Food and Chemical Toxicology 71 (2014) 183–196

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Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate / foodchemtox

Synergistic effect of quercetin and quinic acid by alleviating structuraldegeneration in the liver, kidney and pancreas tissues of STZ-induceddiabetic rats: A mechanistic study

http://dx.doi.org/10.1016/j.fct.2014.06.0100278-6915/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +60 3 7967 5749; fax: +60 3 7967 4964.E-mail addresses: adityaarya18@gmail.com, aditya@um.edu.my (A. Arya).

Aditya Arya a,⇑, Mazen M Jamil Al-Obaidi b, Nayiar Shahid a, Mohamed Ibrahim Bin Noordin a,Chung Yeng Looi c, Won Fen Wong d, Si Lay Khaing e, Mohd Rais Mustafa c

a Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysiab Center of Studies for Periodontology, Faculty of Dentistry, University Technology MAR (UiTM), 40450 Shah Alam, Selangor, Malaysiac Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysiad Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysiae Department of Obstetrics and Gynaecology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

a r t i c l e i n f o

Article history:Received 24 April 2014Accepted 10 June 2014Available online 19 June 2014

Keywords:Synergistic effectQuercetinQuinic acidStreptozotocinDiabetes mellitus

a b s t r a c t

The aim of this study was to investigate the synergistic effects of quercetin (QE) and quinic acid (QA) on aSTZ-induced diabetic rat model to determine their potential role in alleviating diabetes and its associatedcomplications. In our study design, diabetic rats were treated with single and combined doses of QE andQA for 45 days to analyse their effects on liver, kidney and pancreas tissues. The study result showed thatQE and QA treated groups down-regulated hyperglycaemia and oxidative stress by up-regulating insulinand C-peptide levels. Moreover, histological observations of the liver, kidney and pancreas of diabetic ratstreated with single and combined doses of QE and QA showed a significant improvement in the structuraldegeneration. Interestingly, the combination dose of QE and QA (50 mg/kg) exhibited maximum inhibi-tion of the pro-apoptotic protein Bax expression and demonstrate enhancement of the anti-apoptoticprotein Bcl-2 expression in the kidney tissues, suggesting a protective role in the kidneys of diabetic rats.Taken together, these results indicates the synergistic effects of QE and QA in ameliorating hyperglyca-emia, hyperlipidemia and insulin resistance in diabetic rats and therefore, open a new window ofresearch on the combinatorial therapy of flavonoids.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The most common metabolic disorder caused by total or rela-tive insulin insufficiency is diabetes mellitus (DM), which is char-acterised by hyperglycaemia and impaired metabolism ofcarbohydrates, fats, and proteins (Pooja and Samanta, 2011). DMis characterised by high blood glucose levels, which result frominsulin resistance in peripheral tissues or impaired insulin synthe-sis in the pancreas (De Silva et al., 2012). High blood glucose is alsoassociated with the generation of reactive oxygen species and con-sequent oxidative damage in the liver, kidneys, and pancreas(Mohamed et al., 1999). According to the World Health Organisa-tion (WHO), DM is a disease with continuous increasing prevalence(WHO, 2006). This disease affects people worldwide, but the per-centages of diabetics in the global population are particularly high

in Asia and Europe (World Diabetes Foundation (WDF, 2010). Thus,the practice of herbal medicine for the treatment of DM is nearlyuniversal among non-industrialised societies (Singh et al., 2001).According to an estimation published by the WHO in 2008, approx-imately 80% of diabetic patients in the world’s populationpresently rely on herbal medicine for their successive treatments.

Flavonoids are ubiquitously present in common plant-basedfood items and beverages. The first therapeutically beneficial roleof dietary flavonoids was demonstrated by Rusznyak and Szent-Györgyi (1936). Medicinal plants may serve as a good source forthe design of drugs as anti-diabetic agents, and much attentionhas been paid to formulations of herbal medicine (Vishwakarmaet al., 2010). Potent antioxidant properties may be exhibited bymany flavonoids (Rice-Evans, 2001; Taha et al., 2014). In addition,the implication of oxidative stress in the pathogenesis of DM hasbeen proposed to not only generate oxygen free radicals but alsoalter antioxidant enzymes and the formation of lipid peroxides(Ceriello and Motz, 2004). Thus, defence mechanisms against free

184 A. Arya et al. / Food and Chemical Toxicology 71 (2014) 183–196

radicals, such as superoxide dismutase (SOD) and catalase (CAT),the activities of which contribute to the elimination of superoxide,hydrogen peroxide, and hydroxyl radicals, have been investigated.In addition, because DM is considered to be a free radical-mediateddisease, there has been renewed interest in the use of flavonoids,including the flavonol quercetin, in diabetes research (Sanderset al., 2001; Vessal et al., 2003).

Quercetin is a naturally occurring flavonoid that is found inonions and other vegetables and may demonstrate beneficialeffects on disease prevention. It has been reported that quercetinexerts protective effects against b-cell damage in diabetes and iscapable of decreasing serum glucose levels in diabetic rats(Coskun et al., 2005). It has also been reported that quercetin canprotect many types of tissue or organs, including the liver(Yokoyama et al., 2009), kidneys (Yousef et al., 2010) and pancre-atic gland (Coskun et al., 2005), against oxidative damage. Quinicacid derivatives, which are polyphenol-rich compounds, consistof a large family of esters formed between quinic acid and one ormore of several phenylpropanoic acids, such as caffeic acid, ferulicacid, coumaric acid, sinapic acid, and cinnamic acid (Lee et al.,2013). Quinic acid derivatives exhibit various beneficial effects,including antioxidant, anti-inflammatory, anti-HIV, anti-hepatitisB virus, hypoglycaemic, and hepatoprotective activities, as wellas the inhibition of mutagenesis and carcinogenesis (Farah andDonangelo, 2006; Gorzalczany et al., 2008).

However, there has been no further assessment of the anti-hyperglycaemic effect of the combined administration of quercetinand quinic acid on diabetic rats. Thus, this study aimed to investi-gate the potential mechanisms underlying the anti-hyperglycae-mic effect of these two compounds in an STZ-induced diabeticanimal model.

