Introduction

Hyperuricemia is a common biochemical abnormality that has become much more common worldwide and is closely associated with gout, chronic kidney disease (CKD), and metabolic disorders (Benn et al. 2018). It is a condition of abnormal elevation of circulating uric acid as a result of imbalance between its production and elimination (Puig and Martinez 2008). Hyperuricemia can obstruct the renal tubules due to the deposition of urate crystals, resulting in ischemic type of cell injury with collagen deposition and macrophage infiltration (Gude et al. 2013; Mazzali et al. 2001). The mechanisms underlying hyperuricemia-mediated kidney injury include endothelial dysfunction, oxidative stress, and inflammation (Cristóbal-García et al. 2015; Long et al. 2008). In this context, xanthine oxidase (XO) is the key enzyme for the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid which is excreted by kidneys. XO is distributed in mammalian tissues and generates reactive oxygen species (ROS), resulting in oxidative stress (Enroth et al. 2000). Therefore, attenuation of the in vivo production of uric acid by XO inhibitors represents the main treatment strategy for hyperuricemia. Despite that urate-lowering agents are currently available, their long-term use at the recommended dose to lower uric acid levels may cause severe side effects (Chao and Terkeltaub 2009, Yang et al. 2015). Hence, more effective agents with fewer or no adverse effects against hyperuricemia are highly warranted.

Medicinal plants have an important contribution in the treatment of many human diseases, including hyperuricemia (Ajarem et al. 2017; Kamel et al. 2021; Mahmoud et al. 2017; Süntar 2019). Monolluma quadrangula (Forssk.) Plowes is a succulent plant belonging to the family Apocynaceae. It is also called Caralluma quadrangula (Albers and Meve 2002) and members of this genus have been traditionally used for the treatment of various ailments, such as diabetes, skin rashes, inflammation, and cancer (Abdel-Sattar et al. 2009; Ashwini et al. 2017; Bin-Jumah 2019; De Leo et al. 2005; Habibuddin et al. 2008). M. quadrangula has been used as appetite suppressant and traditionally in the treatment of diabetes and gastric ulcers (Abdel-Sattar et al. 2009; Ashwini et al. 2017; Bin-Jumah 2019; De Leo et al. 2005; Habibuddin et al. 2008; Ibrahim et al. 2016). M. quadrangula attenuated ethanol-induced peptic ulcer (Ibrahim et al. 2016) and prevented liver injury in type 2 diabetic rats (Bin-Jumah 2019) via amelioration of oxidative stress and restoration of antioxidants. Although some therapeutic efficacies of M. quadrangula have been demonstrated, its potential renoprotective and XO inhibitory effects in hyperuricemic rats remain unclear. Therefore, this study explored the renoprotective and XO inhibitory action of a flavonoid-rich extract of M. quadrangula in vitro and in potassium oxonate (PO)-induced hyperuricemia in rats. We assummed that M. quadrangula can attenuate hyperuricemia by inhibiting XO activity, and oxidative stress, and modulating urate transorters in the kidny of PO-administered rats.

