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Food Science and Human Wellness 5 (2016) 17–23

Phenolics extract of Tetrapleura tetraptera fruit inhibits xanthine oxidaseand Fe2+-induced lipid peroxidation in the kidney, liver, and lungs tissues

of rats in vitro

Emmanuel Anyachukwu Irondi a,∗, Ganiyu Oboh b, Samson Olalekan Agboola c,Aline Augusti Boligon d, Margareth Linde Athayde d

a Biochemistry Unit, Department of Biosciences and Biotechnology, Kwara State University, Malete, P.M.B. 1530, Ilorin, Nigeriab Functional Foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of Technology, Akure, Nigeria, P.M.B. 704, Akure 340001, Nigeria

c Department of Veterinary Physiology, Biochemistry and Pharmacology, University of Ibadan, Nigeriad Phytochemical Research Laboratory, Department of Industrial Pharmacy, Federal University of Santa Maria, Building 26, Room 1115,

Santa Maria CEP 97105-900, Brazil

Received 2 September 2015; received in revised form 4 November 2015; accepted 6 November 2015Available online 17 November 2015

bstract

The phenolics composition and inhibitory activity of Tetrapleura tetraptera fruit extract on xanthine oxidase (XO) and Fe2+-induced lipideroxidation in the kidney, liver and lungs tissues of rats were evaluated in vitro. Phenolics (flavonoids and phenolic acids) were quantifiedsing reverse-phase high performance liquid chromatrography coupled with diode array detection (HPLC-DAD). The XO and Fe2+-induced lipideroxidation inhibitory abilities of the extract were evaluated using spectrophotometric methods. The extract contained some flavonoids andhenolic acids, including catechin, epicatechin, rutin, quercetin, luteolin, apigenin; gallic, chlorogenic, caffeic and ellagic acids that are beneficialo health. The extract inhibited XO in the kidney, liver and lungs tissues in a dose-dependent manner. The half-maximal inhibitory concentrationsIC50) of the extract on XO from the tissues varied significantly (P < 0.05), and were in the order of liver > kidney > lungs. The extract also inhibitede2+-induced lipid peroxidation in a dose-dependent pattern, having IC50 in the order of liver > lungs > kidney. T. tetraptera fruit extract could be

promising nutraceutical for preventing and managing hyperuricaemia and the associated disease conditions, due to its ability to inhibit XO; thisioactivity is attributable to combined effect of its flavonoids and phenolic acids.

2015 Beijing Academy of Food Sciences. Production and hosting by Elsevier B.V. All rights reserved.

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eywords: Tetrapleura tetraptera; Phenolics; Xanthine oxidase; Hyperuricaem

. Introduction

Experimental evidence has shown that elevated concentra-ion of uric acid, otherwise known as hyperuricaemia, leads tohe deposition of monosodium urate monohydrate crystals inissue, especially joints, thereby resulting in gouty arthritis orric acid nephrolithiasis [1,2]. Gout is a chronic inflammatory

∗ Corresponding author at: Biochemistry Unit, Department of Biosciences andiotechnology, Kwara State University, Malete, P.M.B. 1530, Ilorin, Nigeria.el.: +234 8034870657.

E-mail address: irondi.emmanuel@yahoo.com (E.A. Irondi).Peer review under responsibility of Beijing Academy of Food Sciences.

fRhp

ttp://dx.doi.org/10.1016/j.fshw.2015.11.001213-4530/© 2015 Beijing Academy of Food Sciences. Production and hosting by E

pid peroxidation

rthritis characterized by elevated concentration of uric acidn body fluids, resulting from the over-activity of xanthinexidase (XO) [3]. It is also characterized with severe andpisodic painful inflammation [4]: erythema and swelling [5].pidemiological studies have shown that the overall burdenf the disease is increasing globally [2]. It is more prevalentn men above 30 years of age and in women older than 50ears [2,6]. Moreover, it has the propensity to reduce theuality of life of these individuals [7]. In addition to goutyrthritis, hyperuricaemia is also a well-established causativeactor for uric acid kidney stones and acute kidney failure [8].

ecent epidemiologic studies have also implicated chronic mildyperuricaemia in the development of interstitial nephritis androgressive renal failure [9]. Furthermore, it is an independent

lsevier B.V. All rights reserved.

