Hypouricemic Action of Scopoletin Arising from Xanthine Oxidase Inhibition and Uricosuric Activity

• Abstract
• Materials and Methods
• References

Abstract

Scopoletin exhibited an immediate and dose-dependent hypouricemic effect after intraperitoneal administration (50, 100, 200 mg/kg) in hyperuricemic mice induced by potassium oxonate; however, it did not affect the serum uric acid level in normal mice at the tested doses. For exploring the involved mechanisms of action of scopoletin, potential inhibitory effects on xanthine oxidase and possible uricosuric effects were investigated. Scopoletin (50, 100, 200 mg/kg) significantly inhibited the activity of xanthine oxidase in liver homogenates of hyperuricemic mice although it only showed a relatively weak, albeit competitive-type, inhibition of xanthine oxidase in a commercial assay. Furthermore, a potent uricosuric effect of scopoletin (100, 200 mg/kg) was ascertained. These results demonstrated for the first time that scopoletin exhibits, hypouricemic activities through decreasing uric acid production and as well as a uricosuric mechanism.

Gout is a disorder of urate metabolism characterized by the deposition of monosodium urate crystals in the joints and soft tissues. Recent estimates suggested that gout affects 8.4 per 1000 persons [1]. Gout is currently the most common cause of inflammatory arthritis in men > 40 years of age and is frequently encountered in clinical practice [2]. Current treatment of hyperuricemia associated with gout entails the uses of anti-inflammatory agents to relieve the symptoms of the disease as well as xanthine oxidase inhibitors to block the biosynthesis of uric acid from purine. For patients who underexcrete uric acid, uricosuric drugs such as probenecid are preferentially prescribed.

Scopoletin, a coumarin compound, was isolated from the stems of Erycibe obtusifolia Benth which was usually used in Traditional Chinese Medicine for rheumatic arthritis therapy. It has been proved to possess a strong anti-inflammatory activity in rats and mice [3], [4], [5]. In screening for xanthine oxidase inhibitory compounds, Chang et al. [6], [7] found that scopoletin showed a less strong activity in vitro as compared to other compounds with similar chemical structures such as esculetin. After that, scopoletin failed to attract the interest of researchers. Recently, we screened several coumarins including scopoletin in a search for potential hypouricemic compounds. Interestingly, scopoletin was found to exhibit significant hypouricemic activity in oxonate-induced hyperuricemic mice in spite of a relatively weak inhibition of xanthine oxidase in vitro. To elucidate the underlying mechanisms of action of scopoletin, the present study was undertaken to investigate the in vivo inhibitory potency on xanthine oxidase and the effect on the excretion of uric acid.

The hypouricemic effect of scopoletin is presented in Fig. [1]. Scopoletin (50, 100, 200 mg/kg), 1 h after intraperitoneal injection to hyperuricemic mice, significantly reduced the serum uric acid levels by 35.0, 49.3 and 56.8 %, respectively. But it did not affect the serum uric acid level in normal mice (data not shown). In contrast, allopurinol (20 mg/kg) substantially lowered the uric acid levels in both hyperuricemic and normal mice.

Potassium oxonate pretreatment did not influence the xanthine oxidase activity in liver homogenates of mice. Scopoletin (50, 100, 200 mg/kg) and allopurinol (20 mg/kg) significantly inhibited xanthine oxidase activity in liver homogenates of hyperuricemic mice (Fig. [2]). Commercial enzyme analysis demonstrated scopoletin to be a weak but competitive-type inhibitor of xanthine oxidase (IC₅₀, 164.7 μM; Ki, 103.8 μM).

The urinary urate level in hyperuricemic mice was significantly higher than that in normal mice, whereas urine volumes remained unchanged. Pretreatment with scopoletin (100, 200 mg/kg) or probenecid (100 mg/kg) markedly enhanced the urinary urate level but not urine volume (Fig. [3]).

At doses over 100 mg/kg, scopoletin decreased the serum uric acid level to almost the same extent as allopurinol (20 mg/kg). Such a powerful hypouricemic action may be attributed to inhibition of xanthine oxidase and a uricosuric effect. The inhibitory effect of scopoletin on xanthine oxidase observed from the liver homogenate assay was seemingly stronger than that from the commercial enzyme assay in the present study and previous reports [6], [7]. The precise reason responsible for such a discrepancy remains unclear. Chang et al. [7] reported that group substitution substantially influenced the inhibitory effects of coumarin derivatives on xanthine oxidase. Aesculin, a 7-hydroxycoumarin 6-glycoside derivative, was devoid of activity [8]. We speculated that the metabolites of scopoletin probably exert more intense inhibitory effects on xanthine oxidase than the original substance.

The uricosuric effect of scopoletin might be of special importance, considering that in clinical practice about 90 % of gout patients are ascribed to an underexcretion of urate while only 10 % of the cases are due to an increase of uric acid production [9], and that the renal transport system of urate in mice is similar to that in human beings [10]. In collective consideration, scopoletin should find its way into gout therapy in view of its powerful anti-inflammatory effect [3], [4], [5] and the hypouricemic activity demonstrated for the first time in this paper.

