research article antioxidant and hepatoprotective...

Hindawi Publishing CorporationBiotechnology Research InternationalVolume 2013 Article ID 230142 7 pageshttpdxdoiorg1011552013230142

Research ArticleAntioxidant and Hepatoprotective Properties of Tofu (CurdleSoymilk) against Acetaminophen-Induced Liver Damage in Rats

Ndatsu Yakubu1 Ganiyu Oboh2 and Amuzat Aliyu Olalekan1

1 Department of Biochemistry Ibrahim Badamasi Babangida University Niger State Lapai Nigeria2 Department of Biochemistry Federal University of Technology Ondo State Akure Nigeria

Correspondence should be addressed to Ndatsu Yakubu ndatsuyakubu2011hotmailcom

Received 8 November 2012 Revised 21 January 2013 Accepted 30 January 2013

Academic Editor Gabriel A Monteiro

Copyright copy 2013 Ndatsu Yakubu et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The antioxidant and hepatoprotective properties of tofu using acetaminophen to induce liver damage in albino rats were evaluatedTofus were prepared using calcium chloride alum and steep water as coagulants The polyphenols of tofu were extracted andtheir antioxidant properties were determined The weight gain and feed intake of the rats were measured The analysis of serumalanine aminotransferase (ALT) alkaline phosphatase (ALP) aspartate aminotransferase (AST) and lactate dehydrogenase (LDH)activities and the concentrations of albumin total protein cholesterol and bilirubin were analyzed The result reveals that theantioxidant property of both soluble and bound polyphenolic extracts was significantly higher in all tofus but the steep watercoagulated tofu was recorded higher Rats fed with various tofus and acetaminophen had their serum ALP ALT AST and LDHactivities total cholesterol and bilirubin levels significantly (119875 lt 005) reduced and total protein and albumin concentrationsincreased when compared with basal diet and acetaminophen administered group Therefore all tofus curdled with variouscoagulants could be used to prevent liver damage caused by oxidative stress

1 Introduction

Reactive oxygen species (ROS) have been implicated inmore than 100 diseases [1] Foods (tubers grains fruitsand vegetables) provide a wide variety of ROS-scavengingantioxidants such as phytochemical and antioxidant vitamins[2 3] The increased consumption of fruits and vegetablescontaining high levels of phytochemicals has been recom-mended to prevent or reduce oxidative stress in the humanbody [2ndash4] The natural antioxidant defense mechanismscan be insufficient and hence dietary intake of antioxidantcomponents is important and recommended [5]

Liver disease is a worldwide problem Conventionaldrugs used in the treatment of liver diseases are sometimesinadequate and can have serious adverse effects [6] Soybeansare inexpensive and serve as high quality protein sourceSoymilk and tofu consumption is increasing in Nigeria dueto animal diseases such as mad cow disease global shortageof animal protein strong demand for healthy (cholesterol-free and low in saturated fat) and religious halal food andeconomic reasons [7] The greater acceptance of soy foods

by the general population is due to increased recognitionof the health benefits of soy foods especially by those whowant to reduce their consumption of animal products [8]Tofu also known as soybean curd is a soft cheese-like foodmade by curdling fresh hot soymilk with a coagulant [9]Traditionally in Nigeria it is produced by curdling fresh hotsoymilk with CaCl

2 MgSO

4alum or steep water (effluence

from pap produced from maize) [10ndash12] Tofu is rich inproteins low in saturated fats higher in polyunsaturated fattyacids cholesterol-free and a good source of 120573-vitamin min-erals isoflavones and antioxidants (carotenoids vitamins Cand E phenolic and thiol (SH) compounds and essentialamino acids) [8 12] It has been demonstrated that phenoliccompounds are effective antioxidants due to the formationof stable phenoxyl radical [13] and that flavonoids havepotent antioxidant activities by scavenging hydroxyl radicalssuperoxide anions and lipid peroxy radicals [14] Soybeanproducts reduce the risk of heart diseases by lowering levelsof oxidized cholesterol which is taken up more rapidlyby coronary artery walls to form dangerous plaques [15]Previous research has shown that soy consumption reduces

2 Biotechnology Research International

cholesterol in general while also decreasing the amount ofbad cholesterol (low-density lipoprotein LDL) in the bodyand maintaining the amount of good cholesterol (LDL)[16 17]

Acetaminophen is a commonly used and safe analgesicdrug which is known to cause centribular necrosis uponoverdose [18] Its toxicity accounts for many emergencyhospital admission and continues to be associated withhigh mortality The hepatotoxic effect of acute paracetamoloverdose is well known and has been extensively reviewed[19] However tofu (soybean product) contains compoundsthat are valuable antioxidants and protecting moleculeswhich can trap or destroy free radicals and subsequentlyprotect us from damage due to oxidative stress [9] Inview of this people have started to take an interest in tofuconsumption due to its good nutritional and health benefitto human Therefore this research is designed to evaluateits polyphenol distribution and the antioxidant properties intofu produced using different coagulants and to compareits hepatoprotective properties on acetaminophen-inducedtoxicity in ratrsquos liver

2 Materials and Methods

The soybeans (Glycine max) TGX923-1E variety wereobtained directly from Ibrahim Badamasi Babangida Uni-versity Lapai experimental farm in Niger State Nigeria Thealum and calcium salt (CaCl

2) were industrial grade while

the steep water was collected from domestically processedpap The water used in the analysis was glass distilled Theweaning albino rats used were of the same litter originobtained from the rat colony of the Department of Biochem-istry University of Ilorin Nigeria