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8 E.A. Irondi et al. / Food Science

isk factor for metabolic syndrome, cardiovascular disease,ypertension, obesity, obstructive sleep apnea, stroke, vascularementia, and preeclampsia [10].

Xanthine oxidase (XO) (EC 1.1.3.22) catalyzes the oxidationf hypoxanthine to xanthine and subsequently to uric acid [11]n the purine nucleotides catabolism. Its re-oxidation involves

olecular oxygen which acts as electron acceptor, and duringhis reaction, superoxide radical (O2

•−) and hydrogen perox-de (H2O2) are produced [12]. The O2

•− is transformed into2O2 and O2 either spontaneously or by the catalytic actionf superoxide dismutase. Thus, the over-activity of XO leadso the deposition of uric acid in the susceptible tissues, andhis triggers the inflammatory pathways with a concomitantelease of reactive oxygen species. Hence, gouty arthritis andther inflammatory diseases associated with hyperuricaemia areharacterized by oxidative stress. The kidney, liver and lungsre three major organs in mammals involved in metabolismnd excretion, and previous studies have reported XO activ-ty in the tissues of these organs [6,13,14]. Functionally, inhese organs materials are chemically biotransformed and theetabolic wastes, such as carbon dioxide, water, salt, urea and

ric acid, are removed from the body.Clinically, anti-inflammatory agents are used to relieve the

ymptoms of gout, and XO inhibitors are used to block the syn-hesis of uric acid. These two approaches are common treatmentsf gout. Allopurinol, a purine analog, is the most common XOnhibitor that functions to reduce serum urate level [1]. How-ver, its use has some attendant side effects in patients, and forhis reason it is usually contraindicated in patients with kidneyr heart disease. The side effects include the risk of develop-ng hypersensitivity syndrome that is characterized by renalmpairment, hepatic dysfunction, fever, rashes, leucocytosis andosinophilia [15]. These limitations of allopurinol have necessi-ated research into alternative treatment strategies for gout thatould be safer and effective. In this regard, medicinal plants areidely used to treat gout, as previous studies have demonstrated

hat several of them with high level of flavonoids and otherhenolics compounds possess XO inhibitory activity [16–18].lant-derived polyphenolics with antioxidant potential, includ-

ng flavonoids and phenolic acids, can modulate the expressionf pro-inflammatory signals and ameliorate inflammatory dis-ases such as arthritis [19,20].

Tetrapleura tetraptera, called “aidan” in the South-westernart of Nigeria, and “ihokiriho” by the Ngwa people in the South-astern part of Nigeria, is a deciduous tree belonging to theamily Mimosaceae. It is generally distributed in the lowland for-st of tropical Africa. The fruits, made up of a fleshy pulp withmall, brownish-black seeds, are green when tender and darkrown when fully ripe and possess high nutritional value [21].hen dry, they have a pleasant aroma, and therefore are used as

pice in Central and West Africa [22]. This spicy property makeshem valuable for preparing soup for nursing mothers from therst day of birth to prevent post-partum contraction [23]. Pre-

ious studies have demonstrated that different parts of the plantre used in ethnomedicine for the treatment of several ailmentsncluding diabetes mellitus, hypertension, intestinal parasites,

alaria, asthma, epilepsy, schistosomiasis, wound healing andw(

uman Wellness 5 (2016) 17–23

rthritis [24,25]. Recent studies have also revealed that the podossesses antioxidant and amylase inhibitory activities [21];he fruits and barks extracts also have antioxidant activities22].

The aforementioned health benefits of T. tetraptera maket a promising functional food. Interestingly, functional foodsf plant origin have continued to receive considerable researchttention in the recent time due to their nutritional quality, ther-peutic effects and presumed safety [26]. Hence, to furtherxplore the health benefits of T. tetraptera, the present studyharacterized the phenolics of T. tetraptera fruit, and evaluatedhe XO inhibitory activity of its phenolic extract in the kidney,iver and lungs tissues of rats in vitro.

. Materials and methods

.1. Samples collection and preparation

Ripe fruits samples of T. tetraptera were harvested from thelant in Umueze area of Ekwereazu-Ngwa village in Obi-Ngwaocal government area of Abia State, Nigeria, in November,014. The fruits were botanically identified and authenticatedt the herbarium of the Department of Botany, University ofbadan, Nigeria. Subsequently, they were sun-dried for sevenays, and the seeds were manually removed. The fruits wereater milled into a fine particle size (0.5 mm) powder and packedn air-tight plastic vials, and stored at −4 ◦C until analysis.