Materials and Methods

Reagents: Scopoletin (purity > 98 %) was isolated from the stems of Erycibe obtusifolia Benth under supervision of Professor Zhengtao Wang. Allopurinol (purity > 98 %), probenecid (purity > 98 %), xanthine (purity > 99 %), xanthine oxidase (purity > 99 %) were purchased from Sigma Chemicals (St. Louis, MO, USA). Potassium oxonate was obtained from Tokyo Kassei Industry Co. Ltd. (Tokyo, Japan). All other chemicals were the highest analytical grade available.

Preparation of test samples: Potassium oxonate was suspended in 0.8 % sodium carboxymethyl cellulose (CMC-Na) solution. Scopoletin was dissolved in dimethyl sulfoxide (DMSO) and further diluted with 1/15 mM sodium phosphate buffer (pH 7.5) for in vitro experiments. The final concentration of DMSO in the reaction solution was 0.5 % and this concentration was confirmed in our preliminary experiments not to affect the results. For in vivo experiments, scopoletin was suspended in 0.8 % CMC-Na solution. The volume administered was based on the body weight of animals measured immediately before tests. Allopurinol and probenecid, used as reference compounds, were prepared in the same way as scopoletin.

Animals: Male ICR mice (28 ∼ 32 g) were purchased from the animal center of China Pharmaceutical University and were housed in plastic cages. They were allowed one week to adapt to the environment before experiments. Animals were maintained on a 12 h light/dark cycle in an air-conditioned room and given standard chow and water ad libitum. All animal experiments were conducted under the ethical regulations for animal care and use of China Pharmaceutical University.

Potassium oxonate-induced hyperuricemia in mice: The uricase inhibitor potassium oxonate was used to induce hyperuricemia in mice according to previous reports [8], [11]. Briefly, mice were injected intraperitoneally with potassium oxonate (350 mg/kg). Whole blood samples were collected and serum separated 2 h later. Test samples or allopurinol were intraperitoneally administered 1 h after injection of potassium oxonate. In the uricosuric experiment, mice were given 1 mL of normal saline by gavage as water load right before administration of test samples. Then, urine in 1 h duration was collected in metabolic cages. The concentrations of uric acid in serum and diluted urine were determined by phosphotungstic acid colorimetry using a commercial kit (Uric Acid Test Kit, Nanjing Jiancheng Bioengineering Insititute, Jiangsu, China).

Enzyme assays: Mouse liver was homogenized in 2 mL of PBS buffer (pH 7.4) for 1 min with a homogenizer at a speed of 2000 rpm. The homogenate was centrifuged at 600 × g at 4 °C for 20 min, then at 105,000 × g at 4 °C for 60 min. Xanthine oxidase in the resultant supernatant was measured using a spectrophotometric method [11], [12] with xanthine as substrate. The commercial xanthine oxidase assay was performed by allowing the enzyme to react with xanthine under aerobic conditions following the procedure outlined elsewhere [6], [13]. The inhibitory activity of scopoletin at concentrations of 25, 50, 100, 150, 200 μM was determined and the IC₅₀ value calculated. Allopurinol was used as reference compound to verify the robustness of the enzyme assay.

Statistical analysis: All values are expressed as mean ± standard error of the mean (S.E.M). Student's t-test was applied to the comparison between two groups, while one-way ANOVA followed by LSD test was used for multiple comparisons. Statistical significance was set at p < 0.05.