2. Materials and methods

2.1. Chemicals

Streptozotocin (STZ) was obtained from Sigma Aldrich (USA). Glibenclamidewas acquired from the University Malaya Medical Centre (UMMC). Quinic acidand quercetin were purchased from Sigma–Aldrich USA.

2.2. Animal stock

Healthy, mature Sprague Dawley (SD) male rats were provided by the Experi-mental Animal House of the Faculty of Medicine of University of Malaya (PM/27/7/2014/MMJA (R)). Rats weighing between 200 and 225 g were maintained inwire-bottomed cages at 25 ± 2 �C, administered tap water and a standard pelletdiet, and exposed to a 12-h light/12-h dark cycle at 50–60% humidity in an animalroom. Throughout the experiments, all of the animals received humane care accord-ing to the criteria outlined in the ‘‘Guide for the Care and Use of Laboratory Ani-mals,’’ which was prepared by the National Academy of Sciences and publishedby the National Institute of Health.

2.3. Diabetes mellitus induction by STZ

Diabetes was induced in rats by the intraperitoneal (i.p.) injection of freshlyprepared STZ at a dose of 45 mg/kg of body weight. Three days after the STZ injec-tion, the glucose level of blood collected from the tail vein was determined, andhyperglycaemic rats (blood glucose level >16.7 mmol/L) were considered diabeticrats in the subsequent experiments (Mitra et al., 1996).

2.4. Experimental design

The animals were randomly divided into nine groups containing six rats pergroup as follows:

Group 1: Normal control rats fed normal saline NaCl for 45 days: NC.Group 2: Diabetic control rats treated with saline solution for 45 days: DC.Group 3: Diabetic positive control rats treated with glibenclamide for45 days: PC.Group 4: Diabetic rats treated with quercetin (50 mg/kg/d) for 45 days: QEa.Group 5: Diabetic rats treated with quercetin (100 mg/kg/d) for 45 days:QEb.

Group 6: Diabetic rats treated with quinic acid (50 mg/kg/d) for 45 days:QAa.Group 7: Diabetic rats treated with quinic acid (100 mg/kg/d) for 45 days:QAb.Group 8: Diabetic rats treated with quercetin (50 mg/kg) + quinic acid(50 mg/kg) for 45 days: QEa + QAa.Group 9: Diabetic rats treated with quercetin (100 mg/kg) + quinic acid(100 mg/kg) for 45 days: QEb + QAb.

2.5. In vivo acute toxicity test

The acute toxicity test was performed according to the guidelines delineated bythe Organisation for Economic Co-operation and Development (OECD, 1998). Maleand female healthy adult Sprague Dawley rats were used in this study. The ratswere fasted overnight, divided into three groups (n = 6), and orally fed a combina-tion of quercetin and quinic acid at doses of 10, 100, and 500 mg/kg of body weight(bw). Quercetin and quinic acid were dissolved in distilled water and fed to the ani-mals; the control groups were administered distilled water only. The rats were con-tinuously observed during the first hour, every 1 h for the next 4 h, and every 24 hfor up to 14 days for any physical signs of toxicity, such as writhing, gasping, palpi-tation, decreased respiratory rate, and lethality.

2.6. Measurement of blood glucose

The weekly blood glucose levels of samples obtained from the tail vein of rats inall of the groups were measured using reagent strips (Accu-Check Active Glucosetest strips, Roche, Germany) with a glucometer (Accu-Check Active, Roche,Germany).

2.7. Biochemical parameters assessment

After 45 days of study period all the animals were sacrificed and blood was col-lected for the estimation of insulin, C-peptide, Glycated haemoglobin, Albumin,lipid profile, total antioxidant status (MDA, SOD and CAT). After blood collectionit was allowed to clot and centrifuged at 4000 c for 10 min and the serum obtainedwas used for the further measurement of all the markers.

The serum insulin and C-peptide levels were determined using an ELISA kit(eBio-311 science, San Diego, CA, USA) according to the manufacturer’s protocol.The serum glycated haemoglobin (HbA1c) and total protein levels were determinedas per the methods described by Nayak and Pattabiraman (1981) and Lowry et al.(1951) slight modification respectively. This method is based on the capacity ofthe protein–copper complex to reduce the Folin reagent, which induces blue colour-ation that can be measured at 650 nm. The albumin and lipids, i.e., total cholesterol(TC), high-density lipoprotein cholesterol (HDL-C), and low density lipoproteincholesterol (LDL-C), were measured in triplicate using an automatic biochemicalanalyser (Beckman-700, Fullerton, CA, USA).

2.8. Lipid peroxidation measurement

Serum lipid peroxidation was evaluated by measuring TBARS according to amodified method of Levine et al. (1990). Briefly, 0.2 mL of serum samples wereadded to the reaction mixture containing 1 mL of 1% orthophosphoric acid and0.25 mL alkaline solution of thiobarbituric acid (final volume 2 mL), followed by45 min heating at 95 �C. After cooling, samples and standards of malondialdehydewere read at 532 nm against the blank of the standard curve. The results wereexpressed as nmol MDA/mg protein.

2.9. Antioxidant enzymes (SOD) and (CAT) measurement

Serum SOD activity was estimated by Cayman’s Superoxide Dismutase AssayKit. Tetrazolium salt was used in this assay for the detection of superoxide radicalsgenerated by xanthine oxidase and hypoxanthine at 450 nm. SOD one unit isdefined as the amount of enzyme needed to exhibit 50% dismutation of the super-oxide radicals.

Cayman’s Catalase Assay Kit was used to measure serum CAT activity. Themethod is based on the reaction of the enzyme with methanol in the presence ofan optimal concentration of H2O2. The formaldehyde produced is measured with4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as the chromogen at540 nm.

2.10. Histopathological observation

At the end of experiment, the animals were sacrificed, and the pancreas, liver,and kidney were excised, washed with normal saline, and stored in 10% formalin.The tissue was washed, dehydrated with alcohol, and cleared with xylene, and par-affin blocks were prepared. Serial 5-lm-thick sections were cut using a rotarymicrotome. The sections were then deparaffinised with xylene and hydrated indescending gradients of alcohol. The slides were then transferred into haematoxylin

A. Arya et al. / Food and Chemical Toxicology 71 (2014) 183–196 185

for 10 min and then rinsed several times with water. These sections were examinedand subsequently counterstained with eosin, rinsed with water, dehydrated withascending gradients of alcohol, cleared with xylene, and mounted.