Methanol, formic acid, gallic acid, chlorogenic acid, caf-eic acid and ellagic acid purchased from Merck (Darmstadt,ermany). Catechin, epicatechin, quercetin, rutin, apigenin and

uteolin; xanthine and allopurinol were acquired from Sigmahemical Co. (St. Louis, MO, USA). All other chemicals used

or analysis were of analytical grade.

.2. Preparation of polyphenolics extract

Polyphenolics extract of T. tetraptera fruits was preparedccording to the method described by Kuo et al. [27]. A portionf T. tetraptera fruits powder (100 g) was extracted three suc-essive times with 300 mL of methanol at 50 ◦C for 3 h, andhe samples were filtered after each extraction with WhatmanNo. 2) filter paper. The combined extract was partitioned with00 mL hexane in a separatory funnel to remove the lipids andome of the pigments. The aqueous phase was extracted threeimes with 180 mL ethyl acetate; after which it was evaporated toryness at 45 ◦C under reduced pressure in a rotary evaporator.he residue from this step was redissolved in 250 mL water, and

yophilized to obtain approximately 5 g of T. tetraptera fruitsolyphenolics extract. The percentage yield of the extract was%.

.3. Quantification of phenolic compounds usingPLC-DAD

High performance liquid chromatography (HPLC-DAD)as performed with a Shimadzu Prominence Auto Sampler

SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped

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E.A. Irondi et al. / Food Science

ith Shimadzu LC-20AT reciprocating pumps connected to aGU 20A5 degasser with a CBM 20A integrator, SPD-M20Aiode array detector and LC solution 1.22 SP1 software.

Reverse phase chromatographic analysis was carried outnder gradient conditions using C18 column (4.6 mm × 150 mm)acked with 5 �m diameter particles; the mobile phase wasater containing 1% formic acid (A) and methanol (B), and the

omposition gradient was: 13% of B until10 min, and changedo obtain 20%, 30%, 50%, 60%, 70%, 20% and10% B at 20, 30,0, 50, 60, 70 and 80 min, respectively, following the methodescribed by Menezes et al. [28] with slight modifications.he T. tetraptera fruits extract and mobile phase were filtered

hrough 0.45 �m membrane filter (Millipore) and then degassedy ultrasonic bath prior to use. The extract was analyzed at

concentration of 15 mg/mL. The flow rate was 0.7 mL/min,nd the injection volume was 40 �L. The wavelength were54 nm for gallic acid, 280 nm for catechin and epicatechin,27 nm for chlorogenic, caffeic and ellagic acids, and 365 nmor quercetin, rutin, apigenin and luteolin. Stock solutionsf standards references were prepared in the HPLC mobilehase at a concentration range of 0.030–0.450 mg/mL. Thehromatography peaks were confirmed by comparing theiretention time with those of reference standards and by DADpectra (210–500 nm). The calibration curves were galliccid: Y = 11,964x + 1283.5 (r = 0.9999); chlorogenic acid:

= 13,087x + 1195.4 (r = 0.9998); caffeic acid: Y = 11,962x +273.8 (r = 0.9996); ellagic acid: Y = 12,643x + 1327.6r = 0.9995); catechin: Y = 13,470x + 1195.3 (r = 0.9999);picatechin: Y = 11,786x + 1265.4 (r = 0.9997); quercetin:

= 12,762x + 1373.8 (r = 0.9998); rutin: Y = 13,194x + 1197.0r = 0.9999); apigenin: Y = 13,471x + 1195.6 (r = 0.9994) anduteolin: Y = 12,763x + 1347.9 (r = 0.9999). All chromatographyperations were carried out at ambient temperature and inriplicate.

The limit of detection (LOD) and limit of quantificationLOQ) were calculated based on the standard deviation ofhe responses and the slope using three independent analyti-al curves. LOD and LOQ were calculated as 3.3 and 10 σ/S,espectively, where σ is the standard deviation of the responsend S is the slope of the calibration curve [29].