21 Tofu Preparation Soybeans (20 kg) were soaked in water(6 litres) at 20ndash40∘C for 9 hours The soaked beans weredrained weighed and ground with grinder tap water wasadded at a ratio of 6 1 with raw bean and then filtered to sep-arate soy cake from soymilk The soymilk was subsequentlyheated to 98∘C andmaintained for 1 minute before deliveringto the mix tank When cooled to 87∘C 1 litre of soymilk wasmixed at 420 rpm with each of the coagulants (50mL) Themixed solutions were held for 5 seconds and then filled ontotofu trays and allowed to coagulate for 10 minutes The beancurd was pressed after which the tofu weight was recordedTofu was stored in water at 4∘C overnight prior to analysis

22 Animals Twenty-five of 3-week old strain albino ratswith an average weight of 502 g were used in this studyThey were obtained from the animal house of the Collegeof Medicine University of Ilorin Kwara State Nigeria Theanimals were housed in metabolic cage in the laboratoryunder ambient temperature and 12-hour light and darkperiodicities They were fed with commercial rat pellets(Neimeth Livestock Feeds Ltd Ikeja) and water ad libitumand allowed to acclimatize for 2 weeks Animal experi-ments were conducted in accordance with the internationallyaccepted principle for laboratory animal use and care [20]

23 Extraction of Free Soluble Polyphenols Free soluble phe-nols were extracted using the method modified by Nwannaand Oboh [21] About 200 g of each tofu was homogenizedin 80 acetone (1 2 wv) separately using chilled Waringblender for 5 minutes Thereafter the homogenates were fil-tered through Whatman number 2 filter paper on a Buchnerfunnel under vacuumThe residues were kept for extractionsof bound phenolsThe filtrates were evaporated using a rotaryevaporator under vacuum at 45∘C until 90 of the filtrateshad been evaporated The extracts were frozen at minus40∘C

24 Extraction of Bound Polyphenols These were extractedusing the method modified by Nwanna and Oboh [21] Thebound phenolic contents were extracted from the residuefrom the free soluble polyphenol extracts The drainedresidues from soluble free extractionwere hydrolyzed directlywith 20mLof 4MNaOHat room temperature for 1 hourwithshaking The mixture was acidified to PH 2 with conc HCland extracted six times with ethyl acetate The ethyl acetatefractions were evaporated at 45∘C under vacuum to dryness

25 Antioxidant Activity

251 Total Phenol Content These were determined by themodified method of Nwanna and Oboh [21] About 05mLof each extract was dissolved in 20mL of 70 acetone withequal volume of water 05mL Folin-Cioalteus reagent and25mL of sodium carbonate were subsequently added and theabsorbance was measured after 40 minutes at 725 nm usingtannic acid as standard

252 Reducing Property Thesewere determined by assessingthe ability of the tofu extracts to reduce FeCl

3solution using

modifiedmethod of Nwanna andOboh [21] About 25mL ofeach extract was dissolved in 20mL of methanol and mixedwith 25mL of 200M sodium phosphate buffer (PH 66) and25mL of 100 potassium ferricyanide The mixtures wereincubated at 50∘C for 20 minutes thereafter 25mL 10trichloroacetic acid was added and subsequently centrifugedat 650 rpm for 10minutes 5mL of the supernatant wasmixedwith equal volume of water and 1mL of 01 ferric chlorideThe absorbances were thenmeasured at 700 nm and a higherabsorbance indicates a higher reducing power

253 Free Radical Scavenging Ability The free radical scav-enging ability of the soluble free and bound extracts againstDPPH (1 1-diphenyl-2-picrylhydrazyl) free radical was alsoevaluated [22] About 1mL of the extract was dissolved in20mL methanol and mixed with 1mL of 04M methanolicsolution containing 1 1-diphenyl-2-picrylhydrazyl (DPPH)radicals The mixtures were left in the dark for 30 minutesbefore measuring the absorbance at 516 nm

26 Experimental Design Animals were weighed and ran-domly assigned into five groups namely

normal control group (119899 = 5) was placed on a basaldiet of 20 gday only

Biotechnology Research International 3

negative control group (119899 = 5) was placed on20 gday and 100mg100 gday of basal diet andacetaminophen orally administered respectivelytreated group 1 (119899 = 5) was placed on steep water tofu(20 g) and acetaminophen (100mg100 gday) orallyadministered respectivelytreated group 2 (119899 = 5) was placed on alum tofu(20 g) and acetaminophen (100mg100 gday) orallyadministered respectivelytreated group 3 (119899 = 5) was placed on calcium tofu(20 gday) and acetaminophen (100mg100 gday)orally administered respectively

The experimental periodwas 14 days During the experimentthe weights of the rats were measured two times in a weekduring the 2 weeks of the experiment

27 Measurements The mean average weight of the rats wasdetermined at the beginning of the experiment at every 2days The weight of the rats was determined using weighingscale (OHAUS MODEL Cs 5000 CAPACITY 500 times 2 g)This was done by placing a container on the scale with thebalance adjusted to zero after which the rats in each groupwere placed into container and the measurement taken [23]Total feed consumed in percentage was calculated using thefollowing formula

total feed consumed () = 100 (feed supplied (20 g) minusleftover (g)feed supplied (20 g)

total weight gain (TWG) in percentage was calculated astotal weight gain () = 100 times (final weight minus initial

weight)initial weight [24]On the 15th day the rats were killed by cervical disloca-

tion the bloods collected were centrifuged and the super-natants were collected for the assessment of liver functionIn addition the livers were harvested for histopathologicalanalysis

271 Assessment of Liver Function The serum was used forthe assay of marker enzymes (aspartate aminotransferase(AST) alanine aminotransferase (ALT) alkaline phosphatase(ALP) lactate dehydrogenase (LDH) and bilirubin) concen-trations using Roche Modular Autoanalyzer Total proteinalbumin sugar and total cholesterol were analyzed by usingstandard randox laboratory kits

