Olnrewju Sm OlyerijuMry Tolulope OlleyeOlmide Oljusi CrownKyode KomolfeAline Augusti BoligonMrgreth Linde AthydeAkintunde Afoli Akindhunsi

a Department of Biochemistry,School of Sciences,The Federal University of Technology,PMB 704,Akure,Nigeria

b Department of Biochemistry,Faculty of Science,Federal University Oye-Ekiti,PMB 373,Oye-Ekiti,Nigeria

c Department of Industrial Pharmacy,Federal University of Santa Maria,97105-900 Santa Maria,RS,Brazil

Abstract The effects of ethylacetate extract of red onion(Allium cepa)tunic(ACTE)on some hemodynamic and biochemical parameters were evaluated in normotensive albino rats.Blood pressure parameters were determined in anaesthetized rats orally administered ACTE(10-,20-,or 40 mg/kg)or ramipril (1 mg/kg) once daily for 14 days.Respectively, 10-, 20-, or 40 mg/kg ACTE produced significan (P ＜0.01), dose-dependent fall in systolic blood pressure,SBP(21%,27%,33%),diastolic blood pressure,DBP(6%,10%,16%),pulse pressure,PP(42%,49%,56%),mean arterial blood pressure,MAPB(13%,18%,23%)and heart rate,HR(4%,5%,7%).The highest effective dose(40 mg/kg)compared well with ramipril(1 mg/kg)with regards to SBP(41%),DBP(19%),PP(70%),MABP(29%)and HR(10%).Similar trends(decreases)were recorded for 40 mg/kg ACTE and ramipril,respectively,as regards the activities of serum enzymes:creatine kinase(60%and 65%),ALT(18%and 14%)and ALP(28%and 16%).HPLC fingerprint of the fl vonoid-rich ACTE revealed that fl vonols:quercetin,quercitrin,isoquercitrin,rutin and kaempferol are the active fl vonoids.The results demonstrate the hypotensive effect of A.cepa tunic fl vonoids initiating further investigation of their individual or synergistic contribution(s)to the observed effects.

Keywords: Allium cepa;Extract;Flavonoids;Hemodynamic;Hypotension

1.Introduction

Agents with hypotensive effect could be of therapeutic significanc in the advent of clinical or experimental-induced hypertension.One quarter of the world’s adult population is afflicte by hypertension, and this is likely to increase to 29%by 2025[1].The common progressive disorder leads to several chronic diseases such as cardiovascular disease, stroke, renal disease and diabetes[2].

Plants constitute a rich and diverse source of secondary metabolites that have long contributed to the development of small-molecule based therapeutics[3].Onion(Allium cepaL.),a bulbous herb belonging to the family Alliceae, is a widely consumed vegetable.It is a good source of dietary phytochemicals including organosulphur compounds and fl vonoids[4].As a result of their phytoconstituents,onions exhibit considerable antioxidant properties and could modulate the detoxificatio systems [5].Thus, onion intake is reported to have several beneficia effects on health, such as preventing tumors and cancers [6], cardiovascular diseases [7] and hypertension[8].Most of the documented beneficia effects of onions,including inhibition of platelet aggregation[9],hypoglycaemic,hypolipidemic and antiatherosclerotic[10,11],and antioxidant and antiapoptotic effects[12],have centered on the bulb or other edible parts rather than the tunic which is normally discarded as wastes.We envisaged that this underutilized part of onion,which has not received much attention all the while,may have potential benefit to human health.To the best of our knowledge,there is a dearth of information on effect of red onion(A.cepa)tunic on hemodynamic parameters.This work was therefore initiated to provide scientifi information in this regard.

2.Materials and methods

2.1.Chemicals

Glutathione, 5＇,5＇-dithiobis-(2-nitrobenzoate) DTNB,epinephrine and hydrogen peroxide were purchased from Sigma Chem., Co.(London, UK).Alanine aminotransferase(ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), total protein and total cholesterol kits were obtained from Randox Laboratories,UK.All other chemicals were of analytical grade and were either obtained from Sigma–Aldrich or British Drug Houses,(Poole,UK).

2.2.Plant material

Red onions(A.cepa)were bought from Shasha market,outskirt Akure metropolis,Nigeria,in the month of February 2013.Botanical identificatio and authentication were carried out at the herbarium of the Forestry Research Institute of Nigeria(FRIN),Ibadan,Nigeria.