.4. Handling of experimental animals

Adult male Wistar strain albino rats weighing 200–250 g weresed in this study. The rats were procured from the experimen-al animal breeding unit of Department of Veterinary Medicine,niversity of Ibadan, Nigeria. The animals received humane

are according to the criteria outlined in the Guide for theare and Use of Laboratory Animals prepared by the Nationalcademy of Science and published by the National Institutef Health (USA) [30]. The guidelines were followed to ensurehe protection of the animals’ welfare during the experiment.

he rats were kept in a cage to acclimatize for 7 days, duringhich they were maintained at room temperature under the lab-ratory conditions and were fed with standard diet and waterd libitum.

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uman Wellness 5 (2016) 17–23 19

.5. Preparation of Kidney, liver and lungs tissuesomogenates for XO and lipid peroxidation inhibitionssays

The kidney, liver and lungs tissues homogenates for XO andipid peroxidation inhibition assays were prepared following the

ethod described by Nakamura et al. [31]. The kidney, livernd lungs were rapidly excised after decapitation of the ratsnder mild ether anesthesia. Each tissue was washed in cold.15 mol/L KCl, and blotted dry. Then a portion of 1 g of eachissue was homogenized in 9 volumes of ice-cold 50 mmol/Lris–HCl buffer (pH 7.4) containing 1 mmol/L ethylene diamine

etraacetic acid (EDTA). A portion of the homogenate was cen-rifuged for 10 min at 1400 × g to yield a low-speed supernatanthat was used for the lipid peroxide assay. For the XO assay,nother portion of the homogenate of each tissue was sonicatedwice on ice for 30 sesonds and then centrifuged at 10,000 × gor 20 min at 4 ◦C to obtain the supernatant fraction used.

.6. Xanthine oxidase inhibition assay

The ability of the extract to inhibit XO was determined fol-owing the method reported by Umamaheswari et al. [17] withlight modification. The reaction mixture consisted of 300 �Lf 50 mmol/L sodium phosphate buffer (pH 7.5), 100 �L of thextract at different concentrations (15, 30, 45 and 60 mg/mL) inimethyl sulphoxide (DMSO), 100 �L of freshly prepared tis-ue enzyme preparation and 100 �L of distilled water. The assayixture was pre-incubated at 37 ◦C for 15 min. Then, 200 �L

f 0.15 mmol/L of xanthine solution (substrate) was added tohe mixture which was incubated at 37 ◦C for 30 min; this wasollowed by the addition of 200 �L of 0.5 mol/L HCl to termi-ate the reaction. Allopurinol was used as a positive control forhe assay. A reference test containing 100 �L of DMSO insteadf the extract was carried out in order to obtain the maximumric acid formation. The absorbance was measured at 295 nmn a UV/VIS spectrophotometer against a blank prepared in theame way except that the enzyme solution was replaced withhe phosphate buffer. One unit (U) of this enzyme is defined ashe amount of enzyme required to form 1 mmol of uric acid per

in at the reaction conditions. The percentage XO inhibitoryctivity of the extract was calculated thus:

XO inhibition =[

(A295reference − A295sample)

A295reference

]× 100.

here A295reference is the reference without the extract, and295sample is the absorbance of test containing the extract.

.7. Lipid peroxidation inhibition assay

The ability of the extract to inhibit Fe2+-induced lipid per-xidation in kidney, liver and lungs tissues homogenates of ratsas assayed according to the modified method of Ohkawa et al.

32]. Briefly, to a reaction mixture containing 100 �L of theomogenate supernatant, 30 �L of 0.1 mol/L Tris–HCl bufferpH 7.4) and different concentrations of the extract, 30 �L ofreshly prepared 25 �mol/L ferric sulfate solution was added to

20 E.A. Irondi et al. / Food Science and Human Wellness 5 (2016) 17–23

Fig. 1. Representative high performance liquid chromatography profile ofT. tetraptera fruit. Gallic acid (peak 1), catechin (peak 2), chlorogenic acid((

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Table 1Phenolics composition of T. tetraptera fruits.