28 Statistical Analysis The results were reported as mean plusmnSEM (standard error of mean) One-way analysis of varianceand least significant difference (LSD) test were used toevaluate the significant difference Probability levels of lessthan 005 were considered significant

3 Results

Table 1 shows the result of antioxidants properties of the tofustudied The results revealed that the steep water coagulatedtofu significantly recorded high total phenols (150 plusmn 224)followed by the alum-coagulated tofu (130 plusmn 208) and thecalcium salt coagulated tofu appeared significantly higher in

free radical scavenging activities of the free soluble phenol(655 plusmn 467) The alum coagulated tofu (473 plusmn 397)and the steep water had the least (382 plusmn 357) Converselysteep water coagulated tofu significantly recorded higher freeradical scavenging activity of the bond phenol (691plusmn480)followed by alum coagulated tofu (565plusmn336) and calciumsalt coagulated tofu had the lowest (273plusmn302) free radicalscavenging activity of the bound phenol Also the reducingpower of free soluble and bound phenol of steep water andcalcium salt coagulated tofu were not significantly different(04 plusmn 020) while alum coagulated tofu had the reducingpower of free soluble phenol (03plusmn017) to be lower than thatof bound phenol (05 plusmn 030)

The result of total feed intake and total weight gain arepresented in Table 2 and revealed that there was decrease intotal feed intake (1418plusmn531) and total weight gain (911plusmn135) in rats fed with basal diet and given acetaminophenwhen compared with those rats fed with basal diet withoutacetaminophen (total feed intake 4438 plusmn 940 total weightgain 145 plusmn 170) However there was a significant increase(119875 lt 005) in total feed intake (442 plusmn 210ndash735 plusmn 380) ofrats fed with tofu curdled with various coagulants and givenacetaminophen orally compared to those rats fed with basaldiet and given acetaminophen In addition the total weightgain of rats fed with tofu curdled with various coagulantsplus acetaminophen decreased significantly (minus51plusmn101ndash21plusmn060) when compared with those fed with acetaminophen(91 plusmn 135)

The results of liver function tests of the rats studied areshown in Table 3 and revealed that serum AST ALT ALPand LDH levels of rats treated with basal diet and givenacetaminophen orally were quite higher than that of thegroup treated with basal diet without acetaminophen In con-trast the rats treated with various tofus and acetaminophenorally had significantly lower levels of AST ALT ALP andLDH when compared with the group treated with basal dietand acetaminophen orally (Table 3)

The results of serum chemistry of rats studied arepresented in Table 4 and revealed that bilirubin (0153 plusmn012mgdL) cholesterol (24345 plusmn 700mgdL) and glucose(16268plusmn570mgdL) levels significantly (119875 lt 005) increasedin rats treated with basal diet without acetaminophen intu-bation when compared with those treated with basal diet andacetaminophen intubation Nevertheless the content of totalprotein (345plusmn080mgdL) and albumin (165plusmn057mgdL)respectively significantly decreased (119875 lt 005) In additionrats fed with various coagulated tofu and acetaminophenshowed significant (119875 lt 005) reduction in the levelof bilirubin cholesterol and glucose when compared withrats fed with basal diet and acetaminophen intubation Incontrast the level of total protein and albumin of rats fed withvarious tofus and acetaminophenwere significantly increased(119875 lt 005) when compared with those fed with basal diet andacetaminophen

4 Discussions

Theantioxidant properties of polyphenolic extractions of tofuproduced using different coagulants and the effects of these


Table 1 Antioxidant properties of tofu studied

Sample (tofu) TP FSFP () FSBP RPFP RPBPSWT 150 plusmn 224a 382 plusmn 357c 691 plusmn 480a 04 plusmn 020a 04 plusmn 020b

ALT 130 plusmn 208b 473 plusmn 397b 565 plusmn 336b 03 plusmn 017b 05 plusmn 030a

CST 110 plusmn 191c 655 plusmn 467a 273 plusmn 302c 04 plusmn 020a 04 plusmn 020b

Valueswith the same superscript letter(s) along the same column are not significant different (P le 005) SWT steepwater coagulated tofu ALT alumcoagulatedtofu CST calcium salt coagulated tofursquo TP total phenol FSFP free radical scavenging ability of free soluble phenols FSBP free radical scavenging ability ofbound phenols RPFP reducing power of free soluble phenols RPBP reducing power of bound phenols

Table 2 Total feed intake and weight gain of albino rats fed withcommercial diet and various coagulated tofu (gratsdays)

Sample Total food taken () Total weight gained ()BDA 1418 plusmn 531b 911 plusmn 135b

BDWA 4438 plusmn 940a 145 plusmn 170a

CSTA 647 plusmn 360d minus32 plusmn 080d

SWTA 442 plusmn 210e minus21 plusmn 060e

ALTA 735 plusmn 380c minus51 plusmn 101c

Values representmean of triplicate readingMeans with the same superscriptletter(s) within the same column are not significantly different (P le 005)BDA basal diet with acetaminophen orally administered BDWA basal dietwithout acetaminophen orally administered ALTA alum coagulated tofuwith acetaminophen SWTA tofu coagulated with effluent from pap withacetaminophen CSTA calcium coagulated tofu with acetaminophen orallyadministered