Materials and methods

Extraction and isolation

M. quadrangula was collected from Al-Taif (Saudi Arabia), and the species was identified and authenticated by botanists from the biology department and a voucher specimen was kept at the Herbarium of the College of Science (Jouf University, Saudi Arabia). The plant was dried and powdered using an electric grinder. The grinded air-dried aerial parts (4.5 kg) were defatted with chloroform (3 × 5 L) and exhaustively extracted with 70% ethanol by cold maceration (3 × 10 L). The extract was then filtered, and the solvent was removed under reduced pressure at temperature ≈ 40 °C till complete dryness to afford a dark brown sticky mass residue (234 g). The obtained crude extract was successively partitioned with hexane (1.5 L × 3 times) and ethyl acetate (1.5 L × 3 times). The ethyl acetate fraction (55 g) was carefully chromatographed over a polyamide 6S column and eluted with the solvent system water/ethanol mixture of decreasing polarity at a slow column flow rate. During the elution process, the bands that traveled along the column were tracked using UV light to manage the fractionation process. A total of 24 fractions were collected, dried under vacuum, and examined for their contents by TLC analysis, and the obtained fractions were combined into six main fractions (A1–A6) based on their TLC profile. Fractions A1 and A2 revealed the existence of small amounts of flavonoids and the major content of these fractions was free sugars that were detected by comparative paper chromatography. Fraction A3 was chromatographed over polyamide 6S column using ethanol/water mixture (1:1) as an eluent to produce seven subfractions (B1–B7). Subfractions B2 and B3 were combined and further purified on Sephadex LH-20 column and eluted with methanol/water mixture (1:1) to afford the pure compounds 3 (17 mg) and 4 (21 mg). Subfraction B7 was applied to a Sephadex LH-20 column for purification and eluted with methanol to yield the purified compound 5 (24 mg). Fractions A4 and A5 were combined and applied to the top of polyamide 6S column and then eluted with ethanol/water solvent system (8:2) to give eight subfractions (C1–C8). Subfractions C5–C8 were combined because of their promising similar TLC profile and further chromatographed over polyamide 6S column eluted with ethanol/water (9.5:0.5) to afford 10 subfraction (D1–D10). Subfraction D4 was purified over a Sephadex LH-20 column to afford compound 2 (31 mg), and compound 1 (27 mg) was obtained from subfraction D6 after application to Sephadex LH-20 column eluted with methanol HPLC. Subfractions D9 and D10 were combined and subjected to purification process over a two consecutive Sephadex LH-20 column eluted with methanol/water mixture and methanol to afford the pure compound 6 (32 mg).

Assay of radical-scavenging activity

The contents of polyphenols and flavonoids in the ethyl acetate extract were assayed using Folin Ciocalteu (Singleton and Rossi 1965) and aluminum trichloride methods (Chang et al. 2002), respectively. To determine the radical-scavenging activity of the extract, an in vitro assay was conducted as previously described (Brand-Williams et al. 1995) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) purchased from Sigma (USA).

Assay of XO inhibitory activity in vitro

XO inhibitory activity of MQE was assayed following the method reported by Kong et al. (2000) with few modifications. Different concentrations (0–100 µg/mL) of MQE were prepared, and AP was used as a standard. Hundred µL of each concentration, 300 µL of 50 mM sodium phosphate buffer (pH 7.5), 100 µL XO (0.1 U/mL), and 100 µL distilled water were mixed and incubated at 37 °C for 10 min. Two hundred microliters xanthine (0.15 mM) was added and the mixture was incubated for 30 min, and the reaction was terminated by the addition of 1 N HCl. The absorbance was measured at 295 nm against a blank that was prepared following the same method, but XO was added following the 1 N HCl. The experiment was repeated 3 times (N = 3).

Animals and treatments

Ten-week-old male Wistar rats, weighing 160–180 g, were housed under standard conditions and were supplied a rodent diet and water ad libitum. The anti-hyperuricemia activity of MQE was evaluated using PO-induced rats as previously reported (Liu et al. 2014; Umamaheswari et al. 2007).

Forty-eight rats were divided equally into six groups (N = 8) as follows:

  • Group 1: received 0.5% carboxymethyl cellulose (CMC) orally for 7 days.

  • Group 2: received 600 mg/kg MQE (Bin-Jumah 2018) suspended in 0.5% CMC orally for 7 days.

  • Group 3: received 0.5% CMC and 280 mg/kg PO (Sigma, USA) orally for 7 days (Wang et al. 2016).

  • Group 4: received 10 mg/kg AP (Liu et al. 2014; Umamaheswari et al. 2007) and 280 mg/kg PO (Sigma, USA) suspended in 0.5% CMC orally for 7 days.

  • Group 5: received 300 mg/kg MQE (Bin-Jumah 2018) and 280 mg/kg PO (Sigma, USA) suspended in 0.5% CMC orally for 7 days.

  • Group 6: received 600 mg/kg MQE (Bin-Jumah 2018) and 280 mg/kg PO (Sigma, USA) suspended in 0.5% CMC orally for 7 days.

MQE and AP were administered 1 h after PO. The rats were sacrificed under ketamine anesthesia. Blood was collected via cardiac puncture for the preparation of serum. The rats were immediately dissected, and kidney samples were homogenized in cold PBS (10% w/v), centrifuged, and the clear supernatant was collected for biochemical assays.