Phenolic compound mg/g LOD (�g/mL) LOQ (�g/mL)

Gallic acid 2.95 ± 0.01 0.009 0.030Catechin 0.43 ± 0.03 0.017 0.056Chlorogenic acid 0.21 ± 0.01 0.023 0.076Caffeic acid 3.72 ± 0.02 0.026 0.085Ellagic acid 3.69 ± 0.04 0.008 0.026Epicatechin 1.38 ± 0.01 0.013 0.043Rutin 1.74 ± 0.01 0.015 0.049Quercetin 3.65 ± 0.03 0.029 0.097Luteolin 0.45 ± 0.02 0.011 0.036Apigenin 3.73 ± 0.01 0.018 0.059

Results are expressed as mean ± standard deviations (SD) of triplicate determi-nations.

Table 2Half-maximal inhibitory concentration (IC50) of T. tetraptera fruits extract onxanthine oxidase activity in kidney, liver and lungs tissues of rats.

Tissue IC50 (�g/mL)

Extract Allopurinol

Kidney 39.53 ± 1.02b 4.18 ± 0.11b

Liver 45.71 ± 1.44a 5.41 ± 0.13a

Lungs 33.87 ± 0.96c 3.26 ± 0.10c

Results are expressed as mean ± standard deviations (SD) of triplicate determi-nations. Values with the different lowercase superscript letter along the samecolumn are significantly different (P < 0.05).

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peak 3), caffeic acid (peak 4), ellagic acid (peak 5), epicatechin (peak 6), rutinpeak 7), quercetin (peak 8), luteolin (peak 9) and apigenin (peak 10).

nitiate lipid peroxidation. The volume was made up to 300 �Lith deionized water before incubation at 37 ◦C for 1 h. The

olor reaction was initiated by adding 300 �L of 81 g/L sodiumuodecyl sulphate to the reaction mixture, followed by the addi-ion of 600 �L of acetic acid/HCl (pH 3.4) and 600 �L of 0.8%v/v) TBA (Thiobarbituric acid). This mixture was incubatedt 100 ◦C for 1 h. The absorbance of thiobarbituric acid reac-ive species (TBARS) produced were measured at 532 nm inn UV–Visible spectrophotometer. A reference test without thelant extract was carried out in order to obtain the maximumBARS formation. The decrease in the absorbance of test sam-le in relation to the reference test was used to calculate the %nhibition as follows:

Inhibition =[

(A532reference − A532sample)

A532reference

]× 100.

here A532reference is the absorbance of the reference withouthe extract, and A532sample is the absorbance of test containinghe extract.

.8. Statistical analysis

Results of replicate experiments were expressed asean ± standard deviation (SD). Analysis of variance (ANOVA)as carried out on the result data at 95% confidence levelsing SPSS statistical software package, version 17. IC50 wasalculated from the % inhibition versus extract concentrationon-linear regression curve of the extract.

. Results

The HPLC chromatogram of T. tetraptera fruits (Fig. 1)evealed the presence of the gallic acid (tR = 13.07 min;eak 1), catechin (tR = 15.26 min; peak 2), chlorogenic acidtR = 20.11 min; peak 3), caffeic acid (tR = 24.19; peak 4), ellagiccid (tR = 29.87; peak 5), epicatechin (tR = 35.46 min; peak), rutin (tR = 41.08 min; peak 7), quercetin (tR = 49.23 min;eak 8), luteolin (tR = 55.16 min; peak 9) and apigenin

tR = 62.73 min; peak 10). The phenolics composition of theruits is shown in Table 1. The flavonoids were in the order ofpigenin > quercetin > rutin > epicatechin > luteolin > catechin;

it

ig. 2. Xanthine oxidase activity % inhibition versus T. tetraptera fruit extractoncentration curve in kidney, liver and lungs tissues of rats.

hereas the phenolic acids were in the order of caffeiccid > ellagic acid > gallic acid > chlorogenic acid.

The ability of the extract to inhibit XO in the kidney, livernd lungs tissues homogenates of rats was tested, and the results presented in Table 2 in terms of their half-maximal inhibitoryoncentration (IC50), in relation to allopurinol (positive control).he IC50 of both the extract and allopurinol on the XO in theomogenates of the three tissues varied significantly (P < 0.05)n the order of liver > kidney > lungs. The pattern of inhibitionf XO by the extract was dose-dependent as depicted in Fig. 2.enerally, allopurinol had a stronger inhibitory ability on theO than the extract.

