Protective role of polyphenols from Bauhinia hookeri against carbon tetrachloride-induced hepato-and...

http://informahealthcare.com/rnfISSN: 0886-022X (print), 1525-6049 (electronic)

Ren Fail, Early Online: 1–10! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2015.1061886

LABORATORY STUDY

Protective role of polyphenols from Bauhinia hookeri against carbontetrachloride-induced hepato- and nephrotoxicity in mice

Eman Al-Sayed1*, Mohamed M. Abdel-Daim2*, Omnia E. Kilany3, Maarit Karonen4, and Jari Sinkkonen4

1Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt, 2Department of Pharmacoloy, Faculty of Veterinary

Medicine, Suez Canal University, Ismailia, Egypt, 3Department of Clinical Pathology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia,

Egypt, and 4Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, Turku, Finland

Abstract

The hepatoprotective and nephroprotective activity of a polyphenol-rich fraction (BHPF)obtained from Bauhinia hookeri was investigated against CCl4-induced acute hepatorenaltoxicity in mice. BHPF was administered (100, 200 and 400 mg/kg/day) for 5 days, then CCl4 wasadministered. BHPF pretreatment significantly (p50.001) inhibited the CCl4-induced increase inALT, AST, ALP, LDH, total bilirubin, cholesterol, creatinine, uric acid, urea and malondialdehydein a dose-dependent manner. In contrast, BHPF pretreatment markedly increased the contentsof glutathione and superoxide dismutase in the liver and kidney tissues, indicating the strong invivo antioxidant activity of BHPF. Pretreatment with BHPF preserved the hepatic architectureand conferred marked protection against necrosis and ballooning degeneration. Pretreatmentwith BHPF reduced the inflammatory cell aggregation and degenerative changes in the liningepithelium of the kidney tubules. It can be concluded that BHPF has a remarkable hepato- andnephroprotective activity by enhancing the antioxidant defense status, reducing lipidperoxidation and protecting against the histopathological changes induced by CCl4 in theliver and kidney tissues.

Keywords

Antioxidant, epicatechin gallate, HPLC-PDA-ESI/MS/MS, hepatoprotective, kidney, liver,nephroprotective, proanthocyanidins

History

Received 16 January 2015Revised 17 May 2015Accepted 31 May 2015Published online 6 July 2015

Introduction

The kidney is the main organ involved in the excretion of

xenobiotics.1,2 The liver is the primary organ of metabolism;

therefore, the toxic effects of chemicals usually appear

primarily in the liver and kidney tissues.3,4 Oxidative stress

has a crucial role in many diseases including renal failure,

liver damage, atherosclerosis, inflammation and carcinogen-

esis.5,6 Oxidative stress is caused by cellular excess of

reactive oxygen species (ROS). Lipid peroxidation of cell

membranes induces cell injury.7–9 Under normal homeostasis,

the cells maintain the ROS levels with endogenous non-

enzymatic and enzymatic-antioxidants.10–12 The risk of cell

injury may also be prevented by natural antioxidants

including dietary polyphenols. Dietary polyphenolic com-

pounds have received a great deal of attention because of their

beneficial effects on health, including protection against

oxidative stress and degenerative diseases.13 Dietary poly-

phenolic compounds are known to restore the balance between

the natural antioxidants and free radicals by direct scavenging

of ROS and by enhancing the activity of natural antioxidant

enzymes.13 Proanthocyanidins are naturally occurring antioxi-

dants that are abundant in many plant foods such as fruits,

vegetables and legumes.14 Proanthocyanidins exert a wide

spectrum of pharmacological and therapeutic activities against

oxidative stress. Grape seed proanthocyanidin extract (GSPE)

is marked as a dietary supplement due to its health benefits,

including hepatoprotective, cardioprotective, anti-fibrogenic

and chemopreventive activities.15

The genus Bauhinia (family Fabaceae) comprises 300

species, and is commonly known as ‘‘cow’s paw’’ tree,

because of the shape of their leaves.16 Different species are

used in folk medicine worldwide.16 Diverse range of

pharmacological activities has been reported for many

Bauhinia species, including hepatoprotective, antioxidant,

anti-inflammatory and anti-hyperlipoidemic effects.17,18

Bauhinia hookeri F. Mull, is a small ornamental tree native

to Australia.19 The current study was designed to investigate

the possible mechanisms of the hepato- and nephroprotective

action of a proanthocyanidin-rich fraction obtained from

B. hookeri (BHPF) against the acute toxicity of CCl4 in mice.

*These authors contributed equally to this article.

