine was taken in centrifugal tube and centri- fuged at 2500 rpm for 5 min, urine level of calcium, cre- atinine and uric acid were measured by Prestige Auto Analyzer. Histological examination For histological examination at the end of the experi- ment (the 30st day), all the rats were anesthetized and the kidneys removed and fixed in 10% formaline, dehy- drated in a gradient of ethanol, embedded in paraffin, and then cut in to 5μ serial sections. Then, slides con- taining five actions from each kidney were deparaffin- ized, stained with Hematoxyline and Eosine, and then examined by Olympus light microscope, in each slide 10 microscopic field with a magnification of 10*40 were selected randomly and the aggregation of CaOx deposits were counted in the aforementioned fields. Mean of oxalate crystals, number was reported. Figure1. Figure Representative Microscopic images of kidney sections from, (a) Control Group Shows absence of crystals deposition, (b) The large number and size of calcium oxalate crystals (arrow) in a renal tubule in ethylene glycol group (c,d) The the reduction of calculi number and size of calcium oxalate crystals in (arrow) in a renal tubule preventation and treatment groups. Figure 2. The number of calcium oxalate crystal deposits in the kidneys of the rats at the end of the experiment. Data are expressed as mean ± standard error. Vol 14 No 04 July-August 2017 4026 Effect of Cerasus Avium on nephrolithiasis-Azaryan et al. Data Analysis Data were analyzed with SPSS software (Version 20.0) using One-way ANOVA followed by Duncan’s test for multiple comparisons among all groups. P values less than .05 were considered statistically significant. Data were presented as mean ± standard error. RESULTS Serum parameters The mean level of calcium, creatinine, uric acid and se- rum sodium of the prevention and treatment groups in the 30th day decreased significantly as compared to the EG group.(P < .05). (for details see Table 1).The mean concentration of magnesium and serum potassium of the prevention and treatment group in the 30th day in- creased significantly, as compared to EG control group (P < .05). (for details see Table 1) Urine parameter The calcium, creatinine and uric acid level of urine in prevention and treatment groups in the 30th day, de- creased significantly compared to the EG group (P < .05). (for details see Table 1) Pathology results The examination of kidney sections in control group showed no calcium oxalate deposits or other abnormal- ities in different segments of the nephrons (Figures 1 and 2). But in EG group, calcium oxalate deposits, were found in different segments of the nephron (Figures 1 and 2). In prevention and treatment groups, the num- ber of deposits decreased significantly compared with EG group in both doses of aqueous extract of Cerasus. Avium stem on the 30th day Crystals in different parts of nephrons in the kidney specimens of these groups were also thin, small, and fewer compared with those in group B. (Figures 1 and 2) DISCUSSION Although, in recent years various chemical drugs have become available in the market that may be effective in prevention and treatment, and there is no effective drug therapy without surgery that can lead to complete treatment or prevention of urinary tract stones(2). Sever- al studies reported calcium oxalate crystals are injurious to renal epithelial cells by providing substrates for nu- cleation of crystals aggregation, and exposing sites for the attachment and retention of crystals leading to its adhesion to the epithelial cells, and consequently, the cells may produce some products as well as free rad- icals, inducing heterogeneous crystal nucleation and cause aggregation of crystals.(17,18) Due to the presence of substances such as caffeic acid, ferulic acid, syringic acid, ellagic acid, quercetin, α-tocopherol, pyrogallol, p- hydroxybenzoic acid, vanillin, p-coumaric acid, gal- lic acid and ascorbic acid, the cerasus Avium stems have antioxidant and antiradical properties(19). There- fore, it is speculated that Cerasus Avium stem, could prevent the formation of CaOx calculi and their dis- aggregation through its anti-inflammatory and antiox- idant mechanisms. In the present study, quercetin and tocopherol reduced the morality of tubular and level of free radicals, thereby reducing the formation of calcium oxalate crystals(20,21). In addition, Quercetin, α-tocoph- erol and frolic acid lower cholesterol in serum(19,22,23). According to the study, the increase of fat is one of the causes of kidney stones(24). In this context, oxalate has been reported to induce lipid peroxidation and cause renal tissue damage by reacting with polyunsaturated fatty acids in cell membrane(25). It is possible that one of the reasons for the effect of Cerasus Avium stem on reducing calcium oxalate stones is the anti-fat roles of quercetin , α-tocopherol and frolic acid. Some studies suggest that calcium stones may have an infectious origin. Nano bacterium, an intracellular bacterium, is found in the kidney stones. These bacteria cause an in- crease in calcium stones(26,27). Pyrogallol is a substance found in the stem of Cerasus Avium having anti-bacte- rial and anti-microbial properties(5). Hence, it might be that the anti-bacterial property of Cerasus Avium stem is effective in the treatment of kidney stones in addi- tion to afore-mentioned mechanisms in the formation of CaOx crystals. The present study showed that the major cause of kidney stone is potassium and magnesium re- duction and promising results in preventing recurrence have been shown in patients treated with potassium magnesium citrate. Magnesium complexes with oxa- late, reduce the supersaturation of calcium oxalate and as a consequence reduce the nucleation rate and growth of crystals(28,29,30). The Cerasus Avium stem treatment increased the magnesium and potassium level of serum and thus reduced the growth of calcium oxalate crystals in drug treated animals. In urolithiasis, the glomerular filtration rate (GFR) decreases due to the obstruction to the outflow of urine by stones in urinary system. Due to this, the waste products, particularly nitrogenous sub- stances such as urea, creatinine and uric acid get ac- cumulated in blood(31). The results of this study show that aqueous extract of Cerasus Avium stem reduced serum and urine creatinine and uric acid in prevention and treatment groups. It is not clear which mechanisms in the plants have effects on kidney stones. This may be as a result of its anti-inflammatory, antioxidant, an- ti-bacterial, and anti-fat properties. However, there is need to do more research so as to certain the effects of this plant. CONCLUSIONS The aqueous extracts of Cerasus Avium stem signifi- cantly reduced the elevated level of calcium oxalate ions. The histopathological findings also show sign of improvement after treatment with extract. 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