References

• 1 Lawrence R C, Helmick C G, Arnett F C. et al . Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States.  Arthritis Rheum. 1998;  41 778-99
  CrossrefPubMedSearch in Google Scholar
• 2 Roubenoff R. Gout and hyperuricemia.  Rheum Dis Clin North Am. 1990;  16 539-50
  PubMedSearch in Google Scholar
• 3 Farah M H, Samuelsson G. Pharmacologically active phenylpropanoids from Senra incana .  Planta Medica. 1992;  58 14-8
  PubMedSearch in Google Scholar
• 4 Silvan A M, Abad M J, Bermejo P, Sollhuber M, Villar A. Anti-inflammatory activity of coumarins from Santolina oblongifolia .  J Nat Prod. 1996;  59 1183-5
  CrossrefPubMedSearch in Google Scholar
• 5 Muschietti L, Gorzalczany S, Ferraro G, Acevedo C, Martino V. Phenolic compounds with anti-inflammatory activity from Eupatorium buniifolium .  Planta Medica. 2001;  67 743-4
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Chang W S, Chiang Y H, Lu F J, Chang H C. Inhibitory effects of phenolics on xanthine oxidase.  Anticancer Res. 1994;  14 501-6
  PubMedSearch in Google Scholar
• 7 Chang W S, Chiang H C. Structure-activity relationship of coumarins in xanthine oxidase inhibition.  Anticancer Res. 1995;  15 1969-73
  PubMedSearch in Google Scholar
• 8 Kong L D, Zhou J, Wen Y L, Li J M, Cheng C HK. Aesculin possesses potent hypouricemic action in rodents but is devoid of xanthine oxidase /dehydrogenase inhibitory activity.  Planta Medica. 2002;  68 175-8
  Thieme ConnectPubMedSearch in Google Scholar
• 9 Wright J D, Printo A B. Clinical manifestations and treatment of gout.  Prim Care Update Ob/Gyns. 2003;  10 19-23
  PubMedSearch in Google Scholar
• 10 Dan T, Koga H, Onuma E, Tanaka H, Sato H, Aoki B. The activity of AA-193, a new uricosuric agent, in animals.  Adv Exp Med Biol. 1989;  253A 301-8
  PubMedSearch in Google Scholar
• 11 Hall I H, Scoville J P, Reynolds D J, Simlot R, Duncan P. Substituted cyclic imides as potential anti-gout agents.  Life Sci. 1990;  46 1923-7
  CrossrefPubMedSearch in Google Scholar
• 12 Huh K, Yamamoto I, Gohda E, Iwata H. Tissue distribution and characteristics of xanthine oxidase and allopurinol oxidizing enzyme.  Jap J Pharmacol. 1976;  26 719-24
  PubMedSearch in Google Scholar
• 13 Noro T, Oda Y, Miyase T, Ueno A, Fukushima S. Inhibition of xanthine oxidase from the flowers and buds of Daphne genkwa .  Chem Pharm Bull. 1983;  31 3984-7
  PubMedSearch in Google Scholar

Dr. Yue Dai

Department of Pharmacology of Chinese Materia Medica

China Pharmaceutical University

1 Shennong Road

Nanjing 210038

People's Republic of China

Email: yuedaicpu@hotmail.com

References

• 1 Lawrence R C, Helmick C G, Arnett F C. et al . Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States.  Arthritis Rheum. 1998;  41 778-99
  CrossrefPubMedSearch in Google Scholar
• 2 Roubenoff R. Gout and hyperuricemia.  Rheum Dis Clin North Am. 1990;  16 539-50
  PubMedSearch in Google Scholar
• 3 Farah M H, Samuelsson G. Pharmacologically active phenylpropanoids from Senra incana .  Planta Medica. 1992;  58 14-8
  PubMedSearch in Google Scholar
• 4 Silvan A M, Abad M J, Bermejo P, Sollhuber M, Villar A. Anti-inflammatory activity of coumarins from Santolina oblongifolia .  J Nat Prod. 1996;  59 1183-5
  CrossrefPubMedSearch in Google Scholar
• 5 Muschietti L, Gorzalczany S, Ferraro G, Acevedo C, Martino V. Phenolic compounds with anti-inflammatory activity from Eupatorium buniifolium .  Planta Medica. 2001;  67 743-4
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Chang W S, Chiang Y H, Lu F J, Chang H C. Inhibitory effects of phenolics on xanthine oxidase.  Anticancer Res. 1994;  14 501-6
  PubMedSearch in Google Scholar
• 7 Chang W S, Chiang H C. Structure-activity relationship of coumarins in xanthine oxidase inhibition.  Anticancer Res. 1995;  15 1969-73
  PubMedSearch in Google Scholar
• 8 Kong L D, Zhou J, Wen Y L, Li J M, Cheng C HK. Aesculin possesses potent hypouricemic action in rodents but is devoid of xanthine oxidase /dehydrogenase inhibitory activity.  Planta Medica. 2002;  68 175-8
  Thieme ConnectPubMedSearch in Google Scholar
• 9 Wright J D, Printo A B. Clinical manifestations and treatment of gout.  Prim Care Update Ob/Gyns. 2003;  10 19-23
  PubMedSearch in Google Scholar
• 10 Dan T, Koga H, Onuma E, Tanaka H, Sato H, Aoki B. The activity of AA-193, a new uricosuric agent, in animals.  Adv Exp Med Biol. 1989;  253A 301-8
  PubMedSearch in Google Scholar
• 11 Hall I H, Scoville J P, Reynolds D J, Simlot R, Duncan P. Substituted cyclic imides as potential anti-gout agents.  Life Sci. 1990;  46 1923-7
  CrossrefPubMedSearch in Google Scholar
• 12 Huh K, Yamamoto I, Gohda E, Iwata H. Tissue distribution and characteristics of xanthine oxidase and allopurinol oxidizing enzyme.  Jap J Pharmacol. 1976;  26 719-24
  PubMedSearch in Google Scholar
• 13 Noro T, Oda Y, Miyase T, Ueno A, Fukushima S. Inhibition of xanthine oxidase from the flowers and buds of Daphne genkwa .  Chem Pharm Bull. 1983;  31 3984-7
  PubMedSearch in Google Scholar

Dr. Yue Dai

Department of Pharmacology of Chinese Materia Medica

China Pharmaceutical University

1 Shennong Road

Nanjing 210038

People's Republic of China

Email: yuedaicpu@hotmail.com