Determination of serum uric acid and XO activity

Uric acid and XO activity in serum were assayed using specific kits supplied by Spinreact (Spain) and Sigma (USA), respectively, following the protocols supplied by the manufacturers.

Determination of renal function biomarkers and pro-inflammatory cytokines

Serum uric acid, creatinine, and urea were assayed using kits procured from Spinreact (Spain). Levels of renal tumor necrosis factor (TNF)-α and interleukin (IL)-1β were assayed using R&D Systems (USA) ELISA kits. All assays were performed following the protocols supplied by the manufacturers.

Determination of oxidative stress markers and antioxidants in kidney

MDA, a LPO marker, and NO were measured in the kidney homogenate according to Ohkawa et al. (Ohkawa et al. 1979) and Green et al. (Green et al. 1982), respectively. SOD (Nishikimi et al. 1972) and CAT (Aebi 1984) activities and GSH (Griffith 1980) were estimated in the kidney homogenate.

Gene expression

The effect of MQE on mRNA levels of URAT1, GLUT9, and OAT1 in the kidney of PO-administered rats was determined by qRT-PCR. Total RNA was isolated using TRIzol (ThermoFisher Scientific, USA), purified, and its quantity was determined using a nanodrop at 260 nm. RNA samples with A260/A280 of ≥ 1.8 were selected for cDNA synthesis which was amplified using SYBR green master mix (ThermoFisher Scientific, USA) and the primers in Table 1 (Vivantis Technologies, Malaysia) in a total reaction volume of 20 μL on ABI 7500 real-time PCR System (Applied Biosystems, USA). The obtained data were analyzed using the 2−ΔΔCt method (Livak and Schmittgen, 2001) and normalized to GAPDH.

Table 1 Primers used for qRT-PCR
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In silico molecular docking analysis

The initial geometries of isolated flavonoids (1–6) employed for the docking assessment were fully optimized by the Gaussian 09 software package (Frisch et al. 2009) at the B3LYP level of theory (Becke 1988, 1993; Lee et al. 1988) using the basis set 6-311G (d, p) without constrains (Hehre et al. 1986). The three-dimensional crystal structure of XO (PDB ID: 3NVY) used in this investigation was obtained from the protein data bank (PDB). Autodock Tools (ADT) v1.5.6 and AutoDock Vina software packages were used for carrying out the molecular docking investigation (Trott and Olson, 2010). UCSF Chimera was employed for the macromolecular optimization including solvent and nonstandard residues removal and separation from ligands (Pettersen et al. 2004). The optimized target was prepared for the docking run by means of ADT. The optimization process includes polar hydrogen addition and setting the grid box to the proper configuration of the active site. PyMOL v2.4 was used for image generation and binding site analysis.

Statistical analysis

Data are shown as mean ± standard deviation (SD) or SEM. Statistical significance among groups was tested by one-way ANOVA followed by Tukey’s test using GraphPad Prism 7 (San Diego, CA, USA). A P value less than 0.05 were considered significant.

Results

Phytochemical investigation and radical scavenging activity of M. quadrangula

The phytochemical fractionation of the ethyl acetate fraction of the aerial parts of M. quadrangula led to the isolation of six known flavonoids isolated for the first time from this species (Fig. 1). The chemical structure of the isolated flavonoids was elucidated based on data obtained from spectroscopic analysis and by comparison with those published in the literature. The structure of isolated flavonoids were identified as kaempferol-7-O-β-D-glucopyranoside (1) (Pereira et al. 2012), kaempferol-3-O-β-D-glucopyranosyl (6→1)-α-L-rahmnopyranoside (2) (Mabry et al. 2012), luteolin-3′,4′-di-O-β-D-glucopyranoside (3) (Bader et al. 2003), luteolin 7-O-β-D-glucopyranoside (4) (Orhan et al. 2012), chrysoeriol (5) (Park et al. 2007), and keampferol (6) (Elsayed et al. 2020).

Fig. 1
figure 1

Chemical structure of the isolated compounds

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Determination of total phenolics and flavonoids (Fig. 2A) in MQE revealed 38.45 ± 1.94 mg gallic acid equivalents/g and 16.80 ± 1.71 mg quercetin equivalents/g, respectively. The scavenging activity against DPPH radicals revealed a concentration-dependent efficacy of MQE (Fig. 2B) with IC50 of 32.92 µg.

