Hypouricemic effect of the methanol extract from Prunus mume fruit in mice

Abstract

Context: The fruit of the Prunus mume Sieb. et Zucc (Rosaceae) is used as a health food or medicinal material in traditional herb medicine for a long time in Eastern Asian countries.

Objective: Our present study investigated the hypouricemic effect of the methanol extract from P. mume fruit (MPMF) in mice with potassium oxonate-induced hyperuremia.

Materials and methods: Effect of MPMF (35, 70 and 140 mg/kg, p.o.) administrated for 7 days on the serum, liver, urinary uric acid levels and liver xanthine oxidase (XO) activity were assessed in mice.

Results: Hyperuricemic mice induced by potassium oxonate demonstrated an elevation in serum and liver uric acid levels (11.0 mg/dL and 0.52 mg/g tissue) and a reduction in urinary uric acid levels (49.9 mg/dL). Oral administration of 140 mg/kg MPMF for 7 days reversed the abnormalities in serum, liver and urinary uric acid levels (7.1 mg/dL, 0.37 mg/g tissue and 69.7 mg/dL, respectively). In addition, 70 and 140 mg/kg MPMF (3.1 and 2.9 nmol/min per mg protein) inhibited liver XO activity compared with hyperuricemic mice (3.9 nmol/min per mg protein).

Discussion and conclusion: The results indicated that the beneficial hypouricaemic effect of MPMF may be mediated, at least in part, by inhibiting XO activity in the liver. Our study suggests that P. mume and its extracts may have a considerable potential for development as an anti-gout agent for clinical application.

Keywords::

• Uric acid
• xanthine oxidase
• potassium oxonate
• hyperuremia

Introduction

Gout is caused by elevated levels of uric acid in the blood which crystallize and are deposited in joints, tendons, and surrounding tissues. It is well accepted that hyperuricemia is the underlying metabolic disorder leading to gout (Chen & Schumacher, 2008). Under excretion of urate, the salts of uric acid, has been implicated to produce hyperuricemia (Chen & Schumacher, 2008). Xanthine oxidase (XO) which catalyzes the hypoxanthine and xanthine to uric acid plays an important role in the catabolism of purines and its related metabolic disorders including hyperuricemia and gout (Stamp et al., 2007). As a result, XO is early as one of targets of investigation for pathological mechanism and drug action on hyperuricemia and gout (Osada et al., 1993; Zhu et al., 2004; Wang et al., 2010). For example, allopurinol, one of the most commonly prescribed XO inhibitors for gout worldwide, blocks uric acid production, and long-term therapy is safe and well tolerated (Chao & Terkeltaub, 2009).

Besides chemical medicine, herbal therapy is another effective alternative to treat gout. For example, anthocyanin extracts of purple sweet potato and Hyoscyamus reticulatus L. (Solanaceae) extracts reversed the elevation of serum uric acid in potassium oxonate-induced hyperuricemia (Mohammad et al., 2010; Hwa et al., 2011). In addition, XO inhibitor-like effects have been shown with an extract of leaves of Lagerstromia speciosa (L.) Pers. (Lythraceae) (Unno et al., 2004). Prunus mume Sieb. et Zucc (Rosaceae), a deciduous tree, is widely grown in China and other Eastern Asian countries. The various parts of the P. mume tree (such as fruit, seed, and leaf) have been used as a health food or medicinal material in traditional herb medicine. It has recently been reported that P. mume fruit improved blood fluidity (Chuda et al., 1999), exerted ergogenic and physical fatigue recovery activities (Kim et al., 2008), stimulated the proliferation and osteoblastic differentiation of cells (Kono et al., 2011), enhanced the immune function by stimulating innate immune cells (Tsuji et al., 2011), and exhibited antimicrobial activity against pathogenic oral bacteria (Seneviratne et al., 2011). Moreover, it is particularly used in traditional Chinese medical preparations as a remedy for gout (Zhao & Yang, 2002; Chen et al., 2007). These clinical reports on anti-gout activity suggested us to investigate the function in metabolic diseases improvement. Therefore, the present experiments were designed to verify a possible hypouricemic effect of methanol extract from P. mume fruit (MPMF) in mice with potassium oxonate-induced hyperuremia.

