Urolithiasis (2014) 42:519–526 DOI 10.1007/s00240-014-0695-7
ORIGINAL PAPER
Prophylactic effects of quercetin and hyperoside in a calcium oxalate stone forming rat model Wei Zhu · Yun‑fei Xu · Yuan Feng · Bo Peng · Jian‑ping Che · Min Liu · Jun‑hua Zheng
Received: 3 March 2014 / Accepted: 20 July 2014 / Published online: 2 August 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Quercetin and hyperoside (QH) are the two main constituents of the total flavone glycosides of Flos Abelmoschus manihot, which has been prescribed for treating chronic kidney disease for decades. This study aimed to investigate the effect of QH on calcium oxalate (CaOx) formation in ethylene glycol (EG)-fed rats. Rats were divided into three groups: an untreated stone-forming group, a QH-treated stone-forming group (20 mg/kg/day) and a potassium citrate-treated stone-forming group (potassium citrate was a worldwide-recognized calculi-prophylactic medicine). Ethylene glycol (0.5 %) was administered to the rats during the last week, and vitamin D3 was force-fed to induce hyperoxaluria and kidney calcium oxalate crystal deposition. 24 h urine samples were collected before and
after inducing crystal deposits. Rats were killed and both kidneys were harvested after 3 weeks. Bisected kidneys were examined under a polarized light microscope for semiquantification of the crystal-formation. The renal tissue superoxide dismutase and catalase levels were measured by Western blot. QH and potassium citrate have the ability to alkalinize urine. The number of crystal deposits decreased significantly in the QH-treated stone-forming group as compared to the other groups. Superoxide dismutase and catalase levels also increased significantly in the QH-treated stone-forming group, as compared with the untreated stoneforming group. QH administration has an inhibitory effect on the deposition of CaOx crystal in EG-fed rats and may be effective for preventing stone-forming disease.
W. Zhu, Y. Xu and Y. Feng are equal first contributors for this work.
Keywords Nephrolithiasis · Calcium oxalate · Quercetin · Hyperoside · Oxidative stress
W. Zhu · Y. Xu · B. Peng · J. Che · M. Liu (*) · J. Zheng (*) Department of Urological Surgery, Shanghai Tenth People’s Hospital, Tongji University, Shanghai 200072, China e-mail: [email protected]
Abbreviations QH Quercetin and hyperoside CaOx Calcium oxalate EG Ethylene glycol KCit Potassium citrate SOD Superoxide dismutase NaCl Sodium chloride SDS Sodium dodecyl sulfate PAGE Polyacrylamide gel electrophoresis BSA Bovine serum albumin
J. Zheng e-mail: [email protected] W. Zhu e-mail: [email protected] Y. Xu e-mail: [email protected] B. Peng e-mail: [email protected] J. Che e-mail: [email protected]
Introduction
Y. Feng Department of Nephrology, Nanjing University Affiliated Drum Tower Hospital, Nanjing 210093, China e-mail: [email protected]
Urolithiasis is a multi-factorial urological disorder common all over the world. Calcium oxalate (CaOx) is the most common cause of stones with a frequency of more than 80 %
13
520
[1]. The prevalence of urolithiasis differs in various parts of the world: 1–5 % in Asia, 5–9 % in Europe, 13 % in North America, and 20 % in Saudi Arabia, and its prevalence continues to increase annually [2]. Although this disorder has been known since ancient times, the mechanism of renal stone formation remains unclear and few substances that effectively prevent urolithiasis are available. Although minimally invasive management techniques, such as percutaneous nephrolithtomy, have been developed in the past two decades, the side effects such as hemorrhage, renal fibrosis are serious; therefore the identification of substances that effectively prevent crystal deposition and the development of urolithiasis are urgently required in the clinic. Quercetin is a potent bioflavonoid commonly found in vegetables and fruit. It is a main component of the total flavone glycosides of Flos Abelmoschus manihot, which is a common plant grown in eastern China and south-east Asia [3]. Moreover, quercetin is a potent antioxidant that directly scavenges free radicals, inhibits xanthine oxidase and lipid peroxidation, and alters antioxidant defense pathways both in vivo and in