2.11. Immunohistochemical staining

The staining was performed as previously described (Sohn et al., 2007). The anti-bodies used in this study included rabbit anti-Bax (1:200, Abcam) and rabbit anti-Bcl-2 (1:250, Abcam). To detect Bax and Bcl-2, the kidney sections were incubatedwith the LSAB kit (DAKO K0609, CA, USA) and visualised with 3,30-diaminobenzidin-etetrahydrochloride. The negative controls for immunohistochemistry wereobtained by incubating the sections with non-immune serum instead of the primaryantibody. For morphometric analysis, the positive cell numbers and positive signalsfor each tissue of a total of 40 slides were determined using the Image J software(NIH, Bethesda, MD, USA).

2.12. Statistical analysis

The statistical analysis was performed using the SPSS software package (Version20). The data analyses were performed using one-way analysis of variance (ANOVA)followed by Tukey’s test. All of the results were expressed as the means ± SD fromthe results of six rats in each group. P-values less than 0.05 were consideredsignificant.

3. Results

3.1. Acute toxicity study

At the maximum and minimum doses of the individual andcombined treatments of QE and QA, no lethal or toxic reactionswere observed in the acute oral toxicity study (data not shown),which revealed their non-toxic nature.

3.2. Effects of different doses of quercetin and quinic acid on the bloodglucose levels of fasting diabetic rats

In Table 1, the blood glucose levels of the treated groups arecompared with those of the diabetic control group (P < 0.05), andthe findings reveal that the DC group showed a significantly higherglucose level compared with the NC group. According to the per-centage decrease in the blood glucose level after each treatmentdose shown in parentheses for week 6, the decrease in the glucoselevel due to treatment was less than half of that obtained with thecombined treatment. In addition, increasing the dose of QA and QEdid not result in a marked decrease in the blood glucose levelscompared with the combined treatment of QE + QA. Furthermore,all of the doses of QE and QA significantly decreased the blood glu-cose levels in diabetic rats (P < 0.05) from weeks 4 to 6. However,all of the doses of QEa + QAa produced a significant (P 6 0.05)decrease in the blood glucose level after week 3 and presentedthe highest reduction at week 6 compared with the DC and PCgroups.

Table 1Effects of quercetin and quinic acid on fasting blood glucose levels of diabetic rats.

Group Pretreatment Treatment weeks

Week 0 Week 1 Week 2 We

NC 5.93 ± 0.51 5.98 ± 0.42 5.74 ± 0.61 5.7DC 18.66 ± 1.51# 18.74 ± 2.23# 18.95 ± 1.92# 20.7PC 19.99 ± 0.95 18.48 ± 0.82 16.65 ± 0.56 15.5QEa 19.87 ± 1.13 19.55 ± 1.81 19.21 ± 1.44 19.1QEb 20.43 ± 1.65 18.69 ± 1.78 17.80 ± 1.44 17.4QAa 19.5 ± 1.39 18.71 ± 1.01 18.46 ± 0.97 18.2QAb 20.7 ± 1.41 19.45 ± 0.72 18.83 ± 0.48 17.9QEa + QAa 19.75 ± 2.09 18.71 ± 1.81 17.33 ± 1.47 15.7QEb + QAb 19.68 ± 1.62 19.28 ± 1.59 18.22 ± 1.49 16.4

Values are means ± SD of 6 rats from each group. A Mean values revealed by the Tukey* Significant difference compared to DC (P < 0.05).# Significant difference compared to NC (P < 0.05).

3.3. Effects of different doses of quercetin and quinic acid onbiochemical parameters

The serum insulin, C-peptide, albumin, and total protein levelsin the DC group were significantly declined (Fig. 1A–D), whereasthe HbA1c levels were significantly increased compared to theNC group after the six-week treatment period (Fig. 1E). The QEa,QEb, QAa, and QAb groups showed increases in the serum insulin,C-peptide, total protein, and albumin levels, and the QAa groupshowed the highest increase. Similarly, the QEa, QEb, QAa, andQAb groups showed decreases in the HbA1c levels, and the QAagroup showed the highest decrease. However, the diabetic rats inthe QEa + QAa group displayed a significant increase in the seruminsulin, C-peptide, total protein, and albumin levels, whereas theHbA1c levels significantly decreased after treatment with QEa +QAa compared with those observe in the DC rats and similar tothose observed in the PC group, indicating that the combinedadministration of both compounds to diabetic rats resulted in asignificant improvement in glycaemic control.

3.4. Effects of different doses of quercetin and quinic acid on lipidprofiles

As shown in Fig. 2, the effects of the individual and combinedtreatments of QE and QA on the serum TC (A), LDL-C (B), andHDL-C (C) activities in STZ diabetic rats and normal control ratswere observed. The serum TC and LDL-C levels in the DC groupwere significantly increased compared with those of the NC group(Figs. 2A and B), whereas the HDL-C levels in the DC group weresignificantly decreased compared with those of the NC group(Fig. 2C). Among the individual doses of QE and QA, QAa showedthe highest reduction in the serum TC and LDL-C levels and simi-larly reflected the highest increase in the HDL-C levels. However,there was a significant reduction in the serum TC and LDL-C levelsafter treatment in the PC and QEa + QAa groups, whereas theHDL-C levels were significantly elevated compared with those ofthe DC group after the six-week treatment period. Remarkably,continuous treatment with QEa + QAa decreased these lipidparameters in diabetic rats to near-normal levels.