Address correspondence to Mohamed M. Abdel-Daim, Department ofPharmacology, Faculty of Veterinary Medicine, Suez Canal University,Ismailia 41522, Egypt. Tel/Fax: + 20 643207052; Mobile: + 20107761570; E-mail: abdeldaim.m@vet.suez.edu.eg; abdeldaim.m@gmail.comEman Al-Sayed, Department of Pharmacognosy, Faculty of Pharmacy,Ain-Shams University, 11566 Cairo, Egypt. Tel: +20 2 01001050293;Fax: +20 2 405 1106; E-mail: em_alsayed@pharma.asu.edu.eg;emanalsayedasu@gmail.com

Hepatoprotection was determined by assaying the levels of

alanine aminotransferase (ALT), aspartate aminotransferase

(AST), alkaline phosphatase (ALP), lactate dehydrogenase

(LDH), bilirubin, cholesterol and protein in the serum of

control and treated mice in an acute model of CCl4 intoxica-

tion. Nephroprotection was determined by estimating the

serum level of uric acid, urea and creatinine. The lipid

peroxidation and antioxidant parameters [glutathione (GSH)

and superoxide dismutase (SOD)] were estimated in the liver

and kidney homogenates to determine the possible mechanisms

of the hepato- and nephroprotective activity. A histopatho-

logical examination of liver and kidney sections was conducted

to confirm the hepato- and nephroprotective effects. The

identification of B. hookeri polyphenols was achieved utilizing

the high-performance liquid chromatography coupled with

diode array detection-electrospray ionization tandem mass

spectrometry (HPLC-PDA-ESI/MS/MS) technique.

Material and methods

Chemicals

Carbon tetrachloride (CCl4) was obtained from El Nasr

Pharmaceutical & Chemical Company (Cairo, Egypt).

Silymarin was purchased from Sedico Pharmaceutical

Company (6th October City, Egypt). All the assay kits were

purchased from Biodiagnostics Company (Cairo, Egypt). The

assay kit of LDH was purchased from Randox Laboratories Ltd

(Crumlin, UK). All other chemicals were of analytical grade.

LC–MS grade solvents were used in HPLC-PDA-ESI/MS/MS

analysis. Sephadex LH-20 was obtained from Amersham

Biosciences (Uppsala, Sweden), RP-C18 from Sigma-Aldrich

GmbH (Darmstadt, Germany) and pre-coated silica gel TLC

GF254 was obtained from Riedel-De Haen-AG (Seelze,

Germany).

Plant material

The leaves of B. hookeri were collected in July 2011 from the

botanical garden of the Faculty of Agriculture, Cairo

University, Cairo, Egypt. The plant was botanically identified

by Eng. Therese Labib, the taxonomy specialist at the

herbarium of El-Orman Botanical Garden, Giza, Egypt. A

voucher specimen of B. hookeri was deposited at the herbarium

of the Department of Pharmacognosy, Faculty of Pharmacy,

Ain Shams University, Cairo, Egypt (ASU BHF2011).

Extract preparation and fractionation

The air-dried powdered leaves of B. hookeri (1700 g) were

extracted three times with 80% aqueous ethanol (EtOH). The

total extract was concentrated and freeze-dried to obtain a dry

powder, which was dissolved in absolute EtOH. The EtOH-

soluble portion was concentrated and freeze-dried to obtain a

dry powder (140 g). Column fractionation of part of the

extract (100 g) was performed using Sephadex LH-20

(5� 100 cm), eluted with H2O followed by H2O–MeOH

mixtures of decreasing polarities. Fractions are combined

based on their analytical HPLC profiles to afford eight major

fractions. The fraction eluted with 60% aqueous methanol

(MeOH) was concentrated and freeze-dried to obtain a dry

powder of BHPF (3 g).