As shown in Table 3, the extract also inhibited Fe2+-

nduced lipid peroxidation in the kidney, liver and lungsissues homogenates of rats. Its IC50 values varied significantly

E.A. Irondi et al. / Food Science and H

Table 3Half-maximal inhibitory concentration (IC50) of T. tetraptera fruits extract onFe2+-induced lipid peroxidation in the kidney, liver and lungs tissues of rats.

Tissue IC50 (�g/mL)

Kidney 20.39 ± 1.19c

Liver 36.97 ± 2.06a

Lungs 26.65 ± 1.82b

Data represent the mean ± standard deviation of replicate readings. Values withthe different lowercase superscript letter along the column are significantlydifferent (P < 0.05).

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ig. 3. Fe2+-induced lipid peroxidation % inhibition versus T. tetraptera fruitxtract concentration curve in kidney, liver and lungs tissues of rats.

P < 0.05) such that that of the liver (36.97 ± 2.06 �g/mL) >ungs (26.65 ± 1.82 �g/mL) > kidney (20.39 ± 1.19 �g/mL);nd its pattern of inhibition was also dose-dependent as shownn Fig. 3.

. Discussion

Dietary spices are recognized as natural antioxidant agentsnd inhibitors of pro-oxidant enzymes such as XO. These ben-ficial activities are attributable to the flavonoids and otherolyphenols present in the spices [33]. Hence, we evaluated thenhibitory effect of the phenolic extract of T. tetraptera fruits,

dietary spice, on XO and Fe2+-induced lipid peroxidation inhe kidney, liver, and lungs tissues of rats in vitro. The importantoles the kidney, liver and lungs play as organs of metabolism andxcretion in mammals, and the previously reported XO activityn these organs [6,13], informed the choice of their homogenatess the source of the enzyme.

The chromatographic analysis of phenolic compoundsTable 1) revealed that T. tetraptera fruits are rich in flavonoidsapigenin, quercetin, rutin, epicatechin, luteolin and catechin)nd phenolic acids (caffeic acid, ellagic acid, gallic acid andhlorogenic acid). These two classes of natural phenolic com-ounds are regarded to be of pharmacological importance34], as they possess diverse health benefits, including anti-nflammatory activity [35]. They can modulate the expression ofro-inflammatory signals and ameliorate inflammatory diseasesuch as arthritis like other natural polyphenolics [19,20]. As part

f their anti-inflammatory activity, polyphenolics including theavonoids and phenolic acids have been reported to inhibit XOctivity by recent studies [33,36,37]. Probably, like allopurinol,hey inhibit XO by binding at its purine binding site [38], thereby

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uman Wellness 5 (2016) 17–23 21

locking the ultimate formation of uric acid. In addition to theirbility to inhibit XO, phenolics are well-known for their antiox-dant activities [39], and ability to inhibit Fe2+-induced lipideroxidation [34]. The flavonoids exhibit antioxidant activityy acting as both electrons donors and terminators of chaineactions; and this is due the hydroxyl groups they possess,articularly at the 3′OH and 4′OH of their three-carbon chain40,41]. Phenolic acids, on the other hand, have a phenolic ringnd an organic carboxylic acid function [42], which make themapable of stabilizing and delocalizing unpaired electrons [43].hese distinguishing structural features make flavonoids andhenolic acids prominent antioxidant phenolic compounds.

Inhibitors of XO are used clinically for the treatment of hype-uricaemia and gouty arthritis; as they help in reducing the levelsf uric acid in circulation, and vascular oxidative stress [44]. Inhis regard, the efficacy of plant-derived polyphenolics in inhib-ting XO and alleviating the resultant hyperuricaemia has beeneported; and as natural components of food, they are regardedo be safer than synthetic XO inhibitors such as allopurinol [33].heir inhibitory effect against XO has been attributed to theossibility of the C-5 and C-7 hydroxyl groups of flavones andavonols to replace the C-2 and C-6 ones of xanthine in theO active site [45,46], due to the mutual inter-convertibility of