3.5. Effects of different doses of quercetin and quinic acid on the serumMDA level and antioxidant enzymes

3.5.1. Serum lipoperoxidation (MDA) levelAs shown in Fig. 3A, DC significantly increased the serum MDA

levels (0.46 ± 0.02), and this effect was gradually recovered by anindividual or combined dose of QA and QE; however, the QAagroup presented the lowest MDA levels compared with the other

ek 3 Week 4 Week 5 Week 6

7 ± 0.58 5.67 ± 0.57 5.50 ± 0.38 5.77 ± 0.306 ± 1.10# 20.68 ± 0.80# 20.70 ± 0.70# 19.98 ± 0.45#

5 ± 0.41* 14.74 ± 0.28* 13.29 ± 0.40* 11.00 ± 0.36* (44.94)4 ± 1.69 18.27 ± 1.37* 17.57 ± 1.28* 16.94 ± 1.19* (15.22)5 ± 1.28* 17.03 ± 1.36* 16.75 ± 1.35* 16.51 ± 1.07* (17.37)6 ± 1.01* 17.95 ± 0.90* 17.44 ± 0.56* 16.85 ± 0.49* (15.67)9 ± 0.36* 17.47 ± 0.49* 17.04 ± 0.55* 16.61 ± 0.45* (16.87)6 ± 0.93* 14.21 ± 0.69* 12.99 ± 0.69* 11.36 ± 0.62* (43.14)9 ± 0.92* 15.47 ± 0.50* 14.44 ± 0.69* 13.23 ± 0.49* (33.78)

comparisons test (P < 0.05).

NC DC PC Q

EaQEb

QAa

QAb

QEa+QAa

QEb+QAb0

10

20

30

40

50*

* **

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*

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#

Insu

line

Leve

l uM

/L

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QAa

QAb

QEa+QAa

QEb+QAb0

100

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#

C-P

eptid

e Le

vel p

mol

/LNC DC PC

QEa

QEb Q

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10

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# *

Alb

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NC DC PC Q

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#

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l Pro

ten

gd/l

NC DC PC Q

EaQEb

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5

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15

*

* **

*

*

*

#

Gly

cate

d H

aem

oglo

bin

(HbA

1c) %

A B

C D

E

Fig. 1. Effects of quercetin and quinic acid with different doses on serum biochemical parameters of STZ diabetic rats in comparison with DC rats after the 6-week treatmentperiod. At the end of the treatment, rats were fasted for 12 h and blood was drawn to collect the serum. Panels indicate (A) insulin, (B) C-peptide, (C) albumin, (D) total proteinand (E) HbA1c levels. Data are presented as means ± SD (n = 6). (�) Significant difference compared to DC (P < 0.05). (#) Significant difference compared to NC (P < 0.05).

186 A. Arya et al. / Food and Chemical Toxicology 71 (2014) 183–196

individual-dose treatments. The combined treatment with QEa +QAa completely recovered the increase in the MDA levels(0.27 ± 0.01) (P < 0.05) compared with the DC group.

3.5.2. Serum antioxidant enzyme activitiesThe serum antioxidant enzyme activities (Figs. 3B and C) dem-

onstrated that the DC treatment significantly decreased the hepaticantioxidant enzyme activities, such as SOD and CAT (16.46 ± 0.80)(132.28 ± 1.66), compared with the NC group (P < 0.05). Treatmentwith QE and QA gradually increased the levels of these antioxi-dants, and the QAa group exhibited the highest increase amongthe individual treatment groups. However, QEa + QAa significantlyreversed the STZ-induced decrease in the SOD and CAT activities tolevels similar to those observed in the PC group (27.88 ± 0.82 and193.98 ± 3.50, respectively) compared with the DC group (P < 0.05).

3.6. Histopathological Observation

3.6.1. Histological observations of the liverAs shown in Fig. 4, rat liver sections were stained with haema-

toxylin and eosin, as previously described. The magnification was400� for all of the panels. Panel (A) shows that the NC group

exhibited a normal hepatic architecture with normal hepatocytemorphology, organised hepatic cell (Hc) cords, and a portal vein(Pv) with sinusoidal cords (S). Panel (B) shows that the DC treat-ment resulted in severe pathological changes, such as disorderedhepatic cords, focal necrosis, congestion in the portal vein, infiltra-tion of lymphocytes, increase in Kupffer cells (Kc), and dilatedsinusoids (S). Panel (C) shows the effect of the QEa treatment onthe liver of a diabetic rat, and the results show the attenuation ofSTZ-induced hepatic cellular necrosis and fibrotic changes in therat liver. Sinusoidal dilatation continued to be observed withdecreases in the number of Kupffer cells and RBC infiltration(Rc). Panel (D) shows the effects of QEb treatment on the liver ofa diabetic rat. The sinusoid remained dilated, and RBC was stillobserved. In addition, the central vein was presented in an orderlymanner. Panel (E) shows the effect of QAa treatment on the liver ofa diabetic rat, and the findings show moderate RBC congestion, butthe sinusoid was still dilated. Panel (F) displays the effect of QAbtreatment on the liver of a diabetic rat, and the degeneration ofthe hepatocytes and RBC remained obvious. Panel (G) shows theeffect of the QEa + QAa combined treatment on the diabetic ratliver. The hepatic architecture was similar to the normal hepaticarchitecture and was characterised by a normal central vein,

NC DC PC Q

EaQEb

QAa

QAb

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Tota

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erol

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LDL-

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HD

L-C

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Fig. 2. Effects of quercetin and quinic acid with different doses on serum lipidprofiles of STZ- induced diabetic rats with DC after the 6-week treatment period. Atthe end of the treatment, rats were fasted for 12 h and blood was drawn to collectthe serum. Figure represents TC (A), LDL-C (B), and HDL-C (C), levels in 9 groups.Data are presented as means ± SD (n = 6). (�) Significant difference compared to DC(P < 0.05). (#) Significant difference compared to NC (P < 0.05).

NC DC PC Q

EaQEb

QAa

QAb a

+QAa

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+QAa

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ivity

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+QAa

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250*

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m C

AT

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ivity

nmol

/min

/mL

A

B

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Fig. 3. Effects of QA & QE and combined administration of QA + QE in differentdoses on the serum MDA (A), SOD (B) and CAT (C) levels of in the STZ-induced rats.Each bar was represented as mean ± SD (n = 6). �P < 0.05, compared with DC. (#)Significant difference compared to NC (P< 0.05).

A. Arya et al. / Food and Chemical Toxicology 71 (2014) 183–196 187

normal hepatocytes, no fatty changes, and mild RBC infiltration.Panel (H) demonstrates the effect of the QEb + QAb combinedtreatment on the diabetic rat liver, which is characterised by asmall amount of inflammatory cell infiltration. Panel (I) shows thatthe GL in the PC group exerted a positive effect on the STZ-inducedpathological changes.

