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Anti-Hyperuricemic and Nephroprotective Effects of Smilax China L

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Anti-Hyperuricemic and Nephroprotective Effects of Smilax China L Journal of Ethnopharmacology 135 (2011) 399–405 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Anti-hyperuricemic and nephroprotective effects of Smilax china L. Lvyi Chen a, Huafeng Yin a, Zhou Lan b, Shuwei Ma a, Chunfeng Zhang a,∗, Zhonglin Yang a,∗, Ping Li a, Baoqin Lin c a Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Ministry of Education, No. 24 Tongjia Lane, Nanjing 210009, PR China b Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, PR China c Department of Pharmacology, School of Traditional Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China article info abstract Article history: Ethnopharmacological relevance: Smilax china L., popularly known as “Jin Gang Ten”, has been widely used Received 17 November 2010 as a traditional herbal medicine for the treatment of gout, rheumatoid arthritis and other diseases for a Received in revised form 10 March 2011 long time in China. Accepted 12 March 2011 Aim of study: The present study was carried out to investigate the effect of Smilax china L. on hyperuricemia Available online 21 March 2011 and renal dysfunction in induced hyperuricemic animals. Materials and methods: Five fractions (petroleum ether, chloroform, ethyl acetate, n-butanol and residual Keywords: ethanol fraction) of Smilax china L. were orally administered to potassium oxonate-induced hyper- Smilax china L. Anti-hyperuricemic agents uricemic mice for three days. The xanthine oxidase inhibitory activities and modes of action of nine Hyperuricemia compounds isolated from ethyl acetate fraction (EAF) were then examined in vitro. Finally, different Uric acid dosages of EAF were administered to 10% fructose-induced hyperuricemic rats. Nephroprotection Results: EAF (250 mg/kg) exhibited stronger anti-hyperuricemic activity in hyperuricemic mice compared with the other four fractions. Caffeic acid, resveratrol, rutin and oxyresveratrol isolated from EAF showed different inhibitory activities on xanthine oxidase in vitro, with the IC50 values of 42.60, 37.53, 42.20 and 40.69 ␮M, respectively, and exhibited competitive or mixed inhibitory actions. Moreover, EAF (125, 250 and 500 mg/kg) markedly reversed the serum uric acid level (p < 0.05, p < 0.01 and p < 0.001, respec- tively), fractional excretion of urate (p < 0.05, p < 0.01 and p < 0.01, respectively) and blood urea nitrogen (p < 0.05, p < 0.01 and p < 0.01, respectively) to their normal states, and prevented the renal damage against tubulointerstitial pathologies in hyperuricemic rats. Conclusion: These findings show that Smilax china L. exhibits anti-hyperuricemic and nephroprotective activity in hyperuricemic animals. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction while overproduction is the cause in less than 10% (Wortmann, 2002). Impaired excretion of uric acid is mostly induced by urate Gout is usually characterized by recurrent attacks of acute transporter abnormality in the proximal kidney tubule. While pro- inflammatory arthritis with a red, tender, hot, and swollen joint. duction of uric acid is elevated due to more active XOD and high It is caused by elevated levels of uric acid in the blood which intake of dietary purine (Anzai et al., 2005; Caulfield et al., 2008). crystallize and are deposited in joints, tendons, and surrounding Increasing clinical reports have shown that hyperuricemia asso- tissues. Uric acid, the end product of purine metabolism, is created ciated with an increasing risk of not only gout, but also chronic when xanthine oxidase (XOD) catalyzes the oxidation of hypoxan- nephritis, renal dysfunction, as well as metabolic syndromes (Iseki thine and xanthine in mammal. Renal insufficient excretion of uric et al., 2001, 2004; Ishizaka et al., 2005; Yoo et al., 2005; Cirillo acid is the primary cause of hyperuricemia in about 90% of cases, et al., 2006; Zhou et al., 2006; Weiner et al., 2008). Nowadays, a number of anti-hyperuricemic agents including uricosuric agents and XOD inhibitors have been available in the market (Schlesinger, Abbreviations: BUN, blood urea nitrogen; CF, chloroform fraction; DMSO, 2004). The associated adverse reactions such as gastrointestinal dimethyl sulphoxide; EAF, ethyl acetate fraction; FEUA, fractional excretion of irritation, bone marrow suppression, renal toxicity, hypersensi- urate; HPLC, high-performance liquid chromatographic; NF, n-butanol fraction; PEF, tivity syndromes and so on, however, always limit their clinical petroleum ether fraction; REF, residual ethanol mother solution fraction; Scr, serum uses (Horiuchi et al., 2000; Hammer et al., 2001; Terkeltaub, 2003). creatinine; Sur, serum uric acid; Ucr, urinary creatinine; Uur, urinary uric acid; XOD, Moreover, patients have to use allopurinol which has an higher rate xanthine oxidase. ∗ Corresponding authors. Tel.: +86 25 8327 1426; fax: +86 25 8327 1426. of hypersensitivity reactions