Fig. 2
figure 2

Total phenolics and flavonoids (A) and DPPH•-scavenging activity (B) of M. quadrangula. Data are the mean values of triplicate and expressed as mean ± SD

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In vitro XO inhibitory activity of MQE

Given that uric acid levels depend on a XO-catalyzed reaction, we tested the inhibitory activity of MQE on XO in vitro using AP as a standard. As shown in Fig. 3, MQE showed a XO inhibitory activity in a concentration-dependent manner and the IC50 was 62.01 µg whereas the IC50 of AP was 3.87 µg.

Fig. 3
figure 3

In vitro XO inhibitory activity of MQE. Allopurinol and MQE inhibited XO activity with IC50 values of 3.87 µg and 62.01 µg, respectively. Data are the mean values of triplicate and expressed as mean ± SD

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Molecular docking analysis

Molecular docking analysis was performed to figure out the binding modes of M. quadrangula isolated flavonoids with XO (Fig. 4 and Suppl. Fig. I–II). Polar and hydrophobic interactions of isolated flavonoids (1–6) with the target enzyme along with the drug-enzyme binding affinities estimated by AutoDock Vina are tabulated in Table 2.

Fig. 4
figure 4

The binding interactions of luteolin-3′,4′-di-O-β-D-glucopyranoside and chrysoeriol with XO

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Table 2 Binding energies, polar bonds, and hydrophobic interactions of flavonoids (1–6) isolated from M. quadrangula with XO
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MQE ameliorates serum uric acid and inhibits XO activity in PO-administered rats

To investigate the ability of MQE to inhibit XO activity in vivo, a rat model of hyperuricemia was employed. There was a significant increase in serum uric acid (Fig. 5A) and XO activity (Fig. 5B) of PO-induced hyperuricemic rats when compared with control rats (P < 0.001). MQE ameliorated uric acid and XO activity in serum of PO-induced rats. Normal rats treated with MQE showed no alterations in uric acid levels and XO activity.

Fig. 5
figure 5

MQE decreased serum XO activity (A) and uric acid (B) in PO-administered rats. Data are mean ± SEM, N = 8. ***P < 0.001 versus control and ###P < 0.001 versus hyperuricemic

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MQE ameliorates creatinine and urea in PO-administered rats

The protective effect of MQE against renal dysfunction associated with hyperuricemia in PO-administered rats was evaluated through the measurement of serum creatinine and urea levels. There was a significant increase in serum creatinine and urea levels in PO-induced hyperuricemic rats (P < 0.001) as depicted in Fig. 6A–B. Oral supplementation of either dose of MQE ameliorated serum uric acid (P < 0.001), creatinine (P < 0.001), and urea (P < 0. 01) in hyperuricemic rats.

Fig. 6
figure 6

MQE decreased serum creatinine (A) and urea (B) in PO-administered rats. Data are mean ± SEM, N = 8. ***P < 0.001 versus control. ##P < 0.01 and ###P < 0.001 versus hyperuricemic

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MQE modulates URAT-1, GLUT-9, and OAT-1 in the kidney of PO-administered rats

The administration of PO resulted in significant increase in URAT1 (Fig. 7A) and GLUT9 (Fig. 6B) mRNA abundance in the kidney of rats (P < 0.001). In contrast, OAT1 mRNA abundance (Fig. 6C) was significantly downregulated in the kidney of PO-administered rats when compared with the control (P < 0.001). Treatment of the PO-administered rats with either dose of MQE downregulated URAT1 and GLUT9 and increased OAT1 mRNA abundance. Normal rats that received 600 mg/kg MQE showed no changes in the expression of URAT1, GLUT9, and OAT1.