tofus on acetaminophen-induced hepatotoxicity in albinorats were evaluated Polyphenols particularly the flavonoidsare among the most potent plant antioxidants Polyphenolcan form complexes with reactive metals such as iron zincand copper reducing their absorption [21] It seems to reducenutrients absorption but excess levels of such elements(metals cations) in the body can promote the generation offree radicals and contribute to the oxidative damage of cellmembranes and cellular DNA [25] In addition polyphenolsalso function as potent free radical scavengers within thebody where they can neutralize free radicals before theycan cause cellular damage [26] The results of polyphenolicantioxidant properties of various tofus studied as indicatedin Table 1 revealed that steep water coagulated tofu had asignificantly higher (119875 lt 005) total phenol followed byalum coagulated tofu and calcium salt tofu had the leastvalue of total phenol These values generally compared wellwith what Shokunbi et al [9] reported for some varietiesof commercial mushrooms (001 g100 g) The results arealso in line with the report of Oboh and Rocha [27] onsome hot pepper (Capsicum annum Tepin and Capsicumpubescens) In contrast free radical scavenging activity ofthe free phenols of various types of tofu analyzed had asignificantly higher (119875 lt 005) calcium coagulated tofufollowed by alum coagulated tofu and steep water coagulatedtofu which is the least (Table 1) However the reverse was thecase for the bound phenols where steep water coagulated tofuwas recorded high followed by alum coagulated tofu andcalcium-coagulated tofu had the least value of bound phenol(Table 1)

These values are higher thanwhatOboh andAkindahunsi[28] reported on some commonly consumed green leafy

vegetables in Nigeria Oxidative stress is the state of imbal-ance between the level of antioxidant defense system and theproduction of the oxygen-derived speciesThe increased levelof oxygen and oxygen-derived species causes oxidative stressThe finding suggests that various tofus produced using differ-ent coagulants are good source of antioxidants and could beused to reduce biological oxidative stress and prevent cellulardamage by scavenging free radical activity of the oxidativestress Table 2 shows the results of total feed intake and totalweight gain of rats studied The result reveals that there wasa decrease in total feed intact and total weight gain of therats fed with basal diet and acetaminophen when comparedwith those rats fed with basal diet without acetaminophen(Table 2) This drastic reduction in feed intake and totalweight gain could be because of overdose of paracetamolintake that generated oxidative stress in ratsThe free radicalsproduced in rats might cause certain abnormality in foodconsumed and weight gain It was also observed that ratsfed with alum-coagulated tofu consumed the highest amountof tofu in the duration of the experiment followed bythose fed with calcium chloride coagulated tofu while thosefed with tofu coagulated with steep water consumed thelowest amount of tofu (Table 2) The total weight gain ofrats fed with tofu produced using different coagulants wassignificantly higher (119875 lt 005) in alum-coagulated tofu andtofu coagulated with steep water significantly had the leastweight gain (Table 2) The higher consumption of alum andcalcium chloride coagulated tofu might be because of goodtaste and odour as indicated in the tofu and the negativevalue of weight gain recorded by the rats fed with steep watertofu could be because of very low feed intake While thelow intake of the tofu coagulated with steep water could bebecause of the unpleasant odor imparted by the steep water tothe tofuThe wide variation for tofu consumed by the variousrats could be because of the difference in the taste nutritionalquality and acceptability of the various coagulated tofu [1629] As shown inTable 3 overdosing ratswith 100mgmLdayacetaminophen alone for 14 days caused a significant increase(119875 lt 005) in the serummarker enzymes (AST ALTALP andLDH) (Table 3) Increase in AST levels signified liver damageThis finding suggested that themega doses of acetaminophenadministered induce the production of free radicals whichcause damage to the hepatocytes of rats

This result correlates with the finding of Eriksson et al[30] that toxicity with acetaminophen occurs when toomuch of it is taken The elevations of serum liver enzymesindicate liver damage This correlates with the report ofSai et al [31] that a significant increase in serum AST


Table 3 Serum enzyme marker level of rats studied

Sample (rats) ALP (mgdL) AST (mgdL) ALT (mgdL) LDH (mgdL)BDA 1220 plusmn 493a 1010 plusmn 449a 310 plusmn 249a 15600 plusmn 559a

BDWA 900 plusmn 424c 770 plusmn 392c 260 plusmn 228b 7840 plusmn 396c

SWTA 1100 plusmn 470b 820 plusmn 405b 260 plusmn 228b 8552 plusmn 414c

ALTA 1200 plusmn 490ab 940 plusmn 434b 270 plusmn 232b 9000 plusmn 424b

CSTA 1130 plusmn 475b 800 plusmn 40c 300 plusmn 245ab 9023 plusmn 425b

Means with the same superscript letter(s) along the same column are not significantly different (P le 005) BDA basal diet with acetaminophen orallyadministered BDWA basal diet without acetaminophen orally administered SWTA steep water coagulated tofu with acetaminophen orally administeredALTA alum coagulated tofu with acetaminophen orally administered CSTA calcium chloride coagulated tofu

Table 4 Serum chemistry level of rats studied

Sample (rats) BIL CHL (MgdL) GLU TPN ALBBDA 0153 plusmn 012a 24345 plusmn 700a 16268 plusmn 570a 345 plusmn 083d 165 plusmn 057d

BDWA 0125 plusmn 014b 9520 plusmn 436d 8734 plusmn 418c 775 plusmn 124a 393 plusmn 089a

SWTA 0118 plusmn 004c 14532 plusmn 540c 9350 plusmn 432b 705 plusmn 119b 300 plusmn 077b

ALTA 0122 plusmn 012c 15750 plusmn 561b 9500 plusmn 436b 645 plusmn 114c 260 plusmn 072c

CSTA 0128 plusmn 016b 14843 plusmn 545c 9376 plusmn 433b 674 plusmn 116c 291 plusmn 076c