he carboxyl structures of xanthine to hydroxyl groups [47]. Inhis study, the phenolic extract of T. tetraptera fruits effectivelynhibited XO in the kidney, liver and lungs tissues homogenatesf rats. Interestingly, the IC50 values of the extract on the XOrom these three tissues (kidney: 39.53 ± 1.02 �g/mL; liver:5.71 ± 1.44 �g/mL; lungs: 33.87 ± 0.96 �g/mL) (Table 2) aren the range of the IC50 values earlier reported for Erythrinatricta leaves extract (21.2 �g/mL) [17], and Olea europaeaeaf extract (42 �g/mL) [36] on XO. Furthermore, the order ofhe IC50 values of the extract on the XO from these three tis-ues (liver > kidney > lungs) may suggest a higher activity of theO in the liver homogenate than in the kidney and the lungsomogenates. This supports an earlier report that the distribu-ion XO activity is highest in the liver followed by the kidney andther tissues, as demonstrated by Carro et al. [14]. Similarly, Liut al. [48] reported that XO is present in significant concentrationn the liver. It is noteworthy that apigenin, the most abundant ofhe phenolics in T. tetraptera fruit from our HPLC-DAD result,as earlier reported to possess the strongest XO inhibitory activ-

ty compared with other phenolics in O. europaea leaf extract36].

Elevated XO activity enhances lipid peroxidation due tohe attendant excessive generation of reactive oxygen speciesROS), decreases in the levels of non-protein antioxidantsncluding reduced glutathione (GSH), vitamin C and vitamin; and the inflammation associated with neutrophil infiltration

31]. Lipid peroxidation is the oxidative damage of lipids, espe-ially the polyunsaturated fatty acids that are very susceptibleo oxidative attack, by ROS and transition metal ions; and it isnown to play a key role in cell injury [49]. This is due to the

iverse cytotoxic products, mostly aldehydes such as malon-ialdehyde (MDA) it yields [50]. These cytotoxic aldehydesave been implicated in the pathogenesis of a number of oxida-ive stress-induced inflammatory diseases [51] such as gouty

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2 E.A. Irondi et al. / Food Science

rthritis. Under such condition, the antioxidant status of theffected tissues is attenuated; thereby over-exposing the cells toxidative damage, and aggravating the inflammatory condition.

The peroxidation of membrane lipids and the consequentxidative damage to cell membrane may disrupt the membraneransport, ionic channels, proteins and deactivate membrane-ssociated enzymes; the membrane lipid bilayer itself mayecome more permeable due to oxidative damage [33]. Hence,he ability of the extract to inhibit lipid peroxidation in the kid-ey, liver and lungs tissues homogenates of rats indicates itsbility to prevent and/or ameliorate oxidative damage to theipids in the membranes of the cells of these three importantrgans; thereby maintaining the integrity of their membranetructure and functionality. Furthermore, since the ROS gen-rated by lipid peroxidation are involved in the pathogenesis ofeveral other diseases such as diabetes mellitus, cardiovasculariseases and carcinogenesis, XO inhibitors, including polyphe-olics, could also be useful for the prevention and managementf many other diseases [44]. This may explain why T. tetrapteraruit has other medicinal uses as stated earlier. The lipid per-xidation inhibitory activity of the T. tetraptera fruit extractould be attributed to its flavonoids and phenolic acids. This isn agreement with our earlier report that Mangifera indica and

ucuna urens seeds extracts rich in these two classes of phen-lics inhibited Fe2+-induced lipid peroxidation in rat pancreasomogenate [34].

. Conclusion

Phenolics extract of T. tetraptera fruits inhibited xanthine oxi-ase and F2+-induced lipid peroxidation in the kidney, liver andungs tissues of rats in vitro. These activities could be attributedo the combined effect of the flavonoids and phenolic acidsresent in the fruits. Therefore, T. tetraptera fruits might be

promising functional food that could be explored for the pre-ention and management of hyperuricaemia and its associatedisease conditions.

onflict of interest statement

We declare that we have no conflict of interest regarding thistudy.

cknowledgement

The authors acknowledge the Multi-Disciplinary Centralaboratory, University of Ibadan, Nigeria for giving access to

he facilities in their laboratory to carry out the wet analyses.

eferences

[1] B.T. Emmerson, The management of gout, N. Engl. J. Med. 334 (1996)445–451.

[2] H.M. Kramer, G. Curhan, The association between gout and nephrolithi-asis: the national health and nutrition examination survey III, 1988–1994,

Am. J. Kidney Dis. 40 (2002) 37–42.

[3] A. Burke, E. Smyth, G.A. FitzGerald, Analgesic-antipyretic agents; phar-macotherapy of gout, in: L.L. Brunton, J.S. Lazo, K.L. Parker (Eds.), The

[

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