2.3.Preparation of ethylacetate extract of red onions(A.cepa)tunic

The tunics of onions were removed, cleaned, air-dried and pulverized.The powdered sample (80 g) was macerated in 600 mL of ethyl acetate (Sigma Chemicals; USA) for 4 h at 40°C,with constant agitation.The mixture of the solvent and the ground sample was filtere firs with mesh cloth,and then with filte paper (Whatman No.1) and concentrated using a rotary evaporator.The residue was kept in a refrigerator at 4°C until further uses.Prior to the experiments, the ethylacetate extract was dissolved in distilled water and diluted to the desired concentrations to give a water-soluble fraction(ACTE).

2.4.Phytochemical screening

ACTE was screened for the presence of alkaloids,saponins,tannins, steroids, anthraquinones, terpenoids, fl vonoids and phlobatannins as described in our previous work[13].

2.5.Determination of total flavonoid content

The colorimetric method described by Dewanto et al.[14]was employed in the quantificatio of total fl vonoids in ACTE.To 250 μL of the suitably diluted sample,75 μL of 5%NaNO2solution, 150 μL of freshly prepared 10% AlCl3solution, and 500 μL of 1 mol/L NaOH solution were added.The fina volume was adjusted to 2.5 mL with deionized water and the mixture left to stand for 5 min.The absorption was thereafter measured at 510 nm against the same mixture,without the sample,as blank.The amount of total fl vonoids was expressed asquercetin equivalents(QE,mgquercetin/gsample)through the calibration curveof quercetin.

2.6.Quantification of flavonoids

Reverse phase chromatographic analyses were carried out under gradient conditions using C18column(4.6 mm×150 mm)packedwith5 μm diameter particles; the mobile phase was water containing 2%acetic acid(A)and methanol(B),and the composition gradient was:5%of B until 2 min and changed to obtain 25%, 40%, 50%, 60%, 70% and 100% B at 10 min, 20 min,30 min,40 min,50 min and 60 min,respectively,following the method described by Peroza et al.[15] with slight modifica tions.ACTE was analyzed at a concentration of 12 mg/mL.The presence of fl vonoids was investigated,namely,quercetin,isoquercitrin,quercitrin,rutin and kaempferol.Identificatio of these compounds was performed by comparing their retention time and UV absorption spectra with those of commercially available standards and by DAD spectra(300–600 nm).The fl w rate was 0.7 mL/min, injection volume 50 μL and the wavelength was 356 nm.The samples and mobile phase were filtere through 0.45 μm membrane filte (Millipore)and then degassed by ultrasonic bath prior to use.Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range of 0.050–0.500 mg/mL for quercetin,isoquercitrin,quercitrin,kaempferol and rutin.All chromatography operations were carried out at ambient temperature and in triplicate.

The limit of detection (LOD) and limit of quantificatio(LOQ) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves,as define by Sabir et al.[16].LOD and LOQ were calculated as 3.3 and 10σ/S,respectively,whereσis the standard deviation of the response andSis the slope of the calibration curve.

2.7.Animals

Adult male rats(Wistar strain)weighing 200–220 g,obtained from a private breeder and housed in the primate colony of the Department of Physiology, College of Medicine, University of Lagos, Nigeria were used for this study.The animals were kept in wire mesh cages under controlled light cycle(12 h light/12 h dark), fed with commercial rat chow (Vital Feeds Nigeria Limited)ad libitum,and liberally supplied with water.All animal experiments were conducted according to the guidelines of National Institute of Health (NIH publication 85-23,1985)for laboratory animal care and use.

2.8.Experimental design

Age-matched male rats were divided into fie groups(n=5)and treated as follows:

Group I:control,distilled water(1 mL/kg).

Group II:1 mg/kg ramipril.

Group III:10 mg/kg ACTE.

Group IV:20 mg/kg ACTE.

Group V:40 mg/kg ACTE.

Drugs or distilled water were administered by gavage to normotensive rats once daily for 14 consecutive days.Twenty-four hours after the last administration,animals were anaesthetized with a mixture of urethane(25%)and alpha-chloralose(1%)to determine the hemodynamic parameters.Animals were thereafter sacrificed blood was collected through cardiac puncture and hearts were excised.Sera and heart homogenates were then prepared for biochemical evaluations.