Means with the same superscript letter(s) along the same column are not significantly different (P le 005) BDA basal diet with acetaminophen orallyadministered BDWA basal diet without acetaminophen orally administered SWTA steep water coagulated tofu with acetaminophen orally administeredALTA alum coagulated tofu with acetaminophen orally administered CSTA calcium chloride coagulated tofu BIL bilirubin CHL cholesterol GLU glucoseTPN total protein ALB albumin

ALT and ALP levels suggests liver damage Under normaltherapeutic dose of acetaminophen the excessivemetabolitesproduced by the cytochrome P-450 system can be reducedby glutathione However if acetaminophen is overdosedthe glutathione stores will be depleted and the excessivemetabolites will react with the liver macromolecules andcause hepatic cell death The hepatic cellular enzyme ALPin serum will therefore increase In addition the hepaticmalondialdehyde level will increase invariably hence result-ing in the generation of free radicals in the body [21] Thissuggests that acetaminophen hepatotoxicity appears to becritically dependent on the depletion of cellular glutathioneand a relatively high reduction in the intracellular level ofreduced glutathione leads to a situation of oxidative stress[32 33] Conversely a simultaneous administration of albinorats with 100mgmLacetaminophen alongside various tofusproduced caused a significant decrease (119875 le 005) inserum AST ALT and ALP (Table 3) Decrease in serummarker enzyme levels (liver enzymes) indicates the abilityof various tofus to protect the hepatocytes from oxidativedamage caused by overdosing rats with acetaminophen Thisis an indication that antioxidant mechanismmay be involvedin the protection of the liver cell by the various tofus fromacetaminophen-induced oxidative stress It was suggestedthat soy products contain high antioxidant properties whichserve as an extracellular neutralizer of free radicals [34]

In addition soy food and vegetables had been reported tobe rich in many phenols such as flavonoid [35] Flavonoidshave antioxidants capacity that is much stronger than thoseof vitamins C and E reportedly used to prevent free rad-ical production [35] In contrast the serum LDH level ofrats fed with basal diet and acetaminophen was increasedsignificantly (119875 lt 005) when compared with those fed

with basal diet without acetaminophen (Table 3) This is inline with the findings of Ravikumar et al [36] that theserum LDH level was elevated in rat administered CCl

4 The

increased level of LDH is an indication of abnormality in liverfunctioning which may be due to the formation of highlyreactive free radicals caused by acetaminophen overdoseThe hepatotoxicity of acetaminophen may directly affect thepolyunsaturated fatty acids and alter the liver microsomalmembranes in the rats In a reverse case the LDH levels of ratsfedwith tofu and acetaminophenwere significantly decreased(119875 lt 005) when compared with that of rats in group BDA(Table 3) which is a sign of an improvement by the varioustofus over the damage done to the liver by acetaminophenThe result of serum total cholesterol level as indicated inTable 4 revealed that there was a significant increase in totalcholesterol of rats fed with basal diet and acetaminophenorally when compared to those fed with basal diet withoutacetaminophen orally administered (Table 4)

The elevation in serum total cholesterol level could beattributed to the ability of acetaminophen to induce theproduction of free radicals which results in hypercholes-terolemia and the atherosclerosis This correlates with thefindings of Oboh [15] that an increase in the serum levels ofcholesterol and LDL is associated with hypercholesterolemiaand atherosclerosis respectively It is also supported by thefindings of Ravikumar et al [36] that intoxication of ratwith CCl

4elevated total cholesterol levels which suggests the

inhibition of bile acid synthesis and leads to increased level ofcholesterol However supplementation of tofu curdled withvarious coagulants and acetaminophen orally administeredcauses a significant decrease (119875 gt 005) in serum totalcholesterol compared to those fed with basal diet withacetaminophen orally administered (Table 4) It indicated


that tofus produced using three coagulated agents are capableof preventing acetaminophen inducing oxidative stress andinhibiting bile acids synthesis

This could be attributed to high antioxidant potential ofsoybeans which serve as an extracellular neutralizer of freeradicals [37]This finding is in linewith the study ofOboh [15]that the antihypercholesterolemic effect of soy protein wasfound to decrease the plasma concentrations of LDL as wellas the ratio of plasma LDL to high density lipoprotein (HDL)It was reported that flavonoids could protect membranelipids from oxidation and a major source of flavonoids isvegetables fruits and soybeans [35] The significant (119875 lt005) reduction in total protein and albumin levels in ratsfedwith basal diet with acetaminophen intubation comparedto those fed with basal diet without acetaminophen orallyadministered (Table 4) indicates cellular damage producedThe damage produced might be due to the functionalfailure of endoplasmic reticulum which leads to decreasein protein synthesis and accumulation of triglycerides [36]Increased serum bilirubin level in rats fed with basal dietwith acetaminophen intubation (Table 4) could be lookedupon as a compensatoryretaliatory phenomenon in responseto cellular peroxidative changes which cause damage to thebiliary gland This is because bilirubin functions in vivo asa powerful antioxidant antimutagen and an endogenoustissue protector [37] Reduction of bilirubin and elevation oftotal protein and albumin levels in rats treated with variouscoagulated tofus and acetaminophen orally administered(Table 4) were most effective and stabilized the biliary cellfunction and endoplasmic reticulum leading to bile acidand protein synthesis [38] This indicates hepatoprotectionThe administration of acetaminophen alone may adverselyinterfere with protein metabolism probably by inhibiting thesynthesis of proteins such as albumin in the liver