Materials and methods

Animals

Male Kunming mice (24 ± 2 g) were purchased from Laboratory Animal Centre, Fujian, Medical University, Fujian Province, P. R. China. Animals were housed 8 per cage (320 × 180 × 160 cm) under a normal 12 h light/dark schedule with the lights on at 07:00 a.m. and had free access to tap water and food pellets. Ambient temperature and relative humidity were maintained at 22 ± 2°C and at 55 ± 5%, and given a standard chow and water ad libitum for the duration of the study. The animals were allowed 1 week to acclimatize themselves to the housing conditions before the beginning of the experiments. All procedures were performed in accordance with the published guidelines of the China Council on Animal Care (Regulations for the Administration of Affairs Concerning Experimental Animals, approved by the State Council on October 31, 1988 and promulgated by Decree No. 2 of the State Science and Technology Commission on November 14, 1988).

Preparation of extract and other drugs

The best quality commercial fruit of P. mume was purchased from Fujian Pharmaceutical CO., LTD, R.P.China and authenticated by Cheng-Fu Li, Xiamen Hospital of Traditional Chinese Medicine, P.R.China (voucher specimen number HU/CE-03172). P. mume fruit (500 g), from which seeds were removed, was extracted three times for 12 h with 2 L of methanol by sonication at room temperature (25 ± 2°C). The solutions were combined, filtered, concentrated under reduced pressure and lyophilized into powders (MPMF). The final yield was 4.48% (w/w). Potassium oxonate and allopurinol were purchased from Sigma-Aldrich Co., USA.

Mice model of hyperuricemia and drug administration

Different groups of mice, 8 animals per group, were used for drug treatment. Drugs were dissolved in 0.9% physiological saline. All doses were expressed as milligrams per kilogram body weight of the respective drugs. Food, but not water, was withdrawn from the animals 1 h prior to drug administration. Dose for experimentation was calculated on the basis of body surface area ratio with reference to suggested human doses. Briefly, according to the State Pharmacopoeia of People’s Republic of China (Chinese Pharmacopoeia Committee, 2010), the dosage of P. mume fruit for adults is 6–12 g (the raw material) and the extract yield of MPMF was 4.48%, equivalently for mice, this dosage is 0.70–1.40 mg for 20 g mice calculated by body surface area method (human dose × extract field × conversion factor for 20 g mice). Thus, the dosage of MPMF was 35–70 mg/kg. Therefore, we used three doses of MPMF at 35, 70 and 140 mg/kg in this study. The drug treatment schedule was the same as described in detail elsewhere (Wang et al., 2010). Briefly, six groups of eight mice were administered in a volume of 10 mL/kg by gavage once daily with potassium oxonate (250 mg/kg) or water (vehicle) at 8:00 a.m. for seven consecutive days. MPMF (35, 70 and 140 mg/kg), allopurinol (5 mg/kg) or water were administered by gavage initiated at 9:00 a.m. on the day when the potassium oxonate was given. They were treated for seven consecutive days.

Serum, liver and urine sample collection

One hour later, after final drug administration on the seventh day, mice were sacrificed by decapitation and whole blood samples were collected, allowed to clot for 1 h at ambient temperature, and centrifuged at 10,000× g for 5 min to obtain serum. The serum and urine were stored at −20°C until assayed. Simultaneously, the liver tissues were rapidly and carefully separated on an ice-plate. These tissue samples were stored at −80°C for assays.

Determination of uric acid levels

The liver was homogenized in 10 volumes of 50 mM ice-cold potassium phosphate buffer (pH 7.4). The homogenate was centrifuged at 12,000× g at 4°C for 15 min. The supernatant fraction was used to measure uric acid levels in liver. The uric acid levels in serum, liver and urine were measured using a phosphotungstic acid method (Carroll et al., 1971) with an assay kit (Nanjing Jiancheng Bioengineering Institute, R.P.China).