2.9.Determination of blood pressure

Blood pressure parameters of rats were determined as previously described [17].At state of anaesthesia, the animal were laid supine and carotid artery exposed and cannulated with a small polyethylene catheter connected to a pressure transducer(Becton Dickinson, Sandy, UT, USA) which in turn was connected to a polygraph 7D model of Grass Polygraph (Grass Instrument Company,Quincy,Massachusetts,USA).The speed of the equipment was 10 mm/s.The blood pressure was then calculated as recorded in form of tracing by the polygraph after a 20-min stabilization period.The body temperature of rats was maintained at 37°C with a heating pad throughout the experiment.On the tracing,the values from the baseline to the lowest border of the tracing represent the diastolic pressure while from the baseline to the upper border represent the systolic pressure.Each centimeter(cm)change on the tracing paper corresponds to 20 mm Hg pressure change in the grass polygraph.The mean arterial blood pressure(MAPB)was calculated as follows:MAPB=DP+1/3(SP-DP),where DP=diastolic pressure and SP=systolic pressure.Heart rate(beats/min)corresponds to the number of strokes within a distance of 600 mm(60 cm)on the polygraph recordings.

2.10.Preparation of serum and tissue homogenate

The collected blood was allowed to clot and serum was obtained after centrifugation at 3000 r per minute for 15 min.To prepare the tissue homogenate,excised hearts were rinsed in 1.15% KCl and homogenized in aqueous Tris–HCl buffer(50 mmol/L,pH 7.4).Homogenates were centrifuged at 6000×gfor 20 min at 4°C to obtain the supernatant fractions which were used for analyses.

2.11.Measurement of serum parameters

The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine kinase and total cholesterol concentration were estimated in the serum using assay kits from Randox Laboratories Ltd.,UK according to the instructions of the manufacturer.

2.12.Assessment of antioxidant status

Antioxidant status was determined in the heart homogenates by quantifying the enzymic(superoxide dismutase and catalase)and non-enzymic(reduced glutathione)antioxidants.

2.12.1.Determination of superoxide dismutase(SOD)activity

The activity profil of SOD in the homogenates was determined by the method of Misra and Fridovich [18].Briefl , an aliquot of the diluted sample was added to 2.5 mL of 0.05 mol/L carbonate buffer (pH 10.2) to equilibrate in the spectrophotometer.The reaction was started by the addition of 0.3 mL of freshly prepared 0.3 mmol/L adrenaline to the mixture which was quickly mixed by inversion.The reference cuvette contained 2.5 mL of buffer, 0.3 mL of substrate (adrenaline) and 0.2 mL of water.The increase in absorbance at 480 nm was monitored every 30 s for 150 s.A unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of adrenaline to adrenochrome during 1 min.

2.12.2.Determination of catalase activity

Catalase activity was determined by following the decomposition of H2O2according to the method of Sinha [19].An aliquot (1 mL) of properly diluted enzyme preparation was rapidly mixed with an assay mixture containing 4 mL of H2O2(800 μmol) solution and 5 mL of 0.1 mol/L phosphate buffer(pH 7.0) in a conical flas by a gentle swirling motion.The reaction was run at room temperature.A 1 mL portion of the reaction mixture was withdrawn and blown into 2 mL dichromate/acetic acid reagent at 60 s intervals.The H2O2content of the withdrawn sample was determined by taking the absorbance at 570 nm.Catalase activity was expressed as μmol H2O2consumed/min/mg protein.

2.12.3.Estimation of reduced glutathione(GSH)level

Reduced glutathione (GSH) level was assayed by measuring the rate of formation of chromophoric product in a reaction between 5,5-dinitrobis-2-nitrobenzoic acid and free sulphydryl groups (such as GSH) as described by Beutler et al.[20].Tissue supernatant(1 mL)was precipitated with 5%sulphosalicylic acid(1.5 mL)by gentle mixing and allowed to stand for 5 min before filtering Thereafter,0.5 mL of filtrat was added to 2 mL of 0.1 mol/L phosphate buffer (pH 7.4) followed by Ellman’s reagent (0.25 mL).A blank was prepared with 2 mL of buffer,0.5 mL of diluted precipitating solution(three parts to two parts of distilled water)and 0.25 mL of Ellman’s reagent.The optical density was measured at 412 nm.GSH was proportional to theabsorbance at that wavelength and the estimate was obtained from a GSH standard curve.

Table 1 Phytochemical constituents of ethylacetate extract of Allium cepa tunic(ACTE).

2.13.Protein estimation

Protein concentration was determined by the method described by Lowry et al.[21] using bovine serum albumin(BSA)as standard.

2.14.Statistical analysis

All values are expressed as mean±SD of fie animals.Statistical evaluation was done using One Way Analysis of Variance(ANOVA) followed by Duncan Multiple Range Test.The significanc level was set atP＜0.05.