Fig. 7
figure 7

MQE downregulated mRNA abundance of URAT1 (A) and GLUT9 (B), and upregulated OAT1 (C) in the kidney of PO-administered rats. Data are mean ± SEM, N = 8. ***P < 0.001 versus control and ##P < 0.01 and ###P < 0.001 versus hyperuricemic

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MQE prevents inflammation in PO-administered rats

TNF-α and IL-1β were assayed to evaluate the inflammatory response in PO-administered rats and the protective effect of MQE. As shown in Fig. 8, hyperuricemic rats showed significantly elevated TNF-α (Fig. 8A) and IL-1β (Fig. 8B) as compared to the control rats (P < 0.001). Both doses of MQE were effective in ameliorating TNF-α and IL-1β in the kidney of PO-administered rats. Oral administration of 600 mg/kg MQE did not alter pro-inflammatory cytokines in normal rats.

Fig. 8
figure 8

MQE decreased TNF-α (A) and IL-1β (B) in the kidney of PO-administered rats. Data are mean ± SEM, N = 8. ***P < 0.001 versus control and ###P < 0.001 versus hyperuricemic

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MQE attenuates oxidative stress and enhances antioxidant defenses in PO-administered rats

Since oxidative stress has been demonstrated to be implicated in the development of hyperuricemic nephropathy (Braga et al. 2017; Chen et al. 2019; Dera et al. 2020; Su et al. 2018), we evaluated the effect of MQE on LPO, NO, and antioxidants in the kidney of PO-administered rats. PO induced a significant (P < 0.001) increase in renal MDA (Fig. 9A) and NO (Fig. 9B), and a decrease in GSH (Fig. 9C), SOD (Fig. 9D), and CAT (Fig. 9E). All these changes were significantly attenuated by MQE which decreased MDA and NO, and increased GSH, SOD, and CAT. Normal rats treated with MQE showed no alterations in oxidative stress and antioxidant markers.

Fig. 9
figure 9

MQE attenuates oxidative stress in hyperuricemic rats. MQE ameliorated MDA (A) and NO (B), and increased renal GSH (C), SOD (D), and CAT (E) in the kidney of PO-administered rats. Data are mean ± SEM, N = 8. ***P < 0.001 versus control. #P < 0.05, ##P < 0.01, and ###P < 0.001 versus hyperuricemic

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Discussion

Elevated serum uric acid level has been well acknowledged as an independent risk factor for declined kidney function (Iseki et al. 2001; Obermayr et al. 2008). In people without gout, hyperuricemia is asymptomatic and hence had not drawn much attention for a few decades. Asymptomatic hyperuricemia is frequently associated with kidney impairment and has been attributed to decreased renal clearance (Jung et al. 2020). Given the increased prevalence of several side effects of many medications for hyperuricemia, more effective and safe alternatives are needed. Increasing evidence indicates that medicinal plants are among the potential candidates for the management of hyperuricemia (Kamel et al. 2021; Kong et al. 2000; Liu et al. 2014). Herein, we investigated the XO inhibitory activity of M. quadrangula and its renoprotective effect in a rat model of hyperuricemia.

XO is the key enzyme catalyzing the generation of uric acid through the conversion of hypoxanthine to xanthine and oxidation of the latter. Owing to its role in generating ROS, increased XO activity can lead to hyperuricemia and oxidative stress (Yang et al. 2015). Therefore, attenuation of XO activity is an effective strategy against hyperuricemia and oxidative injury. In this study, MQE exhibited a concentration-dependent XO inhibitory activity in vitro. Molecular docking assessment results revealed the XO inhibitory activity of M. quadrangula isolated flavonoids. This inference is attributed to the obtained low binding energy for the isolated flavonoid-enzyme complexes ranging from − 7.7 to − 8.4 kcal/mol. Interestingly, all isolated flavonoids nearly occupy the same binging pocket in XO with many common amino acid residues involved in polar and hydrophobic interactions of these complexed. These outputs clearly indicated that the isolated flavonoids exhibit thermodynamically favorable binding interactions with XO. For instance, a dense network of hydrogen bonds was detected with compounds 1 and 3 and the large number of hydrophobic interacting residues with compounds 2, 3, 5, and 6. In general, polar and hydrophobic interactions between drugs and proteins are important in maintaining geometrical patterns of these biological macromolecular complexes. Hydrogen bonding, in particular, is known to be a critical mechanism for drug binding into the active regions of proteins. As a result, they help with drug affinity, molecular recognition, and orientation (Abukhalil et al. 2020). In addition, hydrophobic interactions between drug’s lipophilic surfaces and its binding cavity’s hydrophobic regions have a substantial impact on binding energies, and the relative contribution of hydrogen bonding to these affinities is mostly determined by desolvation energy and newly created polar bonds. As a result, adequate geometrical alignment of a drug with the active site is critical for a good protein–ligand interaction. Herein, the molecular modeling analysis of M. quadrangula-isolated phytochemicals-XO systems could provide us with promising paths for the development of potent drugs for the inhibiting and treatment of hyperuricemia and gout.