Simultaneous administration of acetaminophen to ratswith supplementation of various tofus produced reversedthese changes maybe by increasing protein synthesis Stimu-lation has been advanced as a contributory hepatoprotectivemechanismwhich accelerates regeneration of cells [39] LDHactivities all point to the fact that tofu has hepatoprotectivepotential against hepatotoxin caused by acetaminophen Itcould be possible that a probable mechanism of hepato-protection of various tofus against acetaminophen-induceddamage is the antioxidant activity The antioxidant activityof the various tofus supplemented may be attributed tothe presence of phenolics and flavonoids [40] Thereforelower level of serum enzyme markers total cholesteroland bilirubin observed in rats on tofu coagulated withvarious coagulants and acetaminophen orally administeredsuggested liver damage repair but steepwater coagulated tofulooked more promising in terms of liver repair than otherforms of tofu diet It can be concluded from the presentfinding that tofus curdled with various coagulants could beefficiently used to prevent liver damage caused by high dosesof acetaminophen orally administered after successful clinicaltrials In addition the intake of acetaminophen in excessshould be discouraged in homes because of its destructiveeffects on the liver Tofu consumption should be encouragedin diets as it can be used as a functional food to prevent liver

damage due to its antioxidant properties and as such tofucould be recommended for clinical trial

Conflict of Interests

The authors declare that they have no competing interestsapart from the possible ones already acknowledged below

Acknowledgments

The authors would like to acknowledge the Departmentof Biochemistry Federal University of Technology AkureOndo State Nigeria for providing facilities for this workOne of the authors Mr N Yakubu is grateful to the NigerState College of Agriculture Mokwa Niger State Nigeria forfinancial sponsorship that enabled him to participate in thisstudy

References

[1] Y Ali OMunir and B Vahit ldquoThe antioxidant activity of leavesof Cydonia vulgarisrdquo Turkish Journal of Medical Sciences vol 31pp 23ndash27 2001

[2] J Sun Y F Chu XWu andRH Liu ldquoAntioxidant and antipro-liferative activities of common fruitsrdquo Journal of Agriculturaland Food Chemistry vol 50 no 25 pp 7449ndash7454 2002

[3] R H Liu ldquoHealth benefits of fruit and vegetables are from addi-tive and synergistic combinations of phytochemicalsrdquoAmericanJournal of Clinical Nutrition vol 78 no 3 pp 517Sndash520S 2003

[4] Y F Chu J Sun X Wu and R H Liu ldquoAntioxidant andantiproliferative activities of common vegetablesrdquo Journal ofAgricultural and Food Chemistry vol 50 no 23 pp 6910ndash69162002

[5] P DDuh ldquoAntioxidant activity of Burdock its scavenging effecton free radical and active oxygenrdquo Journal of the American OilChemistsrsquo Society vol 75 no 4 pp 455ndash461 1998

[6] T Patel D Shirode S Pal Roy S Kumar and S RamachandraSetty ldquoEvaluation of antioxidant and hepatoprotective effectsof 70 ethanolic bark extract of Albizzia lebbeck in ratsrdquoInternational Journal of Research in Pharmaceutical Sciencesvol 1 no 3 pp 270ndash276 2010

[7] M A Asgar A Fazilah N Huda R Bhat and A A KarimldquoNonmeat protein alternatives as meat extenders and meatanalogsrdquo Comprehensive Reviews in Food Science and FoodSafety vol 9 no 5 pp 513ndash529 2010

[8] V Poysa and L Woodrow ldquoStability of soybean seed composi-tion and its effect on soymilk and tofu yield and qualityrdquo FoodResearch International vol 35 no 4 pp 337ndash345 2002

[9] O S Shokunbi O O Babajide D O Otaigbe and G O TayoldquoEffect of coagulants on the yield nutrient and antinutrientcomposition of Tofurdquo Archives of Applied Science Research vol3 no 3 pp 522ndash527 2011

[10] K Descheemaeker and I Debruyne Clinical Evidence DieteticApplications Garant 2001

[11] P A Murphy and L A Wilson ldquoSoybean protein compositionand tofu qualityrdquo Food Technology vol 51 no 3 pp 86ndash88 1997

[12] G Paganga N Miller and C A Rice-Evans ldquoThe polyphenoliccontent of fruit and vegetables and their antioxidant activitiesWhat does a serving constituterdquo Free Radical Research vol 30no 2 pp 153ndash162 1999


[13] S W Qader M A Abdulla L S Chua N Najim M MZain and S Hamdan ldquoAntioxidant total phenolic content andcytotoxicity evaluation of selectedMalaysian plantsrdquoMoleculesvol 16 no 4 pp 3433ndash3443 2011

[14] A L Miller ldquoAntioxidant flavonoids structure function andclinical usagerdquo Alternative Medicine Review vol 1 no 2 pp103ndash111 1996

[15] G Oboh ldquoCoagulants modulate the hypocholesterolemic effectof tofu (coagulated soymilk)rdquo African Journal of Biotechnologyvol 5 no 3 pp 290ndash294 2006

[16] F M Sacks A Lichtenstein L Van Horn W Harris PKris-Etherton and M Winston ldquoSoy protein isoflavones andcardiovascular health an American Heart Association ScienceAdvisory for professionals from the Nutrition CommitteerdquoCirculation vol 113 no 7 pp 1034ndash1044 2006

[17] J Parma H RShama and R Verma ldquoEffect of source andcoagulants on the Physicochemical and organoleptic evaluationof soy tofurdquo Journal of Dairying Foods ampHome Sciences vol 26no 2 pp 69ndash74 2007

[18] D G Davidson and W N Eastham ldquoAcute liver necrosisfollowing overdose of paracetamolrdquoBritishMedical Journal vol5512 pp 497ndash499 1966

[19] L P James P R Mayeux and J A Hinson ldquoAcetaminophen-induced hepatotoxicityrdquo Drug Metabolism and Disposition vol31 no 12 pp 1499ndash1506 2003