3.Res ults

3.1.Phytochemical constituents of ACTE

The phytochemical analysis of ACTE gave positive result to fl vonoids (425.6±5.92 mg quercetin equivalent/g extract)only, thus indicating the antioxidant phytochemicals are the major constituent of the extract(Table 1).

Fig.1.Representative high performance liquid chromatography profil of fl vonoids in ethylacetate extract of Allium cepa tunic.Rutin (peak 1), isoquercitrin(peak 2),quercitrin(peak 3),quercetin(peak 4)and kaempferol(peak 5).

Table 2 Flavonoid composition of ethylacetate extract of Allium cepa tunic(ACTE).

HPLC fingerprint of the fl vonoids in ACTE revealed that rutin (tR=34.79 min; peak 1), isoquercitrin (tR=39.13 min;peak 2), quercitrin (tR=45.13 min; peak 3), quercetin(tR=50.09 min; peak 4) and kaempferol (tR=52.68 min; peak 5) (Fig.1), are the active fl vonoids.As shown in Table 2,the concentrations (mg/g) of the fl vonols are as follows:rutin (94.31±0.02), isoquercitrin (120.68±0.03), quercitrin(51.27±0.03), quercetin (78.54±0.03) and kaempferol(20.93±0.01).

3.2.Effect on blood pressure parameters

The effect of ACTE on the hemodynamic parameters of rats treated with the extract for 14 days is depicted in Table 3.The hypotensive effect of the extract was significan(P＜0.01/P＜0.001) and dose dependent in experimental animals.Respectively, 10-, 20-, and 40 mg/kg caused significan(P＜0.05) decreases (%) in systolic blood pressure, SBP (21,27, 33), diastolic blood pressure, DBP (6, 10, 16), pulse pressure,PP(42,49,56),mean arterial blood pressure,MABP(13,18,23)and heart rate,HR(4,5,7)when compared with the control.The results suggest that the highest effica y was recorded for 40 mg/kg ACTE, which compares well with the reference drug, ramipril, on SBP (41), DBP (19), PP (70), MABP (29)and HR(10).

3.3.Influence on serum biochemical indices

The effect of ACTE on serum biochemical indices of normotensive rats is depicted in Table 4.Significan decrease(P＜0.001) in the activity of serum cardiac marker, creatine kinase, was noticeable in experimental rats administered 10- (52%), 20- (58%) and 40 mg/kg (60%) of ACTE or 1 mg/kg ramipril(65%).Serum activity of alanine aminotransferase (ALT) was decreased by treatment with 10-, 20 mg/kg ACTE (P＜0.01), 40 mg/kg ACTE (P＜0.001) and 1 mg/kg Ram(P＜0.001).Similarly,serum activity of aspartate aminotransferase (AST) was decreased by 40 mg/kg (P＜0.01) and 1 mg/kg Ram (P＜0.001).Significan (P＜0.001) lowering of alkaline phosphatase(ALP)activity was observed in rats administered all dosages of ACTE and 1 mg/kg Ram.It could also be observed that 40 mg/kg ACTE produced the highest decreases in the activities of serum ALT (18%) and ALP (28%).Concurrently, serum total cholesterol concentration was reducedby ACTE (10 mg/kg;P＜0.01 and 20 mg/kg;P＜0.001) and ramipril(1 mg/kg;P＜0.001).

Table 3 Effects of ethylacetate extract of Allium cepa tunic(ACTE)on blood pressure parameters of rats.

3.4.Effect on cardiac antioxidant status

Cardiac activities of superoxide dismutase and catalase were respectively increased(P＜0.001)in rats treated with 10 mg/kg(53%and56%),20 mg/kg(83%and150%)and40 mg/kg(111%and 197%) dosages of extract.Treatment with 1 mg/kg Ram also resulted in similar effects on cardiac activities of antioxidant enzymes.On the contrary, the level of GSH was reduced in experimental animals treated with all dosages of extract or ramipril(Fig.2).

4.Discussion

The result of phytochemical screening revealed the overwhelming presence of fl vonoids in the ethylacetate extract ofA.cepatunic(ACTE).The findin is in agreement with previous works[22,23]which showed that ethylacetate fractions contain high amount of phenolics/fl voniods, thus initiating their uses as fl vonoid-rich sources.Flavonoids have aroused considerable interest recently because of their potential beneficia effects on human health.In line with previous finding on onions[5,24],HPLC screening of fl vonoids in ACTE revealed the fl vonols:kaempferol, quercetin and quercetin glycosides (isoquercitrin,quercitrin and rutin)as the active fl vonoids.