HPLC-PDA-ESI/MS/MS method

Part of the fraction was dissolved in 20% MeOH (20 mg/mL)

and the solution was filtered through 0.2 lm PTFE

membranes. LC–HRESIMS was performed on a Bruker

micrOTOF-Q quadrupole time-of-flight mass spectrometer

(Bremen, Germany), coupled to a 1200 series HPLC system

(Agilent Technologies, Waldbronn, Germany), equipped

with an auto sampler, a binary pump and a diode-array

detector. Chromatographic separation was performed on an

XBridge C18 (2.1 mm� 100 mm; 3.5 lm) column (Waters,

Dublin, Ireland). The mobile phase consisted of acetonitrile

(A) and 0.1% formic acid (B). The elution profile was

0–3 min, 100% B (isocratic); 3–30 min, 0–30% A in B;

30–45 min, 30–70% A in B; 45–55, 70% A in B (isocratic)

with a flow rate of 0.2 mL/min. The HPLC system was

controlled by Hystar software (version 3.2; Bruker BioSpin,

Rheinstetten, Germany). The mass spectrometer was con-

trolled by the Compass 1.3 for micrOTOF software package

(Bruker Daltonics). The ionization technique was an

electrospray. The mass spectrometer was operated in

negative mode. Mass detection was performed in full scan

mode in the mass range m/z 50–2000. The following

settings were applied to the instrument: capillary voltage,

4000 V; end plate offset, �500 V. Heated drying gas (N2)

flow rate was 12 L/min; the drying gas temperature was

200 �C. The gas flow to the nebulizer was set at a pressure

of 1.6 bar. For collision-induced dissociation MS/MS

measurements, the voltage over the collision cell varied by

collision sweeping mode from 20 to 70 eV. Argon was used

as a collision gas. Sodium formate was used for calibration

at the end of each LC/MS run. The data were analyzed

using Compass Data Analysis Software (version 4.0 SP5;

Bruker Daltonics).

Animals

Male Swiss Albino mice weighing 27.5 ± 2.5 g were pur-

chased from the Animal House Facility, Faculty of Veterinary

Medicine, Suez Canal University, Ismailia, Egypt. The mice

were housed in cages in a ventilated room under the

controlled laboratory conditions of temperature (25 ± 2 �C)

and under 12 h light/dark cycles. The mice were fed a

standard rodent pellet chow and water ad libitum. The animals

were acclimatized for at least l week before use. All the

animal experiments were approved by the ethical committee

of the Faculty of Veterinary Medicine, Suez Canal University,

Ismailia, Egypt (201405).

Acute toxicity study

Male Swiss Albino mice weighing 27.5 ± 2.5 g were used to

determine the acute oral toxicity of BHPF according to the

reported method.20 BHPF was diluted in saline, and the mice

(n¼ 8 per group) were treated with high doses (500, 1000

and 2000 mg/kg body weight p.o.). The animals were

observed for 24 h to record toxicity symptoms and mortality

rates. The animals were also observed for possible toxico-

logical or behavioral changes for further 14 days after BHPF

treatment.

2 E. Al-Sayed et al. Ren Fail, Early Online: 1–10

Experimental design

Forty-eight Swiss Albino mice were divided into six groups

(n¼ 8 per group). Group (I) served as the normal control and

received saline only. Group II was treated intraperitoneally

with a sublethal dose of CCl4 at a dose of 0.5 mL/kg body

weight (20% v/v in corn oil) at the end of the experiment and

served as the positive control. The mice in groups (III, IV and

V) were treated orally with 100, 200 and 400 mg/kg body

weight of BHPF, respectively. Group VI was treated with

standard silymarin at a daily dose of 200 mg/kg.21 All tested

material and silymarin were administered orally, once daily,

for 5 consecutive days. On the sixth day, liver and kidney

injury was induced in animals (all groups except the normal

group) by a single i.p. injection of 20% CCl4 v/v in corn oil

(0.5 mL/kg bw).21 Blood collection was done by direct cardiac

puncture under diethyl ether anesthesia 24 h after the last

treatment dose. The collected blood was left to clot at room

temperature and the sera were separated by centrifugation at

3000 rpm for 15 min, then the sera were stored at �20 �C for

biochemical analysis. The animals were then sacrificed by

decapitation under diethyl ether anesthesia and the liver and

kidney were excised rapidly. One part of each tissue

was perfused with a 50 mM ice-cold phosphate buffered

saline (pH 7.4), containing 0.1 mM ethylenediaminetetraace-

tic acid to remove any red blood cells and clots. These tissues

were then homogenized ice-cold phosphate buffered saline

in (5–10 mL/g tissue), centrifuged at 5000 rpm for 30 min

and stored at �80 �C for the estimation of lipid peroxidation,

GSH content and SOD activity. Another hepatic and renal

tissue part was preserved in 10% formalin for the

histopathology.