when uricosuric drugs lose their effi- E-mail address: [email protected] (Z. Yang). cacy in the case of concurrent renal insufficiency. Therefore, it is 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.03.033 400 L. Chen et al. / Journal of Ethnopharmacology 135 (2011) 399–405 very urgent and important to search for better anti-hyperuricemic the Provision and General Recommendation of Chinese Experimen- medicines. tal Animals Administration Legislation and were approved by the Smilax china L., popularly known as “Jin Gang Ten” in China, Science and Technology Department of Jiangsu Province. belongs to the Liliaceae family. It is prescribed to treat gout and rheumatoid arthritis by dispelling wind-evil and eliminat- ing dampness according to the theory of Chinese medicine 2.4. Anti-hyperuricemic effect of Smilax china L. fractions in (State Administration of Traditional Chinese Medicine of People’s potassium oxonate-induced hyperuricemic mice Republic of China, 1999; Chen et al., 2008; State Pharmacopoeia Commission of People’s Republic of China, 2010). It has also been 2.4.1. Preparation of plant extracts used for syphilis, acute bacillary dysentery, tumor (Wu et al., 2010) The dried powder of Smilax china L. rhizome (10 kg, 40-mesh) × and inflammation (Shu et al., 2006) for more than 1000 years was percolated with 80% EtOH (1 100 L) for six days at room and virtually no toxicity was reported. Stilbenes, flavonoids and temperature. The pooled extracts upon solvent were concentrated steroidal saponins are reported as the main active components of under partial vacuum to obtain 1200 g of ruddy residue (12%, w/w). Smilax china L. (Ruan et al., 2002; Xu et al., 2008). Although there are The residue was extracted successively with equal volumes (9.6 L, many reports of biological activities on gout, it is still little known for three times) of petroleum ether, chloroform, ethyl acetate and about the anti-hyperuricemic mechanisms and active ingredients n-butanol. Each sub-fraction was then concentrated under reduced of Smilax china L. Therefore, the research was cried out to study the pressure to obtain petroleum ether fraction (PEF, 96 g, 8%, w/w), anti-hyperuricemic mechanisms and active components of Smilax chloroform fraction (CF, 192 g, 16%, w/w), EAF (300 g, 25%, w/w), n- china L. butanol fraction (NF, 480 g, 40%, w/w) and residual ethanol mother Anti-hyperuricemic fractions of Smilax china L. were inves- solution fraction (REF, 132 g, 11%, w/w). tigated in potassium oxonate-treated mice through examining serum uric acid (Sur) level. Subsequently, compounds were isolated 2.4.2. Mice model of hyperuricemia and drug administration from the anti-hyperuricemic fraction, and their XOD inhibitory All the mice were divided at random into eight groups of activities were assayed in vitro. Finally, the anti-hyperuricemic ten mice each. Hyperuricemic mice model induced by potas- mechanism and nephroprotective effect of ethyl acetate frac- sium oxonate (uricase inhibitor) was used to study drug action tion (EAF) were also investigated through evaluating the indexes (Stavric et al., 1975; Zhu et al., 2004). Briefly, PEF (80 mg/kg), CF of fractional excretion of urate (FEUA), blood urea nitrogen (160 mg/kg), EAF (250 mg/kg), NF (400 mg/kg), REF (110 mg/kg) (BUN), XOD and renal tubulointerstitial pathological changes in and allopurinol (10 mg/kg) were dissolved in 0.3% CMC–Na aque- fructose-induced hyperuricemic rats. The final results provided a ous solution, respectively. The dosage of each fraction corresponds pharmacological basis on hypouricemia and renal protection of to 8.33 g crude drug/kg. All drugs were given orally once daily at Smilax china L. 8:00–9:00 a.m. for three consecutive days. Mice were intraperi- toneally injected with potassium oxonate (250 mg/kg) 1 h before 2. Materials and methods the final drug administration to increase the Sur level. Food, but not water, was withdrawn from the animals 1.5 h prior to drug 2.1. Plant materials administration. The raw materials of the rhizome of Smilax china L. were col- lected in October 2008 in Anhui province, China, provided by 2.4.3. Sample collection and measurements Simcere Drugstore in Nanjing, China. The materials were identified Blood samples were collected from mice by tail vein bleeding 1 h by Professor Ping Li, School of Traditional Chinese Medicine of China after the final drug administration on the third day. The blood was Pharmaceutical University. A voucher specimen was deposited allowed to clot for approximately 1 h at room temperature and then under the number no. 25441 in the herbarium of China Phar- centrifuged at 2500 × g for 10 min to obtain the serum. The serum maceutical University. All the materials were dried at the room was stored at −20 ◦C until use. The Sur level was measured using temperature to constant weight. standard diagnostic kits. Each assay was performed in triplicate. 2.2. Reagents and drugs 2.5. Isolation and identification of compounds from EAF All chemicals were of analytical grade.
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