In the present study,we observed marked decreases in the systolic and diastolic pressure,mean arterial blood pressure,pulse rate and heart rate of rats administered ACTE.The hypotensive effect of ACTE could be of therapeutic benefi in the event of clinical or drug-induced elevated blood pressure or hypertension[25]where reduction in blood pressure is highly desirable.The result was comparable to that recorded for the popular angiotensin-converting enzyme (ACE) inhibitor and hypotensive drug, ramipril.It might be impossible to categorically ascertain the mechanism underlying the hypotensive effect of ACTE from the data obtained in this study.It is however known that ACE inhibitors such as ramipril and catopril lower blood pressure and bring about fewer cardiovascular events in high risk populations by binding a zinc molecule at the active site of ACE,thereby slowing down the conversion of angiotensin I to the potent vasoconstrictor,angiotensin II[26].Quercetin and its glycosides are often the most abundant polyphenolic compounds found in the human diet,and richly found in the peel of onions [4,5,24].Several beneficia cardiovascular effects have been ascribed to this phytochemical, including antihypertensive effect and ability to improve endothelial function[27,28].Flavonoids are known to bind metal ions, such as zinc, and there is evidence that quercetin may inhibit ACE activity via this mechanism[29].Olaleye et al.[30]reported that rutin and quercetin reduced the blood pressure of animals induced with high salt diet.Overstimulation of the renin-angiotensin system(RAS),and by extension ACE,is one means by which high salt concentration induces hypertension [31].Going by the aforementioned, it is safe to hypothesize that ACTE could exert its hypotensive effect by interfering with ACE through one or more of its constituent fl vonoids.

In the present study, ACTE did not adversely affect serum total cholesterol and markers of tissue injury: creatine kinase CK, aspartate aminotransferase AST and alanineaminotransferase ALT.Serum CK and AST have been used as indicators of the stage of myocardial injury[13].The activities of these cellular enzymes present in the blood are directly related to the intactness of the plasma membrane of the cardiac cells.Therefore, the inhibition of the release of these enzymes into the serum by ACTE could be due to the ability of some of the fl vonoids present in the extract to maintain cardiac membrane integrity and restrict the leakage of these enzymes [32].Conversely,the ability of the ACTE to reduce serum cholesterol in treated animals portrays good signal for cardioprotection taken into consideration the role hyperlipidemia plays in the etiology and progression of many cardiovascular diseases[33,34].

Table 4 Effects of ethylacetate extract of Allium cepa tunic(ACTE)on serum biochemical indices of rats.

Fig.2.Effects of ethylacetate extract of Allium cepa tunic(ACTE)on cardiac activities of superoxide dismutase(A)and catalase(B),and level of reduced glutathione(C)in rats.P value:***＜0.001 compared with the normal control group.ACTE:ethylacetate extract of Allium cepa tunic;Ram:ramipril.

Superoxide dismutase (SOD) and catalase are endogenous antioxidant enzymes responsible for the dismutation of superoxide radicals to H2O2and detoxificatio of H2O2to water,respectively.Coupled with the actions of the master endogenous antioxidant molecule,GSH,these enzymes offer protection against tissue damage [35].In the present study, we observed an increase in the activities of cardiac SOD and catalase with concomitant decrease in GSH.The observed effect on GSH is a deviation from the well-established antioxidant activity of fl vonoids [36].It is however known thatin vivo, some fl vonoid metabolites exhibit prooxidative tendencies [37],some of which proceed via concentration-dependent stimulation of H2O2production [35,38].Such prooxidative tendency does not necessarily indicate toxicity in its entirety but could be beneficia and capable of causing overall cytoprotection [35],since levels of some antioxidant defenses and biotransformation enzymes might be elevated due to the mild degree of oxidative stress ensuing[39].Oxidative metabolites of fl vonoid such as quercetin are preferably inactivated by the major intracellular reducing agent GSH, to form two non-reactive products [40].The reduced level of GSH could thus be due to the modulation of several biological processes by fl vonoids[41].

5.Conclusion

The present study demonstrates the hypotensive effect of red onion(A.cepa)tunic which is often discarded as wastes.Further investigation is however in progress,in the case of this study,to ascertain the effect of chronic consumption of ACTE and evaluate the individual or synergistic contribution(s)of its fl vonoids to the observed hypotensive effect.