Serum biochemical analysis

The already separated sera were used for the estimation of

serum liver biomarkers according to the manufacturer’s

protocol and previously reported methods for ALT, AST,22

ALP,23 LDH,24 total bilirubin,25 cholesterol26,27 and total

proteins.28 The biochemical markers of kidney damage were

estimated according to reported methods for creatinine,29

urea30 and uric acid.31

Evaluation of tissue lipid peroxidation andantioxidant parameters

Lipid peroxidation was evaluated by measuring the MDA

content in hepatic and renal tissues.32 The antioxidant

parameters were assayed according to previous methods for

the SOD activity33 and GSH.34

Histopathological examination

Liver and kidney specimens from all experimental groups

were fixed in 10% formol saline for 24 h. Washing was done

by the tap water followed by serial dilutions of alcohol

(methyl, ethyl and absolute ethyl). The specimens were

cleared in xylene and embedded in paraffin at 56 �C in a hot-

air oven for 24 h. Sections were prepared (4 lm thick) by a

sledge microtome and then stained with hematoxylin and

eosin. The liver and kidney sections were examined for

pathological changes by a light microscope.

Statistical analysis

All the data were expressed as the means ± SEM. The

statistical analysis of the data was performed using the one-

way ANOVA test followed by Tukey’s post hoc test to

determine the difference between the mean values of the

different groups. All statistical analyses were performed using

the GraphPad InStat software (Version 3.06, La Jolla, CA).

p-Values50.05 were considered statistically significant.

Results

Identification of the constituents of BHPF byHPLC-PDA-ESI/MS/MS

The individual constituents of BHPF were identified using the

HPLC instrument coupled to a PDA detector and a mass

spectrometer. The combination of the PDA and mass spec-

trometry (MS/MS) data provided a sensitive method for the

characterization of the constituents of BHPF (Table 1 and

Figure 1). Compounds 1–3 were tentatively identified based on

their molecular ions [M�H]� at m/z 729.16 and 881.18,

respectively, and the MS/MS ions at m/z 407.09, 289.08,

245.08, 169.02, 125.03, of which the first three ones are typical

for dimeric procyanidins.35,40 The MS/MS ions at m/z 169.02

indicated the presence of galloyl groups.41 Compounds 1 and 2produced the MS2 base peak at m/z 407.09 by the loss of a

gallic acid moiety (�170 amu) followed by a retro-Diels-Alder

fragment (�152 amu).42 The position of the galloyl residue

was confirmed to be attached to the C30 of the base unit of the

procyanidin molecule based on the presence of the fragment

ion at m/z 407.08, while the fragment ions at m/z 425, 577, 559

were not detected. These fragments are more predominant if

the galloyl residue is attached to the C3 of the top unit. The

molecule loses the galloyl residue or gallic acid and produces

the MS2 base peak at m/z 577 and a secondary peak at m/z 559,

respectively.42 Similarly, compounds 4–9 were identified

based on their [M�H]� and fragment ions as well as from

the distinctive PDA data.36–38 Compounds 10 and 12 were

tentatively identified based on their molecular ions and their

distinctive PDA of flavonoids.39

Acute toxicity

No adverse behavioral changes, toxicity symptoms or mor-

tality were observed in mice at doses up to 2000 mg/kg of

BHPF. Based on these findings, the oral 50% lethal dose

(LD50) value of BHPF is greater than 2000 mg/kg.

Hepato- and nephroprotective effects

A significant increase in the activity of the serum markers of

liver damage ALT, AST, ALP and LDH (p50.001) was

observed in the CCl4-intoxicated group compared with the

negative control group (Figure 2). The pretreatment of

intoxicated mice with BHPF produced a marked hepatopro-

tective effect and reduced the activity of serum hepatic

biomarkers in a dose-dependent manner. The percentage

decrease in the liver marker enzymes at the treatment doses

(100, 200 and 400 mg/kg/day) was 16%, 42% and 53% for

ALT, 20%, 45% and 58% for AST, 21%, 49% and 57% for

ALP and 22%, 49% and 60% for LDH, respectively, compared

DOI: 10.3109/0886022X.2015.1061886 Protective role of polyphenols from B. hookeri 3

with the CCl4-treated group. Notably, BHPF pretreatment at

a dose of 400 mg/kg markedly reduced the levels of ALT

and AST, which were comparable and non-significant to

those in the silymarin-treated group (Figure 2). The levels of

the ALP and LDH in the group treated with 400 mg/kg of

BHPF were comparable and not significantly different from

the normal control and silymarin groups (Figure 2).