Hepatic XO activity assay

The liver was homogenized in 10 volumes of 50 mM ice-cold potassium phosphate buffer (pH 7.4) containing 5 mM ethylenediamine tetraacetic acid disodium salt (EDTA-Na) and 1 mM phenylmethanesulfonyl fluoride (PMSF). The homogenate was centrifuged at 12,000× g at 4°C for 15 min. The supernatant fraction was centrifuged at 12,000× g at 4°C for 15 min once again and the supernatant fraction was used to detect XO activity by an assay kit (Nanjing Jiancheng Bioengineering Institute, R.P. China). Protein concentration was determined by the method of Lowry et al. (1951) using an assay kit (Nanjing Jiancheng Bioengineering Institute, R.P. China). The liver XO enzyme activity was expressed as nmol/min/mg protein.

Statistical analyses

All data were expressed as mean ± S.E.M. To compare experimental and control groups, we used one-way ANOVA, followed by post hoc Tukey test using the SPSS software (SPSS Inc., Chicago, USA). A value of p < 0.05 was considered statistically significant for analysis.

Results

Effects of MPMF on serum, liver and urinary uric acid in mice with potassium oxonate-induced hyperuremia

Effects of MPMF on uric acid levels in serum, liver and urine of hyperuricemic mice are shown in Figure 1. Compared to normal-vehicle mice, serum uric acid levels of mice were significantly increased by potassium oxonate (p < 0.01) (Figure 1A). A one-way ANOVA revealed a significant effect after drug treatment (p < 0.01). Post hoc analysis indicated a significant decrease in the serum uric acid levels elicited by the administration of MPMF at 70 and 140 mg/kg (p < 0.05, p < 0.05, respectively). Allopurinol (5 mg/kg), the positive control, also reduced the serum uric acid levels after 7 days of treatment (p < 0.01).

In addition, as shown in Figure 1B, potassium oxonate induced a remarkable elevation in liver uric acid levels in mice compared to normal-vehicle mice (p < 0.01). A one-way ANOVA revealed a significant effect after drug treatment (p < 0.05). Post hoc analysis indicated MPMF only at 140 mg/kg and allopurinol reduced the liver uric acid levels (p < 0.05, p < 0.05, respectively).

Figure 1.  Effects of methanol extract from Prunus mume fruit (MPMF) on uric acid levels in serum (A), liver (B) and urine (C) in mice with potassium oxonate-induced hyperuremia. Data were expressed as the mean ± S.E.M. ^##p < 0.01 vs vehicle-control group (Con); ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs vehicle with potassium oxonate group (Mod).

As shown in Figure 1C, in potassium oxonate-induced hyperuricemic mice, urinary uric acid levels were significantly lower than that of normal-vehicle (p < 0.01). A one-way ANOVA revealed a significant effect after drug treatment (p < 0.001). Post hoc analysis indicated MPMF at 70 and 140 mg/kg (p < 0.01, Pp < 0.001, respectively) and allopurinol at 5 mg/kg (p < 0.01) exhibited a significant increase in urinary uric acid levels in hyperuricemic animals.

Effects of MPMF on hepatic XO activity in mice with potassium oxonate-induced hyperuremia

Potassium oxonate induced a remarkable elevation of liver XO activity compared to normal-vehicle mice (p < 0.01) as shown in Figure 2. A one-way ANOVA revealed a significant effect after drug treatment (p < 0.001). A reduction in liver XO activity was observed following administration with 70, 140 mg/kg MPMF (p < 0.05, p < 0.01, respectively). Allopurinol at 5 mg/kg (p < 0.001) also suppressed liver XOD activity of hyperuricemic mice.

Figure 2.  Effects of methanol extract from Prunus mume fruit (MPMF) on liver xanthine oxidase (XO) activity in mice with potassium oxonate-induced hyperuremia. Data were expressed as the mean ± S.E.M. ^##p < 0.01 vs vehicle-control group (Con); ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs vehicle with potassium oxonate group (Mod).