vitro [4, 5]. Quercetin is an efficient antioxidant that has been implicated as in inhibitor of oxidative damage in renal tubular cells and renal tissues [6]. However, to our knowledge, Park et al. have investigated the effect of quercetin on oxalate-induced urinary stone formation [7]. Hyperoside is a major pharmacologically active constituent of the flavonoid glycosides found in natural plants, and recently has attracted increasing attention due to its diverse antioxidant, anti-inflammatory, diuretic, anti-hyperglycemic, hypo-uricemic, hepatoprotective and anti-fungal properties. However, the anti-urolithiasis effects of this substance have not been confirmed in vivo. Previous studies conducted in our laboratory demonstrated that the combination of quercetin and hyperoside derived from the traditional Chinese herb, Abelmoschl manihot, showed satisfactory anti-proliferative activities in the human renal cancer cell line 786-O [8]. However, to our knowledge, there is no evidence of the prophylactic effects of the two compounds in urolithiasis. Therefore, the present study was undertaken to determine the effect of quercetin and hyperoside (QH) on urinary excretion factors and to determine the anti-oxidative effects on calcium oxalate urolithiasis in a rat hyperoxaluria-induced stone model, compared with those of sodium potassium citrate. This information is important in evaluating the potential of QH for the treatment of renal calculi in the clinic.
Materials and methods Preparation of QH for gastric feeding Polyphenolics were extracted from a standardized hyperoside and quercetin dehydrate supplement (ratio 1:1) in
13
Urolithiasis (2014) 42:519–526
capsule form provided by Suzhong Pharmaceutical Co. (Taizhou, Jiangsu, China). The dried samples were ground to a powder for administration by gastric intubation. Dry powder of QH stored at room temperature was dissolved in 500 ml of heated double-distilled water to prepare a stock solution that was stored at room temperature. The feeding solution contained QH at a concentration of 2 mg/ml was prepared immediately before use. Stone‑forming rat model and experimental design All animal experimental protocols were approved by the Animal Research Committee of the Tongji University. Sprague–Dawley rats (n = 18; aged 7 weeks; weight 180–200 g, were acclimated to room temperature for more than 4 weeks and then fed a standard commercial rat chow during the study. Ethylene glycol (EG 0.5 %) was administered to the rats in drinking water during the last week of the 3-week study. Rats were force-fed with 0.5 μmol of vitamin D3 dissolved in 1 ml of salad oil by gastric intubation to induce hyperoxaluria and kidney calcium oxalate crystal deposition. The rats were divided into three groups. All groups were acclimated for 1 week before the start of the experiment. Group 1, EG group (n = 6): animals received normal drinking water for 2 weeks and crystal deposits were induced in week 3. Group 2, potassium citrate (KCit) group (n = 6): the animals were force-fed KCit (100 mg/kg daily) by gastric intubation for 2 weeks before the induction of crystal deposits in week 3 [9, 10]. Group 3, quercetin and hyperoside group (n = 6): the animals were force-fed QH (20 mg/ kg body weight daily in week 3 [11, 12]. The experimental period was 3 weeks. At the end of the experiment, all rats were euthanized with an overdose of anesthesia (diethyl ether) and both kidneys were harvested. One kidney was treated with 10 % formalin, embedded in paraffin, and strained with hematoxylin and eosin solution, while the other kidney was immediately stored at −80 °C for further study. Measurement of urinary variables Twenty-four hour urine samples were collected on day 14 before the start of crystal deposit induction and before euthanization. Rats were kept individually in metabolic cages for 24 h urine collection, which was either analyzed immediately or stored at −80 °C until further analysis. Drinking volume, urine volume, and urine pH were measure manually. Urinary calcium, potassium, sodium, phosphate, creatinine, and magnesium levels were measured using a Model 705 automate analyzer (SRL, Tokyo, Japan). A portion of the urine sample was acidified with 6 mol/L HCl to maintain urine pH at