Besides its ability to suppress XO, MQE exhibited a radical scavenging activity manifested by inhibition of the DPPH radicals. In support of the in vitro results, the effect of MQE on XO activity and free radicals was tested in a rodent model of PO-induced hyperuricemia. This is a frequently used experimental model for the assessment of the efficacy of anti-hyperuricemia candidates. PO is a selective competitive inhibitor of uricase that inhibits the activity of hepatic uricase, resulting in increased uric acid levels (Stavric et al. 1975; Wang et al. 2016). Consistent with previous studies (Chen et al. 2019; Liu et al. 2014; Zhao et al. 2006), PO-administered rats in the present investigation exhibited an increase in uric acid and XO activity. Uric acid is produced through purine catabolism and hyperuricemia occurs as a result of excessive production and/or inadequate excretion (Ichida et al. 2012). Treatment of the PO-administered rats with MQE decreased uric acid levels and suppressed XO activity, demonstrating its anti-hyperuricemia efficacy. The XO inhibitory activity of MQE could be attributed to its constituents, in particular phenolics and flavonoids. We have recently demonstrated the ability of plant-derived phenolics to suppress XO both in vitro and in vivo (Kamel et al. 2021).

Next, we investigated the effect of MQE on urate transporters in order to further explore its anti-hyperuricemia efficacy. Urate under-excretion, regulated by organic anion transporters in kidney, is implicated in hyperuricemia (Mandal and Mount 2015). These transporters include URAT1, localized in the apical membrane of proximal tubular cells, GLUT9, localized in apical and basolateral membranes of the distal nephron, and the basolateral urate/dicarboxylate exchangers OAT1 (Habu et al. 2003; Tan et al. 2016; Vitart et al. 2008). GLUT9 and URAT1 promote urate reabsorption whereas OAT1 is involved in urate excretion (Habu et al. 2003; Tan et al. 2016; Vitart et al. 2008). In the present study, GLUT9 and URAT1 were upregulated and OAT1 was downregulated in the kidney of PO-administered rats as previously reported in rodent models of hyperuricemia (Su et al. 2018; Wang et al. 2016). Treatment with MQE decreased GLUT9 and URAT1 and increased OAT1 expression in the kidney of PO-induced rats. Therefore, M. quadrangula exerted its anti-hyperuricemic activity by inhibiting XO and modulating renal URAT1, GLUT9, and OAT1 expression. The lack of Western blotting data could be a limitation of this study; however, MQE effectively modulated the mRNA abundance of GLUT9, URAT1, and OAT1.

Elevated uric acid has been implicated in the development and progression of kidney diseases (Sah and Qing, 2015). Here, PO-administered rats exhibited renal dysfunction manifested by elevated circulating levels of creatinine and urea. Serum creatinine and urea are markers of renal function, and their elevation indicates altered renal function (Higgins 2016). As an index of renal function, creatinine is inversely correlated with the true glomerular filtration rate (GFR) (Lopez-Giacoman and Madero 2015), and urea is a metabolic product of protein breakdown that is used as a biochemical index to detect abnormal renal function (Walmsley et al. 2010). Experimental hyperuricemia induced by oxonic acid exacerbated the decline in GFR and albuminuria associated with tubulointerstitial nephritis, glomerular hypertrophy, and other renal damages (Mazzali et al. 2002; Nakagawa et al. 2003). Treatment of the oxonate-induced rats with MQE decreased serum creatinine and urea, demonstrating its beneficial effect against renal impairment.