Discussion

Hyperuricemia is the main pathological feature of gout. Since serum uric acid levels are closely linked with hypertension, cardiovascular disease, kidney disease (Kutzing & Firestein, 2008), the discovery of hypouricaemic agents that could further improve current gout therapies are welcomed (Dalbeth & So, 2010). In the past few years, the pharmaceutical industry has seen a shift from the search for specifically target drugs to the pursuit of combination or herb therapies that comprise more than one active ingredient (Qiu, 2007). More important, encouraging data support the hypouricaemic effectiveness of some traditional prescriptions or products such as Sanmiao wan and cassia oil (Zhao et al., 2006; Wang et al., 2010).

Our current study assessed the hypouricaemic effect of MPMF in mice with potassium oxonate-induced hyperuremia. Potassium oxonate, a well-known selectively competitive uricase inhibitor, which blocks the effect of hepatic uricase and produces excessive serum uric acid levels in rodents, is the most frequently employed to perform the hyperuricemic mouse model because of a short time and cost less (Hall et al., 1990). The measurement index serum uric acid can be decreased by therapeutically-effective drugs for gout (Mayer et al., 2005). In our study, the results that serum uric acid levels were markedly higher than that of control, indicated potassium oxonate successfully induced hyperuricemia. MPMF at 70 and 140 mg/kg treatment for 7 days significantly decreased serum uric acid in potassium oxonate-induced mice. However, it should be noted that the extent of reduced effect on serum uric acid by MPMF was lower than that by allopurinol. In addition, our data also found that MPMF decreased liver uric acid and elevated urinary uric acid in the potassium oxonate-treated mice. Consistent with our study, an early study performing with the same protocol showed that potassium oxonate induced the abnormality of uric acid in serum, liver and urine, and allopurinol reversed the changes (Wang et al., 2010). Our present findings showed that MPMF, in addition to having many previously described biological activities (Chuda et al., 1999; Kim et al., 2008; Kono et al., 2011; Tsuji et al., 2011; Seneviratne et al., 2011), may be a potent uric acid-lowering agent.

The identification of new hypouricaemic drugs, such as acting by inhibiting uric acid synthesis (XO inhibitors), is definitely needed (Dalbeth & So, 2010). The enzyme XO oxidizes hypoxanthine and xanthine to uric acid in the purine catabolic pathway. It is well known that liver XO play a key role in the pathophysiology of hyperuricemia and gout (Carcassi et al., 1969; Pacher et al., 2006). It is evident that XO is increased in the liver of the patients affected by primary gout, in comparison with the controls (Carcassi et al., 1969). The inhibition of XO can reduce the uric acid levels (Pacher et al., 2006). In this study, we further evaluated the XO activity in potassium oxonate-treated mice after MPMF treatment. Our current study showed that the abnormality of XO activity was suppressed by MPMF and suggested that MPMF may serve as a XO inhibitor.

MPMF mainly contains several triterpenoids such as oleanolic acid, ursolic acid, lupeol and α-amyrin (Kawahara et al., 2009; Yamai et al., 2009). It has been reported that lupeol and its extract decreased serum uric acid in hyperuricemia-like rats and nephrolithiasis patients (Sudharsan et al., 2006; Singh et al., 2011), oleanolic acid and ursolic acid possessed XO inhibition activity in vitro (Yin & Chan, 2007). Therefore, we speculated that the hypouricemic effect of MPMF could be attributed to triterpenoids.

Conclusion

The present results clearly demonstrated that MPMF was able to significantly decrease serum and liver uric acid levels, and elevate urinary uric acid levels in potassium oxonate-treated mice. This beneficial hypouricaemic effect may be mediated, at least in part, by inhibiting XO activity in the liver. Further work, including clinical application, will be necessary to determine whether MPMF produces a similar therapeutic efficacy as was observed in the present study.

Declaration of interest

The project was co-financed by the Natural Science Foundation of Fujian Province of China (No. 2011J05081) and Huaqiao University (No. 09BS507) to Li-Tao Yi (L.T. Yi).