ORIGINAL PAPER
Prophylactic effects of quercetin and hyperoside in a calcium oxalate stone forming rat model Wei Zhu · Yun‑fei Xu · Yuan Feng · Bo Peng · Jian‑ping Che · Min Liu · Jun‑hua Zheng
Received: 3 March 2014 / Accepted: 20 July 2014 / Published online: 2 August 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Quercetin and hyperoside (QH) are the two main constituents of the total flavone glycosides of Flos Abelmoschus manihot, which has been prescribed for treating chronic kidney disease for decades. This study aimed to investigate the effect of QH on calcium oxalate (CaOx) formation in ethylene glycol (EG)-fed rats. Rats were divided into three groups: an untreated stone-forming group, a QH-treated stone-forming group (20 mg/kg/day) and a potassium citrate-treated stone-forming group (potassium citrate was a worldwide-recognized calculi-prophylactic medicine). Ethylene glycol (0.5 %) was administered to the rats during the last week, and vitamin D3 was force-fed to induce hyperoxaluria and kidney calcium oxalate crystal deposition. 24 h urine samples were collected before and
after inducing crystal deposits. Rats were killed and both kidneys were harvested after 3 weeks. Bisected kidneys were examined under a polarized light microscope for semiquantification of the crystal-formation. The renal tissue superoxide dismutase and catalase levels were measured by Western blot. QH and potassium citrate have the ability to alkalinize urine. The number of crystal deposits decreased significantly in the QH-treated stone-forming group as compared to the other groups. Superoxide dismutase and catalase levels also increased significantly in the QH-treated stone-forming group, as compared with the untreated stoneforming group. QH administration has an inhibitory effect on the deposition of CaOx crystal in EG-fed rats and may be effective for preventing stone-forming disease.
W. Zhu, Y. Xu and Y. Feng are equal first contributors for this work.
Keywords Nephrolithiasis · Calcium oxalate · Quercetin · Hyperoside · Oxidative stress
W. Zhu · Y. Xu · B. Peng · J. Che · M. Liu (*) · J. Zheng (*) Department of Urological Surgery, Shanghai Tenth People’s Hospital, Tongji University, Shanghai 200072, China e-mail: [email protected]
Abbreviations QH Quercetin and hyperoside CaOx Calcium oxalate EG Ethylene glycol KCit Potassium citrate SOD Superoxide dismutase NaCl Sodium chloride SDS Sodium dodecyl sulfate PAGE Polyacrylamide gel electrophoresis BSA Bovine serum albumin
J. Zheng e-mail: [email protected] W. Zhu e-mail: [email protected] Y. Xu e-mail: [email protected] B. Peng e-mail: [email protected] J. Che e-mail: [email protected]
Introduction
Y. Feng Department of Nephrology, Nanjing University Affiliated Drum Tower Hospital, Nanjing 210093, China e-mail: [email protected]
Urolithiasis is a multi-factorial urological disorder common all over the world. Calcium oxalate (CaOx) is the most common cause of stones with a frequency of more than 80 %
13
520
[1]. The prevalence of urolithiasis differs in various parts of the world: 1–5 % in Asia, 5–9 % in Europe, 13 % in North America, and 20 % in Saudi Arabia, and its prevalence continues to increase annually [2]. Although this disorder has been known since ancient times, the mechanism of renal stone formation remains unclear and few substances that effectively prevent urolithiasis are available. Although minimally invasive management techniques, such as percutaneous nephrolithtomy, have been developed in the past two decades, the side effects such as hemorrhage, renal fibrosis are serious; therefore the identification of substances that effectively prevent crystal deposition and the development of urolithiasis are urgently required in the clinic. Quercetin is a potent bioflavonoid commonly found in vegetables and fruit. It is a main component of the total flavone glycosides of Flos Abelmoschus manihot, which is a common plant grown in eastern China and south-east Asia [3]. Moreover, quercetin is a potent antioxidant that directly scavenges free radicals, inhibits xanthine oxidase and lipid peroxidation, and alters antioxidant defense pathways both in vivo and in vitro [4, 5]. Quercetin is an