U J 02 - Spring 2012 - All 008- without adv.pdf 472 | Effect of Hydroalcoholic Extract of Hypericum Perforatum L. Leaves on Ethyl- ene Glycol-Induced Kidney Calculi in Rats Mohsen Khalili,1 Mohammad Reza Jalali,2 Mohammad Mirzaei-Azandaryani3 Purpose: To investigate the effects of the hydroalcoholic extract of Hypericum perforatum (H. perforatum) leaves on the kidney calculi in rats. Materials and Methods: - H. Perforatum solution was fed at the same time of EG application and was repeated once for two days EG-ammonium chloride-added drinking water and was fed with normal chow. H. Perforatum in low were removed and prepared for histologic evaluation of calcium oxalate deposits. Results: H. perforatum (300 H. perforatum - creased compared to controls (P < .01; P < .05; and P - ment of the rats with high dose of H. Perforatum decrementing effect of EG on serum level of free calcium (P experiments showed that chronic feeding of H. perforatum (300 and 500 mg/kg, in EG group. Conclusion: Chronic treatment of rats with hydroalcoholic extract of H. perfo- ratum reduced the size and number of calcium oxalate deposits in EG-induced calculi. Keywords: hypericum perforatum, kidney calculi, ethylene glycol, calcium oxalate Corresponding Author: Mohammad Mirzaei- Azandaryani, MD Department of Physiology, Medicinal Plant, School of Medicine, Shahed Univer- sity of Medical Sciences, Tehran, Iran Tel: +98 21 8896 4792 Fax: +98 21 8896 6310 E-mail: mohammadmir- zaei89@yahoo.com Received December 2010 Accepted June 2011 1Department of Physiolo- gy, Medicinal Plant, School of Medicine, Shahed Uni- versity of Medical Sciences, Tehran, Iran 2Department of Pathol- ogy, School of Medicine, Shahed University of Medi- cal Sciences, Tehran, Iran 3Shahed University Stu- dent Research Committee (SHUSRC), School of Medi- cine, Shahed University of Medical Sciences, Tehran, Iran Endourology and Stone Disease Endourology and Stone Disease 473Vol. 9 | No. 2 | Spring 2012 |U R O LO G Y J O U R N A L Hypericum Perforatum on Kidney Calculi | Khalili et al INTRODUCTION Urinary stone is a prevalent disorder in the urinary system. Genetics, low activity, and diet are the most common factors that could lead to calculi formation by high satura- tion of calcium and production of calcium oxalate Calcium oxalate and calcium phosphate are the most common kid- ney stones and may cause various adverse effects, such as obstruction, infection, hemorrhage, and pain resulted from passage of stones in the urinary tract system. Unfortunately, chemical drugs for prevention or treatment of the kidney stones could not success- fully dissolve the problem. Meanwhile, if laser therapy as a cost-effective procedure is used for dis- ruption of calculi, many severe complications may yield. Therefore, the wisely clinical method for pre- vention, disaggregation, or disruption of calculi is the usage of a safe, cheap, and with low side effects medication, like medicinal plants. Hypericum perforatum L., commonly known as St. especially grown in Iran, India, China, Turkey, and some other countries. The most common SJW preparations used are hydroalcoholic extracts of the aerial portion of the plant that contain at least ten different kinds of biochemical compounds. The essential and active ingredients of this herb include - vorable effects have been reported. Many stud- ies show that SJW could relieve mild to moderate forms of depression. Furthermore, antioxidant, urinary system relaxant effects, and inhibition of calcium crystallization in the urinary system have been reported. The present study has experimen- tally evaluated the effect of SJW extract on ethylene glycol-induced CaOx crystallization in rat model. MATERIALS AND METHODS Animals were procured from Pasteur Institute in Tehran, four per cage in a temperature-controlled colony they were given free access to water and kept at 80% to 85% of their free-feeding body weight throughout the experiment. This study was conducted in accordance with the policies set forth in the Guide for the Care and the Research Council of Shahed University of Medical Sciences. Preparation of Plant Hydroalcoholic Extract Hypericum perforatum (H. perforatum) was pro- University. The hydroalcoholic extract was pre- pared as described elsewhere. Experimental Procedure inducer in the rats. This