Similarly, a remarkable and dose-dependent decrease of

the serum bilirubin and cholesterol levels was observed in

the BHPF pretreated groups (Table 2). The percentage

decrease in the serum bilirubin and cholesterol of the treated

groups compared with the CCl4-treated group is listed in

Table 2. In contrast, the total protein level was significantly

increased in the groups treated with BHPF in a dose-

dependent manner. The total protein level in the groups

treated with 200 and 400 mg/kg of BHPF were comparable

and not significantly different from the normal control and

silymarin groups (Table 2). The biochemical markers of

kidney damage, uric acid, urea and creatinine were markedly

reduced in the BHPF treated groups compared with the

CCl4-intoxicated group (Table 2). These results indicated

that BHPF effectively reduced the CCl4-induced hepatorenal

toxicity.

Antioxidant activity and lipid peroxidation

A significant reduction in hepatic and renal GSH and SOD

contents was observed in CCl4-intoxicated mice. In contrast, a

significant increase in the MDA levels was evident as

compared to the normal control group (Figure 3).

Administration of BHPF at the treatment doses (100, 200

and 400 mg/kg/day) produced a marked increase in hepatic

GSH (by 42%, 78% and 106%, respectively), as well as in

renal GSH (by 22%, 45% and 74%, respectively). In addition,

BHPF pretreatment improved the activity of hepatic SOD (by

18%, 61% and 105%, respectively), and the activity of renal

SOD (by 44%, 72% and 91%, respectively) relative to the

CCl4-intoxicated group. In contrast, the elevated hepatic level

of MDA was reduced by 19%, 45% and 50% and the renal

MDA by 22%, 35% and 51% at the tested doses, respectively,

compared with the CCl4-intoxicated group (Figure 3).

Notably, the liver GSH content in the groups treated with

200 and 400 mg/kg of BHPF was comparable and insignifi-

cant to those in the normal control and silymarin groups.

BHPF pretreatment at a dose of 400 mg/kg markedly reduced

the hepatic and renal MDA levels, which were comparable

and non-significant to those in the normal and silymarin-

treated groups. These results clearly indicated the strong

in vivo antioxidant activity provided by BHPF.

Histopathological observations

Liver sections of the normal control group showed normal

histological structure of the central vein and intact surround-

ing hepatocytes in hepatic parenchyma. CCl4 induced severe

loss of hepatic architecture with multiple focal necrosis,

ballooning degeneration in the hepatocytes all over the

hepatic parenchyma. The pathological changes induced by

CCl4 were markedly ameliorated in the groups treated with

BHPF at the three treatment doses. Pretreatment with 200 andTab

A[M�

6407.0

in-30 -

0273.0

5437.1

3[M�

ethyla

Figure 1. HPLC chromatogram of BHPF and the fragmentation pattern of its constituents.

Figure 2. Effect of BHPF and silymarin on the hepatic function tests of CCl4-intoxicated mice. Data are expressed as the means ± SEM (n¼ 8). BHPF1, 2 and 3: 100, 200 and 400 mg/kg of BHPF, respectively. Values having different superscripts are significantly different at p50.05.

400 mg/kg of BHPF conferred marked protection against liver

damage as evidenced by the intact hepatic architecture and

absence of histopathological lesions. Diffuse Kupffer cells

proliferation in between the hepatocytes was observed in

the group treated with 100 mg/kg of BHPF (Figure 4).

Histological examination of kidney sections revealed normal

histological structure of the glomeruli and tubules at the

cortex with absence of histopathological alterations in the

normal control group. CCl4 induced marked inflammatory

cell aggregation in between the tubules, associated with

marked degeneration in the lining epithelium of all the

tubules, along with blood vessel congestion. The aforemen-

tioned renal pathological changes induced by CCl4 were

markedly reduced in the groups treated with BHPF at the

three treatment doses. Notably, pretreatment with BHPF at a

dose of 400 mg/kg markedly reduced all the degenerative

changes in the lining epithelium of the tubules. Mild

inflammatory cell infiltration was noticed in between the

tubules in the groups pretreated with 100 and 200 mg/kg of

BHPF (Figure 5).