3.6.2. Histological observations of the pancreasAs shown in Fig. 5, the staining of the rat pancreas sections with

haematoxylin and eosin was performed as previously described inthe methods section. In panel (A), the NC pancreatic sectionshowed normal islets of Langerhans, a normal pancreatic structure,and normal acini tissues. Panel (B), which corresponds to the DCgroup, shows a disorganisation of the structure of the endocrineand exocrine cells with damaged and necrotic pancreatic aciniand islet shrinkage. In panel (C), the QEa treatment exertedminimal restoration of pancreatic endocrine cells and expansionof pancreatic islets, which showed a prominent hyperplastic islet.As shown in Panel (D), QEb treatment resulted in a disorganised

cell structure, and the degeneration of some of the pancreatic aciniwas still observed. Panel (E) shows the effect of QAa treatment onthe diabetic rat pancreas, which resulted in less change, a reduc-tion in vacuolation and lesions, and an expansion of pancreaticislets. As shown in Panel (F), the effect of QAb treatment on the dia-betic rat liver displays a lower restoration of pancreatic cells. InPanel (G), the effect of the QEa + QAa combined treatment on thediabetic rat liver demonstrates that these factors better promotecell survival, provide a more healthy structure, and present aprominent hyperplastic islet and improved islet morphology. Theeffect of the QEb + QAb treatment on the diabetic rat pancreas,which is shown in Panel (H), exhibits a moderate restoration ofpancreatic cells. As shown in Panel (I), the PC groups exhibitedthe most interesting aspect of the examined pancreatic sectionstreated with positive control drug, Glibenclamide which showednormal islets of Langerhans.

3.6.3. Histological observations of the kidneyAs shown in Fig. 6, the rat kidney sections were stained with

haematoxylin and eosin as described in the methods section. The

RBC Hc

Kc

S

Cv

S

HcPv

Kc

S

B

Hc

C

Cv

Hc

Kc

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D

Cv

Hc

S

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S

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RBC

F

G

Cv Cv

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HcHc

H

I

A

Fig. 4. H & E staining of different group of rats liver (400�). (A): NC (B) DC (C) QEa (D) QEb (E) QAa (F) QAb (G) QEa + QAa (H) QEb + QAb (I) PC. Panel (A) shows that NC had anormal hepatic architecture. Panel (B) shows that DC resulted in severe pathological changes, such as increase in Kuppfer cells (Kc), and dilation sinusoids (S). Panel (G) showsthe highest effect obtained by QEa + QAa treatment on the liver of diabetic rat. The hepatic architecture was similar to the normal hepatic architecture and was characterisedwith a normal central vein, normal hepatocytes, no fatty changes and mild RBC infiltrations, which were similar in the structure compare to positive control (I).

188 A. Arya et al. / Food and Chemical Toxicology 71 (2014) 183–196

magnification was 400� for all of the panels. As shown in Panel (A),the NC rat kidney presents a normal glomerulus surrounded byBowman’s capsule, proximal and distal convoluted tubules withoutany inflammatory changes, normal capillaries (CP), and normalpodocytes (Pc). Panel (B) shows the kidneys of the DC-treated rats,which present degenerated glomeruli infiltrated by inflammatorycells and a thickening of the basement membrane. The proximalconvoluted tubule exhibited oedematous changes with a deposi-tion of mucopolysaccharide and hyaline substances. Furthermore,it exhibited severe pathological damages, such as mesangial

expansion and glomerular hypertrophy (Gh). Panel (C) shows theeffect of QEa treatment, and the findings present features of heal-ing, i.e., less expansion of the glomerulus, the presence of fewinflammatory cells, mildly dilated capillaries, and decreased muco-polysaccharide and hyaline deposits. Panel (D) shows that the QEbtreatment results in less changed morphology and a decreasedexpansion of the glomerular and mesangial matrix. Panel (E) showsthe effect of QAa treatment on the diabetic rat liver with moderatechanges in the kidney. As shown in Panel (G), the analysis of therenal histology after the QEa + QAa combined treatment showed

Fig. 5. H & E staining of different group of rats pancreas (400�). Panel (A): NC (B) DC (C) QEa (D) QEb (E) QAa (F) QAb (G) QEa + QAa (H) QEb + QAb (I) PC. In panel (A), the NCpancreatic section showed normal islets of Langerhans. Panel (B) DC shows a disorganisation of the structure of the endocrine and exocrine cells with damaged and necroticpancreatic acini and islet shrinkage. In Panel (G), the effect of QEa + QAa on diabetic rats liver demonstrates that these factors better promoted the cells and provides a morehealthy structure and shows a prominent hyperplastic islet and improved islet morphology. Treatment of QEb + QAb in Panel (H) exhibited moderate restoration of pancreaticcells on the diabetic rat. The treatment of positive control (Glibenclamide) in Panel (I), demonstrated the most interesting aspect of the examined pancreatic sections, whichshowed normal islets of Langerhans.

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Bc

Pc

Gh

Bc

Pc

Gh

Cp

Gh

Pc

Gh

Cp

Pc

Cp

Gh

Pc Bc

Mc

E F

A B

C D

CpGh

Pc

Gh

G H

I

Gh

Fig. 6. H & E staining of different group of rats kidney (400�). Panel (A): NC (B) DC (C) QEa (D) QEb (E) QAa (F) QAb (G) QEa + QAa (H) QEb + QAb (I) PC. In diabetic rats (B),there is a significant damage to the glomeruli (hypertrophy, hypercellularity, tubules dilatation and atrophy) compared to the normal control (A). Treatment of QE and QAattenuated the kidney alteration (C,D,E,F,G,H) in dibetic rats, which are comparable with the positive control (I) on the STZ-induced pathological changes.

190 A. Arya et al. / Food and Chemical Toxicology 71 (2014) 183–196

no clear histopathological abnormalities and a decreased expan-sion of the glomerular and mesangial matrix. Panel (I) shows the

PC group. These broad changes caused the loading of the Bowman’scapsule space and an adhesion of the capillaries to the wall.