[20] NIH Publication no 85-23 ldquoRespect for liferdquo National Instituteof Environmental Health Sciences-NIEHS 1985 httpwwwniehsnihgov

[21] E E Nwanna and G Oboh ldquoAntioxidant and hepatoprotectiveproperties of polyphenol extracts from Telfairia occidentalis(Fluted Pumpkin) leaves on acetaminophen induced liverdamagerdquo Pakistan Journal of Biological Sciences vol 10 no 16pp 2682ndash2687 2007

[22] F Ursini M Maiorino P Morazzoni A Roveri and G PifferildquoA novel antioxidant flavonoid (IdB 1031) affecting molecularmechanisms of cellular activationrdquo Free Radical Biology andMedicine vol 16 no 5 pp 547ndash553 1994

[23] O A Semeon ldquoHaematological Characteristics of Clariasgariepinus (Buchell 1822) Juveniles Fed on Poultry HatcheryWasterdquoAmerican-Eurasian Journal of Toxicological Sciences vol2 no 4 pp 190ndash195 2010

[24] H Obek S Ugras I Bayram et al ldquoHepatoprotective effect ofFoeniculumvulgare essential oil a carbon-tetrachloride inducedliver fibrosis model in ratsrdquo Scandinavian Journal of LaboratoryAnimal Science vol 31 no 1 pp 201ndash211 2004

[25] O Y Okafor O L Erukainure J A Ajiboye R O Adejobi FOOwolabi and S BKosoko ldquoPineapple peel extractmodulateslipid peroxidation catalase activity andhepatic biomarker levelsin blood plasma of alcohol-induced oxidative stressed ratsrdquoAsian Pacific Journal of Tropical Medicine vol 1 no 1 pp 11ndash14 2011

[26] N Ara and H Nur ldquoIn vitro antioxidant methanolic leavesand flowers extracts of Lippia albardquo Research Journal of MedicalSciences vol 4 no 1 pp 107ndash110 2009

[27] G Oboh and J B T Rocha ldquoPolyphenols in red pepper[Capsicum annuum var aviculare (Tepin)] and their protectiveeffect on some pro-oxidants induced lipid peroxidation in brainand liverrdquo European Food Research and Technology vol 225 no2 pp 239ndash247 2007

[28] G Oboh and A A Akindahunsi ldquoChange in the ascorbic acidtotal phenol and antioxidant activity of sun-dried commonly

consumed green leafy vegetables in Nigeriardquo Nutrition andHealth vol 18 no 1 pp 29ndash36 2004

[29] K G Aning A G Ologun A Onifade J A Alokan A IAdekola and V A Aletor ldquoEffect of replacing dried brewerrsquosgrainwith rsquosorghum rootletsrsquo on growth nutrient utilisation andsome blood constituents in the ratrdquo Animal Feed Science andTechnology vol 71 no 1-2 pp 185ndash190 1998

[30] L S Eriksson U Broome M Kalin and M Lindholm ldquoHepa-totoxicity due to repeated intake of low doses of paracetamolrdquoJournal of Internal Medicine vol 231 no 5 pp 567ndash570 1992

[31] K Sai A Takagi T Umemura and Y Kurokawa ldquoToxicologyrdquoJournal of Environmental Pathology vol 11 pp 139ndash143 1992

[32] S L Arnaiz S Llesuy J C Cutrin and A Boveris ldquoOxidativestress by acute acetaminophen administration in mouse liverrdquoFree Radical Biology and Medicine vol 19 no 3 pp 303ndash3101995

[33] M A Tirmenstein and S D Nelson ldquoAcetaminophen-induced oxidation of protein thiols Contribution of impairedthiol-metabolizing enzymes and the breakdown of adeninenucleotidesrdquo Journal of Biological Chemistry vol 265 no 6 pp3059ndash3065 1990

[34] TAnderson andA JTheron ldquoAntioxidant and tissue protectivefunction of ascorbic acidrdquo World Review of Nutritional andDiabetes vol 62 pp 37ndash38 1990

[35] M Alıa C Horcajo L Bravo and L Goya ldquoEffect of grapeantioxidant dietary fiber on the total antioxidant capacity andthe activity of liver antioxidant enzymes in ratsrdquo NutritionResearch vol 23 no 9 pp 1251ndash1267 2003

[36] S Ravikumar M Gnanadesigan J Seshserebiah and S JacobInbanseon ldquoHepatoprotective effect of an Indian salt marshherb Suaeda monoica Forsk Ex Gmel against concanavalin aninduced toxicity in ratsrdquo Life Sciences and Medicine Researchvol 2010 p LSMR-2 2010

[37] K Pratibha U Anand and R Agarwal ldquoSerum adenosinedeaminase 51015840 nucleotidase and malondialdehyde in acuteinfective hepatitisrdquo Indian Journal of Clinical Biochemistry vol19 no 2 pp 128ndash131 2004

[38] S V Sureshkumar and S H Mishra ldquoHepatoprotective activityof extracts from Pergularia daemia Forsk against carbon tetra-chloride induced toxicity in ratsrdquo Pharmacognosy Magazinevol 3 no 11 pp 187ndash191 2007

[39] D Awang ldquoMilk Thistlerdquo Canadian Pharmacists Journal vol23 pp 749ndash754 1993

[40] O M Iniaghe S O Malomo J O Adebayo and R O AriseldquoEvaluation of the antioxidant and hepatoprotective propertiesof the methanolic extract of Acalypha racemosa leaf in carbontetrachloride-treated ratsrdquoAfrican Journal of Biotechnology vol7 no 11 pp 1716ndash1720 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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Zoology


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GenomicsInternational Journal of