Oxidative stress and inflammatory response have been suggested to contribute to renal dysfunction in hyperuricemia. Uric acid can elicit inflammatory reactions by acting as a danger signal (Xiao et al. 2015), and hyperuricemia can provoke or potentiate inflammation in both healthy and diseased kidneys (Martinon et al. 2006). Here, PO-administered rats exhibited an increase in IL-1β and TNF-α, demonstrating a triggered inflammatory response. Uric acid crystals have been reported to cause gout by activating NLRP3 inflammasome and the production of IL-1β and IL-8 in cultured monocytes and macrophages (Martinon et al. 2006). Not only the crystals but also soluble uric acid activated NLRP3 inflammasome and promoted inflammation in macrophages (Kim et al. 2015), and bone marrow-derived macrophages under hypoxia through induction of mitochondrial ROS generation (Braga et al. 2017). In support of these findings, MDA and NO were increased and GSH and antioxidant enzymes were declined in the kidney of PO-administered rats. Similar findings have been demonstrated in multiple experimental studies (Chen et al. 2019; Dera et al. 2020; Su et al. 2018). Increased LPO deteriorates the membrane fluidity and permeability and inactivates the membrane-bound enzymes (Ramana et al. 2013). Moreover, LPO-derived aldehydes can diffuse across the membranes and modify any protein in the cytoplasm and nucleus, resulting in cell destruction (Ayala et al. 2014; Ramana et al. 2013). Elevated uric acid induces renal oxidative stress and eventually culminating in kidney damage (Dera et al. 2020, Meneshian and Bulkley 2002). XO generates superoxide anions and hydrogen peroxide (H2O2) (Meneshian &Bulkley 2002), and uric acid induces mitochondrial and NADPH oxidase-mediated ROS generation (Braga et al. 2017; Sanchez-Lozada et al. 2019), and provokes oxidative stress through the stimulation of renin-angiotensin system (Corry et al. 2008). Given that XO generates uric acid together with ROS, inhibition of XO and activation of antioxidant defenses were assumed to mediate the protective effect of MQE against hyperuricemia and renal impairment in PO-administered rats.

MQE attenuated oxidative stress and inflammatory response and augmented antioxidant defenses in the kidney of hyperuricemic rats. The dual antioxidant and anti-inflammatory efficacy of M. quadrangula was in accordance with the in vitro results showing the ability of MQE to scavenge free radicals and inhibit XO activity, effects that could be directly attributed to the contained flavonoids. Accordingly, MQE decreased MDA and increased SOD and CAT in the stomach of a rodent model of gastric ulcer induced by ethanol (Ibrahim et al. 2016) and in the liver and heart of hypercholesterolemic rats (Bin-Jumah 2018). Attenuation of inflammation has also been reported in hypercholesterolemic rats treated with MQE (Bin-Jumah 2018). Previous studies have demonstrated the amelioration of macrophage infiltration and tubulointerstitial inflammation in murine diabetes as a result of lowering uric acid levels by XO inhibitors (Kim et al. 2015; Kosugi et al. 2009). Therefore, XO inhibitory activity of MQE plays a central role in attenuating hyperuricemia and its associated oxidative stress and inflammation in PO-administered rats.

Conclusion

This study confers new information on the anti-hyperuricemia and nephroprotective efficacies of M. quadrangula. MQE inhibited XO and scavenged free radicals in vitro. In a rat model of hyperuricemia, MQE suppressed XO activity, and decreased serum uric acid, urea, and creatinine. In addition, MQE attenuated oxidative stress and inflammatory response, enhanced antioxidant, and modulated URAT1, GLUT9, and OAT1 in the kidney of hyperuricemic rats (Fig. 10). These findings provide insights for the beneficial efficacies of M. quadrangula, which may lead to the development of effective therapeutic regimens against hyperuricemia, pending further studies to explore the exact underlying mechanism(s).

Fig. 10
figure 10

A schematic diagram illustrating the beneficial effect of M. quadrangula extract against hyperuricemia. M. quadrangula extract suppressed XO activity, decreased uric acid, attenuated ROS generation, oxidative stress, and inflammation, and prevented kidney impairment, effects that were associated with modulation of urate transporters. XO, xanthine oxidase; O2−, superoxide radical; PCT, proximal convoluted tubule; URAT1, urate transporter 1; GLUT9, glucose transporter 9; OAT1, organic ani-on transporter 1

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