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3.7. Immunohistochemistry evaluation

3.7.1. Effect of quercetin and quinic acid on the Bcl-2 expression levelin the renal glomeruli

The Bcl-2 protein expression in the kidney tissues was qualita-tively analysed using immunohistochemistry, as shown in Fig. 7A,and the findings show that the level of Bcl-2 protein expressionwas markedly reduced in the DC group compared with the NCgroup. However, this decrease in the Bcl-2 level was graduallyrecovered after the administration of different doses of QE andQA. The arrows indicating QAa and QAb show an increase inprotein expression compared with QEa or QEb. However, theQEa + QAa combined treatment (Panel G) exhibited a significantincrease in Bcl-2 protein expression compared with the DC andPC groups (Panel I). The histological results were confirmed usinga quantitative and histomorphometry study, as shown in Fig. 7B,which shows that the DC groups (0.002 ± 0.001) exhibits a signifi-cant decrease in Bcl-2 expression compared with the NC group(0.035 ± 0.001). However, different doses of QE and QA signifi-cantly enhanced the expression of Bcl2 compared with the DCgroup (P < 0.05), and the QEa + QAa group displayed the highestincrease in the expression level of this protein (0.032 ± 0.001).

3.7.2. Effect of quercetin and quinic acid on Bax expression level inrenal glomeruli

The Bax protein expression in the kidney tissues was qualita-tively analysed using immunohistochemistry. As shown inFig. 8(A), the level of Bax protein expression was increased in theDC group compared with the NC group, and this increase was grad-ually recovered with different individual doses of QE and QA. Thecomparison of the individual treatment groups revealed that theQAa and QAb groups showed a higher reduction in the Bax expres-sion levels compared with the QEa and QEb groups, as shown bythe two arrows in each panel. No arrows are shown in Fig. 8(A)G, which indicates that the QEa + QAa treatment significantlydecreased Bax protein expression compared with the DC and PCgroups. These histological results were confirmed using a histo-morphometry study, which revealed a significant increase in Baxexpression in the DC group (0.041 ± 0.001) compared with theNC group (0.001 ± 0.001) (Fig. 8B). Furthermore, different dosesof QE and QA can significantly inhibit the Bax expression levelcompared with the DC treatment. However, Bax expression wasmore significantly reduced by the combined administration ofQEa + QAa (0.002 ± 0.001) compared with the DC treatment.

4. Discussion

Flavonoids are a group of naturally occurring compounds thatare widely distributed as secondary metabolites in the plantkingdom. It has been demonstrated that these compounds haveinteresting clinical properties, such as anti-inflammatory, anti-allergic, antiviral, antibacterial, and antitumoural activities(Middleton, 1998). One of these flavonoids, namely quercetin(3,5,7,3,4-pentahy-droxyflavone), can prevent oxidant injury andcell death by several mechanisms, such as the scavenging of oxygenradicals (Inal et al., 2002), which protect against lipid peroxidation(Laughton et al., 1991). Furthermore, quercetin has been proven tobe a potent anti-diabetic agent (Mahesh and Menon, 2004) withan antioxidant and anti-inflammatory profile (Bureau et al., 2008).Another polyphenol-rich compound is quinic acid, which has beenpreviously reported to be an active compound with significant anti-oxidant and anti-hyperglycaemic properties (Pero et al., 2009). Inaddition, this compound is capable of reducing the oxidative stressburden in cells (Saltiel and Kahn, 2001). Because there is no scien-tific evidence of the combined effect of quercetin and quinic acid

on experimental diabetic rats, this study aimed to investigate thesynergistic anti-hyperglycaemic activity of these two flavonoidson STZ-induced diabetic rats.

Our study demonstrated no signs of toxicity with QE or QA. TheNational Toxicology Program performed a renal toxicity study ofquercetin and found an increase in the severity of chronic nephrop-athy, hyperplasia, and neoplasia of the renal epithelium in malerats treated with 2000 mg/kg of body weight, but no adverseeffects were observed with lower doses of 50 and 500 mg/kg ofbody weight (Aguirre et al., 2011). According to our results, thecombined administration of QEa + QAa suggested a markedimprovement in hyperglycaemia and hyperlipidemia after fourweeks of administration. The decrease in the elevated blood glu-cose in the DC group and the normalisation of the glucose levelsin the QEa + QAa group are shown in Table 1, and these resultsmay be due to the stimulatory activity of quercetin and quinic acid,both of which have nearly no effect on the glycaemic status andoxidative stress markers in normal rats but have significant effectson the glucose level parameters in STZ-treated rats (Arya et al.,2012a; Prince and Kamalakkannan, 2006). The hypoglycaemiceffect of both compounds may be due to their antioxidant proper-ties (Sanders et al., 2001; Pero et al., 2009; Arya et al., 2012b; Tahaet al., 2014).

Until recently, insulin was the sole pancreatic b-cell hormonethat is known to lower blood glucose levels (Brezar et al., 2011).Healthy individuals sustain stable blood glucose levels throughbasal insulin secretion (Raghavan and Garber, 2008), but hyper-glycaemia in diabetes is the main symptom caused by a lack ofinsulin and/or insulin resistance (Ke et al., 2009). Consistent withthese findings, we showed reduced insulin levels in the DC group,but this reduction was restored by treatment with QEa + QAa to alevel similar to that observed in the PC group (Fig. 1A). In addition,Vessal et al. (2003) suggested that quercetin supplementation isbeneficial for decreasing the blood glucose concentration, therebypromoting the regeneration of pancreatic islets and increasinginsulin release in STZ-induced diabetic rats and thus exerting itsbeneficial anti-diabetic effects. The most reliable primary outcomeof the investigation of diabetes treatment is the C-peptide levels,which further validated the assessment of b-cell function andaimed to preserve or improve endogenous insulin secretion(Palmer et al., 2004). The serum C-peptide levels were significantlyincreased in the QEa + QAa group, which is was consistent with theresults reported by Taha et al. (2014), and may be due to theincreased secretion of insulin from remnant b-cells (Fig. 1B).