component in the body is broken down to some organic acids, especially oxalic acid, which precipitates as CaOx crystals in the kidney. However, concomitant adminis- by EG application solely. In the present study, the animals were randomly - - ed drinking water throughout the entire experi- H. Perforatum solutions were fed by gastric gavage at the same time of EG application and repeated once for two days till the end of the experimental period. access to food and normal drinking water. Group chloride-added drinking water and was fed with 474 | H. Per- foratum doses, respectively. Serum and Urine Analysis At the beginning and end of the experiment, the - pose, each rat was individually kept in a meta- The blood serum was obtained for biochemical th week. Blood was collected from retro-orbital plexus by capillary tube at the beginning and from the heart follow- ing anesthesia, at the end of the experiments. Laboratory examination included measurement of free calcium, phosphorus, magnesium, potas- sium, and sodium. Evaluation of the Severity of Renal Crystal Deposition At the end of the experiment after blood sample collection, the rats were killed by carbon dioxide inhalation. Thereafter, the right and left kidneys were removed from the body and weighed. The kidneys were kept in formalin and then blocked - tally right and left kidney sections in 5 μm were prepared by microtome and then the slides were stained by Hematoxylin and Eosin. Finally, a light microscope was used in order to examine the presence of crystal depositions in his- tological sections. For this purpose, aggregation of CaOx deposits were counted in 10 microscopes each group. For accuracy in counting of CaOx de- posits, we hypothetically divided each sagittal re- nal specimen into 10 equal–square size regions by Table 1. Biochemical data of rats at baseline. Baseline data, mmol/dL Group 1 Control (n = 10) Group 2 EG (n = 12) Group 3 EG + HP (low dose) (n = 15) Group 4 EG + HP (high dose) (n = 15) Urine level Phosphorus 117.15 ± 21.20 120.00 ± 15.90 145.00 ± 21.14 115.75 ± 26.60 Free calcium 51.10 ± 9.3 54.66 ±10.10 44.02 ± 12.77 Magnesium 113.16 ± 15.17 127.00 ± 13.14 131.75 ± 24.25 Sodium 111.00 ± 3.71 Potassium 59.00 ± 7.07 72.25 ± 7.33 Serum level Phosphorus Free calcium 11.24 ± 0.11 10.93 ± 0.23 11.10 ± 0.23 11.43 ± 0.35 Magnesium 2.21 ± 0.43 2.71 ± 0.50 Sodium 150.51 ± 1.21 152.33 ± 1.45 151.50 ± 1.32 149.50 ± 0.95 Potassium 6.35 ± 0.73 7.10 ± 0.07 Hypericum perforatum. Data are expressed as mean ± standard error. Endourology and Stone Disease 475Vol. 9 | No. 2 | Spring 2012 |U R O LO G Y J O U R N A L Data Analysis One-way ANOVA and post-hoc Tukey tests were used for analysis of serum and urine parameters. The non-parametric Kruskal-Wallis test and Mann-Whitney U test were used for comparison of CaOx deposites. P values of less than .05 were RESULTS Biochemical Analysis in baseline biochemical parameters among con- there were no differences in serum and also urine levels of phosphorus, free calcium, magnesium, sodium, and potassium at the beginning of the study. parameters after the experiment are shown. As in- dicated, urine level of free calcium in groups EG H. Perforatum and phosphorous in H. Perforatum compared to controls (P < .01; P < .05; and P rats have markedly raised the urine level of mag- nesium (P administration of H. Perforatum serum levels of phosphorus and free calcium in to normal rats (P < .05 and P However, treatment of rats with high dose of H. Perforatum decremented effect of EG on serum level of free calcium (P body and kidney weights gain or loss between Histological Examination As Figure 1 shows, no CaOx deposits were found Table 2. Effect of H. Perforatum on urine and serum biochemical data in rats.