efficient antioxidant that has been implicated as in inhibitor of oxidative damage in renal tubular cells and renal tissues [6]. However, to our knowledge, Park et al. have investigated the effect of quercetin on oxalate-induced urinary stone formation [7]. Hyperoside is a major pharmacologically active constituent of the flavonoid glycosides found in natural plants, and recently has attracted increasing attention due to its diverse antioxidant, anti-inflammatory, diuretic, anti-hyperglycemic, hypo-uricemic, hepatoprotective and anti-fungal properties. However, the anti-urolithiasis effects of this substance have not been confirmed in vivo. Previous studies conducted in our laboratory demonstrated that the combination of quercetin and hyperoside derived from the traditional Chinese herb, Abelmoschl manihot, showed satisfactory anti-proliferative activities in the human renal cancer cell line 786-O [8]. However, to our knowledge, there is no evidence of the prophylactic effects of the two compounds in urolithiasis. Therefore, the present study was undertaken to determine the effect of quercetin and hyperoside (QH) on urinary excretion factors and to determine the anti-oxidative effects on calcium oxalate urolithiasis in a rat hyperoxaluria-induced stone model, compared with those of sodium potassium citrate. This information is important in evaluating the potential of QH for the treatment of renal calculi in the clinic.
Materials and methods Preparation of QH for gastric feeding Polyphenolics were extracted from a standardized hyperoside and quercetin dehydrate supplement (ratio 1:1) in
13
Urolithiasis (2014) 42:519–526
capsule form provided by Suzhong Pharmaceutical Co. (Taizhou, Jiangsu, China). The dried samples were ground to a powder for administration by gastric intubation. Dry powder of QH stored at room temperature was dissolved in 500 ml of heated double-distilled water to prepare a stock solution that was stored at room temperature. The feeding solution contained QH at a concentration of 2 mg/ml was prepared immediately before use. Stone‑forming rat model and experimental design All animal experimental protocols were approved by the Animal Research Committee of the Tongji University. Sprague–Dawley rats (n = 18; aged 7 weeks; weight 180–200 g, were acclimated to room temperature for more than 4 weeks and then fed a standard commercial rat chow during the study. Ethylene glycol (EG 0.5 %) was administered to the rats in drinking water during the last week of the 3-week study. Rats were force-fed with 0.5 μmol of vitamin D3 dissolved in 1 ml of salad oil by gastric intubation to induce hyperoxaluria and kidney calcium oxalate crystal deposition. The rats were divided into three groups. All groups were acclimated for 1 week before the start of the experiment. Group 1, EG group (n = 6): animals received normal drinking water for 2 weeks and crystal deposits were induced in week 3. Group 2, potassium citrate (KCit) group (n = 6): the animals were force-fed KCit (100 mg/kg daily) by gastric intubation for 2 weeks before the induction of crystal deposits in week 3 [9, 10]. Group 3, quercetin and hyperoside group (n = 6): the animals were force-fed QH (20 mg/ kg body weight daily in week 3 [11, 12]. The experimental period was 3 weeks. At the end of the experiment, all rats were euthanized with an overdose of anesthesia (diethyl ether) and both kidneys were harvested. One kidney was treated with 10 % formalin, embedded in paraffin, and strained with hematoxylin and eosin solution, while the other kidney was immediately stored at −80 °C for further study. Measurement of urinary variables Twenty-four hour urine samples were collected on day 14 before the start of crystal deposit induction and before euthanization. Rats were kept individually in metabolic cages for 24 h urine collection, which was either analyzed immediately or stored at −80 °C until further analysis. Drinking volume, urine volume, and urine pH were measure manually. Urinary calcium, potassium, sodium, phosphate, creatinine, and magnesium levels were measured using a Model 705 automate analyzer (SRL, Tokyo, Japan). A portion of the urine sample was acidified with 6 mol/L HCl to maintain urine pH at