As shown in Fig. 1C, the level of albumin was markedly reducedin the DC group, which may be due to impaired tubular reabsorp-tion or leakage of albumin due to glomerular basement membranedamage combined with an increase in transglomerular filtrationpressure. However, the QEa + QAa combined treatment signifi-cantly increased the serum albumin towards the NC and PC levels,which was reflected in the reduction of kidney damage due to STZ-induced hyperglycaemia. Consistent with our results, Kandasamyand Ashokkumar (2012) concluded that flavonoids restore thereduced albumin level in diabetic nephrotoxic rats. Thus, QE andQA, particularly at a dose of 50 mg/kg, demonstrate a beneficialpharmacological effect on diabetic, hepatic, renal functionalmarkers and protein levels. Persistent hyperglycaemia results inthe glycation of Hb, which results in the formation of HbA1C(Yabe-Nishimura, 1998). Furthermore, the QEa + QAa combinedtreatment significantly decreased the levels of HbA1c andincreased the level of serum insulin in experimental diabetic rats.Agents with antioxidant or free radical-scavenging properties havebeen shown to inhibit oxidative reactions associated with glyca-tion (Elgawish et al., 1996). Hii and Howell (1984) reported thatthe exposure of isolated rat islets to specific flavonoids, such asquercetin, enhances insulin release by 44–70%.

E F

A B

C D

G H

I

NC DC PC Q

EaQEb

QAa

QAb a

+QAa

QE

b

+QAb

QE

0.000

0.010

0.020

0.030

0.040

*

* **

*

*

*

#

Kid

ney

Bcl

2 (D

OI)

A

B

Fig. 7. (A) Effect of quercetin and quinic acid on Bcl-2 expression level in the diabetic renal rat. Immunohistochemical staining (400�). Panel (A): NC (B) DC (C) QEa (D) QEb(E) QAa (F) QAb (G) QEa + QAa (H) QEb + QAb (I) PC (Bcl-2 expression shown with arrows). (B) Representative immunohistochemical staining and quantitative analysis ofdifferent doses of quercetin and quinic acid treatment on Bcl-2 expression showed in kidney of diabetic rats. Data were analysed by Image analysis software as mean ± SD byone-way ANOVA (Tukey), (�) indicated P < 0.05 different from DC (n = 6 per group). (#) Significant difference compared to NC (P < 0.05).

192 A. Arya et al. / Food and Chemical Toxicology 71 (2014) 183–196

E F

A B

C D

G H

I

NC DC PC Q

EaQEb

QAa

QAb a

+QAa

QE

b

+QAb

QE

0.000

0.010

0.020

0.030

0.040

0.050

*

* **

*

**

#

Kid

ney

Bax

(DO

I)

A

B

Fig. 8. (A) Effect of quercetin and quinic acid on Bax expression level in the diabetic renal rat. Immunohistochemical staining (400�). Panel (A): NC (B) DC (C) QEa (D) QEb (E)QAa (F) QAb (G) QEa + QAa (H) QEb + QAb (I) PC. (Bax expression shown with arrows). (B) Representative immunohistochemical staining and quantitative analysis of differentdoses of quercetin and quinic acid treatment on Bax (B) expression showed in kidney diabetic rats. Data were analysed by Image analysis software as mean ± SD by one-wayANOVA (Tukey), (�) indicated P < 0.05 different from DC (n = 6 per group). (#) Significant difference compared to NC (P < 0.05).

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194 A. Arya et al. / Food and Chemical Toxicology 71 (2014) 183–196

According to the data shown in Fig. 2A and B), the levels ofserum TC and LDL-C were significantly elevated in the DC group,whereas the levels of HDL-C were decreased (Fig. 2C). Thus, thesuccessive combined administration of QEa + QAa produced anobvious reduction in the serum TC and LDL-C levels and anincrease in HDL-C, which is consistent with the findings reportedby Kandhare et al. (2012). Furthermore, the flavonoid-richcompound quercetin exerted a significant reduction in the serumcholesterol and LDL concentrations in alloxan-diabetic rats(Nuraliev and Avezov, 1991). In addition, the QEa + QAa combinedtreatment attenuated the disorder of lipid metabolism, similarly tothe effect observed in the study conducted by Wang et al. (2012).

It has been pre-clinically and clinically proven that the level oflipid peroxidation is significantly increased in the plasma of dia-betic rats and diabetic patients (Sato et al., 1979). Furthermore,lipid peroxidation, which is a free radical-mediated oxidation ofpolyunsaturated fatty acids, is increased in the DC group, as isevident from the elevated MDA levels. The QEa + QAa combinedtreatment effectively reverts the serum MDA to normal levels(Fig. 3A), suggesting the antioxidant functions of the combinationof these flavonoids, which is comparable to the findings reportedby Machha et al. (2007). In addition, the imbalance between theformation of reactive oxygen species, i.e., free radicals, and theendogenous antioxidant enzymes results in the generation of oxi-dative stress (Cotter and Cameron, 2003). SOD is responsible forthe conversion of superoxide anions to H2O2 and thus reduces oxi-dative stress. In our results, the QEa + QAa combined treatmentefficiently restored the depleted status of the antioxidant enzymesSOD and CAT (Fig. 3B and C), indicating the combined antioxidativeproperties of the two polyphenols. The results of the present inves-tigation are consistent with the findings reported by Arya et al.(2012c) and Dias et al. (2005).

In our histopathology study, the DC group showed many path-ological changes, such as degenerated hepatocytes with polymor-phic nuclei, focal necrosis, congestion in the portal vein,infiltration of lymphocytes, increases in Kupffer cells, dilated sinu-soids, and mononuclear cell infiltrates extending throughout thehepatic tissue, which is similar to the effects observed in previousdiabetic animal models (Motshakeri et al., 2014). The unorganisedhepatocytes, microvesicular vacuolisation, granular degeneration,and necrosis are marked changes observed in the diabetic liver(Zhou et al., 2008). Previous histological studies have shown thatquercetin rescued the liver damage of diabetic rats by increasingthe activity of antioxidant enzymes and by scavenging hydroxyl,superoxide, alkoxyl, and peroxyl radicals (Kandasamy andAshokkumar, 2012). Consistent with our findings, the severity ofhepatic injuries were reduced with the QA and QE individual treat-ments and with the QEa + QAa combined treatment; no consider-able fatty changes were observed, indicating the protective effectof these two flavonoids against the hepatic complications of diabe-tes, as shown in Fig. 4.