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Stem CellsInternational



Enzyme Research



Microbiology


cholesterol in general while also decreasing the amount ofbad cholesterol (low-density lipoprotein LDL) in the bodyand maintaining the amount of good cholesterol (LDL)[16 17]

Acetaminophen is a commonly used and safe analgesicdrug which is known to cause centribular necrosis uponoverdose [18] Its toxicity accounts for many emergencyhospital admission and continues to be associated withhigh mortality The hepatotoxic effect of acute paracetamoloverdose is well known and has been extensively reviewed[19] However tofu (soybean product) contains compoundsthat are valuable antioxidants and protecting moleculeswhich can trap or destroy free radicals and subsequentlyprotect us from damage due to oxidative stress [9] Inview of this people have started to take an interest in tofuconsumption due to its good nutritional and health benefitto human Therefore this research is designed to evaluateits polyphenol distribution and the antioxidant properties intofu produced using different coagulants and to compareits hepatoprotective properties on acetaminophen-inducedtoxicity in ratrsquos liver

2 Materials and Methods

The soybeans (Glycine max) TGX923-1E variety wereobtained directly from Ibrahim Badamasi Babangida Uni-versity Lapai experimental farm in Niger State Nigeria Thealum and calcium salt (CaCl

2) were industrial grade while

the steep water was collected from domestically processedpap The water used in the analysis was glass distilled Theweaning albino rats used were of the same litter originobtained from the rat colony of the Department of Biochem-istry University of Ilorin Nigeria

21 Tofu Preparation Soybeans (20 kg) were soaked in water(6 litres) at 20ndash40∘C for 9 hours The soaked beans weredrained weighed and ground with grinder tap water wasadded at a ratio of 6 1 with raw bean and then filtered to sep-arate soy cake from soymilk The soymilk was subsequentlyheated to 98∘C andmaintained for 1 minute before deliveringto the mix tank When cooled to 87∘C 1 litre of soymilk wasmixed at 420 rpm with each of the coagulants (50mL) Themixed solutions were held for 5 seconds and then filled ontotofu trays and allowed to coagulate for 10 minutes The beancurd was pressed after which the tofu weight was recordedTofu was stored in water at 4∘C overnight prior to analysis

22 Animals Twenty-five of 3-week old strain albino ratswith an average weight of 502 g were used in this studyThey were obtained from the animal house of the Collegeof Medicine University of Ilorin Kwara State Nigeria Theanimals were housed in metabolic cage in the laboratoryunder ambient temperature and 12-hour light and darkperiodicities They were fed with commercial rat pellets(Neimeth Livestock Feeds Ltd Ikeja) and water ad libitumand allowed to acclimatize for 2 weeks Animal experi-ments were conducted in accordance with the internationallyaccepted principle for laboratory animal use and care [20]

23 Extraction of Free Soluble Polyphenols Free soluble phe-nols were extracted using the method modified by Nwannaand Oboh [21] About 200 g of each tofu was homogenizedin 80 acetone (1 2 wv) separately using chilled Waringblender for 5 minutes Thereafter the homogenates were fil-tered through Whatman number 2 filter paper on a Buchnerfunnel under vacuumThe residues were kept for extractionsof bound phenolsThe filtrates were evaporated using a rotaryevaporator under vacuum at 45∘C until 90 of the filtrateshad been evaporated The extracts were frozen at minus40∘C

24 Extraction of Bound Polyphenols These were extractedusing the method modified by Nwanna and Oboh [21] Thebound phenolic contents were extracted from the residuefrom the free soluble polyphenol extracts The drainedresidues from soluble free extractionwere hydrolyzed directlywith 20mLof 4MNaOHat room temperature for 1 hourwithshaking The mixture was acidified to PH 2 with conc HCland extracted six times with ethyl acetate The ethyl acetatefractions were evaporated at 45∘C under vacuum to dryness

25 Antioxidant Activity

251 Total Phenol Content These were determined by themodified method of Nwanna and Oboh [21] About 05mLof each extract was dissolved in 20mL of 70 acetone withequal volume of water 05mL Folin-Cioalteus reagent and25mL of sodium carbonate were subsequently added and theabsorbance was measured after 40 minutes at 725 nm usingtannic acid as standard

252 Reducing Property Thesewere determined by assessingthe ability of the tofu extracts to reduce FeCl

3solution using

modifiedmethod of Nwanna andOboh [21] About 25mL ofeach extract was dissolved in 20mL of methanol and mixedwith 25mL of 200M sodium phosphate buffer (PH 66) and25mL of 100 potassium ferricyanide The mixtures wereincubated at 50∘C for 20 minutes thereafter 25mL 10trichloroacetic acid was added and subsequently centrifugedat 650 rpm for 10minutes 5mL of the supernatant wasmixedwith equal volume of water and 1mL of 01 ferric chlorideThe absorbances were thenmeasured at 700 nm and a higherabsorbance indicates a higher reducing power

253 Free Radical Scavenging Ability The free radical scav-enging ability of the soluble free and bound extracts againstDPPH (1 1-diphenyl-2-picrylhydrazyl) free radical was alsoevaluated [22] About 1mL of the extract was dissolved in20mL methanol and mixed with 1mL of 04M methanolicsolution containing 1 1-diphenyl-2-picrylhydrazyl (DPPH)radicals The mixtures were left in the dark for 30 minutesbefore measuring the absorbance at 516 nm

26 Experimental Design Animals were weighed and ran-domly assigned into five groups namely

normal control group (119899 = 5) was placed on a basaldiet of 20 gday only











3 Results






4 Discussions





































4 The












Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











3 Results






4 Discussions





































4 The












Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































4 The












Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Acknowledgments


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article antioxidant and hepatoprotective...

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