†£ Baseline data, mmol/dL Group 1 Control (n = 10) Group 2 EG (n = 12) Group 3 EG + HP (low dose) (n = 15) Group 4 EG + HP (high dose) (n = 15) Urine level Phosphorus 114.11 ± 19.2 117 ± 13.9 95.75 ± 16.65 a* Free calcium 29.54 ± 9.14 a** 34.30 ± 11.67 a* 41.32 ± 9.21 Magnesium 110.13 ± 14.14 147 ± 12.12 a* 141.75 ± 14.2 a* a* Sodium Potassium 47.70 ± 05.32 64.31 ± 2.27 Serum level Phosphorus 9.75 ± 0.90 a* Free calcium 10.72 ± 0.41 6.6 ± 0.24 a** 5.1 ± 0.33 a* 9.3 ± 0.43 b* Magnesium 3.39 ± 0.06 2.95 ± 0.17 Sodium 150.75 ± 2.13 151.66 ± 1.66 151.25 ± 1.1 Potassium 7.45 ± 0.92 6.75 ± 0.26 7.12 ± 0.06 6.90 ± 0.2 † Hypericum perforatum. £ Data are expressed as mean ± standard error. a and b show significant group compared to normal and EG groups, respectively. *P < .05 and **P < .01. Hypericum Perforatum on Kidney Calculi | Khalili et al 476 | in control animals. But due to EG application, a in the proximal tubules, loops of Henle, distal tubules, and collecting ducts (P - ment of the EG group with low and high doses of H. Perforatum P < In addition to decrement of the number of calcu- H. Perforatum groups, we also found a marked reduction in the animals which were treated with H. Perforatum DISCUSSION In the present study, we successfully induced CaOx formation in the rat’s kidney by adding EG to dinking water, which is in line with other studies. We added ammonium chloride to EG drinking water for masking the metabolic acido- sis induced by EG-derivative acids, such as gly- colaldehyde acid, glycolic acid, glyoxylic acid, and oxalic acid. Our data showed that ethanolic extract of H. Per- foratum had a preventive effect on CaOx calcu- lus formation in the rat’s kidney. The low and high doses of extract also reduced the number of Complementary data indicated a low level of free calcium in urine and serum in EG-group in comparison with control animals. This is because most of the serum or urine calcium was bound with oxalate to form crystals; hence, very little free calcium was detected. To the best of our knowledge, the present study of the H. Perforatum on the prevention and treat- ment of CaOx kidney calculus. The exact mechanisms through which H. Perfo- ratum affects CaOx calculi are still under debate. Table 3. Effect of H. Perforatum on the body and kidney weight of rats.†£ Group 1 Control (n = 10) Group 2 EG (n = 12) Group 3 EG + HP (low dose) (n = 15) Group 4 EG + HP (high dose) (n = 15) Body weight gain, g* 175.11 ± 16.22 157.63 ± 23.9 166.25 ± 17.65 Right kidney weight, g 1.99 ± 0.31 1.66 ± 0.47 Left kidney weight, g 1.91 ± 0.5 1.74 ± 0.21 1.79 ± 0.49 † Hypericum perforatum. £ Data are expressed as mean ± standard error. * Body-weight gain is computed as the final body weight of each rat minus the baseline body weight of the same rat. Figure 1. Calcuim oxalate deposition in study groups.£¥ £ Bars represent the mean number of calculi in each group. ¥ Hypericum perforatum. * and # show difference compared to control and EG groups, respectively. **P < 0.01, ***P < .001, and # P < .05. Endourology and Stone Disease 477Vol. 9 | No. 2 | Spring 2012 |U R O LO G Y J O U R N A L Calcium oxalate crystals and high levels of ox- alate in the nephrons can damage the epithelial cells, and consequently, the cells may produce some products as well as free radicals, inducing heterogeneous crystal nucleation and cause ag- gregation of crystals. H. Perforatum quercetin and quercetin-3. Several studies - - fects. Therefore, it is speculated that H. Per- foratum could prevent the formation of CaOx calculi and their disaggregation through its anti- Furthermore, H. Perforatum extract may interfere with the process of epithelial cell damage induced by crystals. The reports about the suppression of each step of CaOx crystal formation, growth, and aggregation in vitro are concomitant with the same results in vivo trial in our experiment. Therefore, prevention of crystal nucleus produc- tion by H. Perforatum can be speculated. The most important protein that plays the role of crys- tal nucleus is chondroitin sulphate. In addition to afore-mentioned mechanisms in the formation of CaOx crystals, the role of bacterial origin, such as nanobacteria, should also be men- tioned. However, the powerful antibacterial effect of H. Perforatum could yield its antilithi- atic effect. Of notice is the diuretic effect of H. Perforatum, which could help in prevention of calculi. Figure 2. (A) Normal medullary and papil- lary tubules are shown in a con- trol rat’s kidney. (B) The large size of calcium oxa- late crystals (arrow) in a renal tu- bule in ethylene glycol-indoced calculi in rats. (C) Arrow shows the reduction of calculi size in ethylene glycol + Hypericum Perforatum group. Hypericum Perforatum on Kidney Calculi | Khalili et al 478 | CONCLUSION The oral feeding of the alcoholic extract of H. 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