Roat et al. (2014) suggested that any alteration to the structure,size, and function of islets indicates metabolic changes related toinsulin secretion, insulin sensitivity, and loss of glycaemic control.In the study conducted by Motshakeri et al. (2014), theSTZ-induced diabetic SD rats presented small islets, includingdegenerated cells and reduced b cells, which is consistent withthe findings obtained by Roat et al. (2014). Similarly, the pancreastissues of diabetic rats revealed a reduction in the number of islets,degeneration of b cells, hydropic degeneration, clumping of b cells,pyknosis, and necrosis, which caused a change in the morphologyof the cells due to the STZ-induced partial damage of some b cells(Fig. 5). In the present study, the islet cells were restored by QE andQA, which recovered the degenerated cells. This result was consis-tent with those obtained by Rifaai, who reported that quercetinimproves degenerated cells with no inflammatory cell infiltration

(Rifaai et al., 2012). However, our studies showed that the admin-istration of an increased individual dose of QEb and QAb to diabeticanimal may cause reversible effects, unlike the combined adminis-tration of QEa + QAa, suggesting that there were more functionalcells in the QEa + QAa group compared with the individual treat-ment groups the group with the higher combination dose (QEb +QAb). This finding may explain the higher level of insulin thatwas found (Ong et al., 2011). In addition, the QE and QA treatmentsrestore the pancreas histology by alleviating oxidative stress,which is in tune with the results reported by Fernandez-Alvarezet al. (2004).

One of the major complications associated with diabetes is dia-betic nephropathy (DN), which results from the expansion of mes-angial cells, a hallmark of diabetic rats (Matsubara et al., 2006), theaccumulation of extracellular matrix protein, the thickening of theglomerular and tubular basement membranes, tubulointerstitialfibrosis, glomerulosclerosis, renal endothelial dysfunction, albu-minuria, proteinuria, and a reduction in the glomerular filtrationrate (Balakumar et al., 2009). In Fig. 6, the DC group showed acuteswelling of cells, hydropic degeneration of tubules, widening ofBowman’s space, glomerular atrophy, congestion of capillaries,and tubular necrosis. The severe pathological changes observedinvolved focal mesangial matrix expansion, proteinuria, and a sig-nificant increase in albuminuria compared with the NC group. Areduced degree of expansion and degeneration was observed fromPanel C to H compared to Panel I (the QE and QA treatments),which is consistent with the results obtained by Bashir et al.(2014), who showed that the quercetin-treated diabetic rats dem-onstrated a recovery of the normal kidney structure with intactglomerular epithelial cells and tubules. However, the QEa + QAacombined treatment ameliorated the mesangial expansion, pro-teinuria, and albuminuria, and this dosage combination was themost beneficial dose for alleviating histological injuries comparedwith the other doses.

Cell apoptosis is directly affected by multiple genes inside cells.Among these genes, the most representative are the pro-apoptotic(Bax) and anti-apoptotic (Bcl-2) genes (Lian et al., 2011). In thisstudy, we found that the QEa + QAa combined treatment amelio-rated the increased pro-apoptotic Bax protein level and thedecreased Bcl-2 protein level in the accumulated glomeruli ofSTZ-induced diabetic rats. Bcl-2 proteins play an important rolein b-cell physiology and apoptosis due to their anti-apoptotic prop-erties. Thus, the presence of these genes can result in the increasedsurvival of b cells (Gurzov and Eizirik, 2011). According to Li et al.(2005), an upregulated expression of Bcl-2 proteins is induced bytreatment with high glucose, which suppresses apoptosis inVSMCs. Diabetes is the result of reduced insulin secretion by b cellsand thus reduced expression of Bcl-2 genes, as observed in the DCgroup. The decreased Bcl-2 expression was completely restored bythe QEa + QAa combined treatment, which is comparable to theresults obtained by Gurzov and Eizirik (2011). However, Bax ispro-apoptotic, which indicates that it promotes the apoptosis ofb cells and exhibits increased expression in diabetes, but itsexpression is downregulated by treatment with QEa + QAa. Ourresults, which were confirmed through a quantitative analysis(Fig. 8A and B), were consistent with previous reports that showedthat diabetic kidney cells undergo apoptosis both in vitro andin vivo via the activation of Bax protein and downstream targets,which support the treatment of diabetes via the prevention ofapoptosis (Eslami and Sharifi, 2012). It has been reported thatthe QEa + QAa combined treatment demonstrates a protectiveeffect against cell apoptosis (Sohn et al., 2010). The accumulationof QEa + QAa in the kidney and their anti-apoptotic effects onpodocytes may ameliorate diabetic effects. As a result, the com-bined flavonoid approach is an effective treatment for diabetes inthe kidney and may be a valuable therapeutic approach.

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Taken together, our study revealed the hypoglycaemic activityof quercetin and quinic acid, but the precise mechanism underly-ing this phenomenon remains unclear. However, it can be assumedthat the two promising phytochemicals, quercetin and quinic acid,protect pancreatic b cells due to their antioxidant properties, asevidenced by an increase in antioxidant status and a decrease inlipid peroxidation, which resulted in insulin release in thediabetic-treated rats and may reduce the risk of diabetic complica-tions. Similar results have been reported with flavonoids, such asquercetin and rutin, which showed nearly no effect on the glycae-mic status and oxidative stress markers in normal rats but havesignificant effects on antioxidant parameters in STZ-treated rats(Prince and Kamalakkannan, 2006). However, our results for thecombined low dose of quercetin and quinic acid showed themaximum protective effect compared with the individual dosetreatments and the higher-dose combination therapy.

5. Conclusions

In summary, the combined administration of QEa + QAa exertedan anti-hyperglycaemic effect in a STZ-induced diabetic rat model.Consistent with previous studies describing increased insulinsecretion by flavonoids, we propose that the increase in insulin lev-els induced by treatment with QEa + QAa is due to the protectiveeffect of quercetin and quinic acid on the liver, kidney, and pancre-atic b-cells. Furthermore, the QEa + QAa combined treatment alsoshowed a positive modulation of the lipid profile, antioxidants,and Bcl-2 defence. Taken together, these biological effects maybe attributed to the combinatorial effects of both the compounds.Thus, further studies are warranted to investigate the deep mech-anism and synergistic effects of the flavonoids to create possibility.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

Acknowledgements

This research is financially supported by the University ofMalaya research grant; HIR: (H-20001-E00002) and UMRG:RP001D-13BIO & RG360/11HTM. The funding sources were notinvolved in the study design, collection, analysis, interpretationof data, writing of the report or the decision to submit the articlefor publication.

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