Discussion

CCl4 is a potent environmental hepatotoxin. CCl4 also causes

disorders in kidneys, lungs and brain. In addition, it may lead

to acute and chronic renal injuries.43 CCl4 is widely used in

experimental models to induce liver and kidney damage.44,45

The highly reactive trichloroethyl radicals (CCl3. and

CCl3O2.) are formed from CCl4 by cytochrome P-450.

These radicals initiate lipid peroxidation, necrosis and fatty

changes of the liver. The trichloroethyl radicals also change

the cellular antioxidant capacity by deactivating GSH and the

defense antioxidant enzymes.21,44 In this study, the acute

model of CCl4-intoxication was used because it resembles the

acute intoxication in human.43 The present study indicated

that CCl4 administration significantly increased the serum

levels of ALP, AST, ALT, LDH, cholesterol and total

bilirubin. Moreover, it reduced the total protein level, which

indicated severe loss of liver function. Histopathological

examination revealed severe loss of hepatic architecture

associated with multiple necrosis, marked ballooning degen-

eration of hepatocytes along with marked inflammatory cell

infiltration and degeneration of the kidney tissues. CCl4intoxication also induced severe oxidative stress as evidenced

by the marked increase in hepatic and renal MDA levels. The

SOD and GSH levels in the liver and kidney tissues were

markedly reduced in response to CCl4 intoxication. The

results of the present study indicated that pretreatment with

BHPF restored the increased MDA levels to their normal

values. The inhibitory effect against lipid peroxidation

suggested that BHPF could prevent the hepatorenal damage

induced by free radicals, along with the subsequent histo-

pathological alterations in the liver and kidney. In contrast,

BHPF pretreatment significantly enhanced the GSH and SOD

levels as compared to the CCl4-intoxicated group. Modulation

of these antioxidant defenses clearly contributed to the

hepato- and nephroprotective activity of BHPF. Based on

the results of this study, the hepato- and nephroprotective

effect of BHPF is attributed to its ability to reduce the rate of

lipid peroxidation, to enhance the antioxidant defense statusTab

and to guard against the pathological changes of the liver and

kidney induced by CCl4 administration. The remarkable

hepato- and nephroprotective effect of BHPF may, at least

partly, be due to the antioxidant effect of its bioactive

constituents. The HPLC-PDA-ESI/MS/MS analysis of BHPF

revealed the presence of proanthocyanidins and epicatechin

gallate (ECG) as the major components. Experimental

evidence suggests that whole plant fractions usually have

much better pharmacological activities than their single

isolated ingredients due to synergistic interactions between

the individual components.46 It is also proved that mixtures of

antioxidant compounds are more active than the individual

components of these mixtures.47

Proanthocyanidins are complex polymers of polyhydroxy

flavan-3-ol constitutive units, and are classified according to

the hydroxylation pattern of their constitutive units and the

linkages between them.35 The most common units are

(+)-catechin, (�)-epicatechin in the case of procyanidin

type, or (+) gallocatechin and (�)-epigallocatechin, for the

prodelphinidin structure.35 Previous studies demonstrated that

proanthocyanidins confer greater protection against free

radicals and lipid peroxidation than vitamins C, E and

b-carotene.14 Many studies confirmed the protective effect of

GSPE against chemical assaults in various organs using

different models. GSPE exhibited a strong protective effect on

thioacetamide-induced hepatic fibrosis in mice,15 and pro-

duced hepatoprotective as well as anti-fibrogenic effects

against dimethylnitrosamine-induced liver injury in rats.48 In

addition, GSPE significantly protected against acetamino-

phen-induced liver and kidney injury by reducing oxidative

stress, ALT activity and by inhibiting apoptotic and necrotic

cell death.14 GSPE also conferred protection against cyclo-

sporine A- and cisplatin-induced nephropathy in rats and

recovered the kidney functions. The nephroprotective activity

Figure 3. Effect of BHPF and silymarin on lipid peroxidation and antioxidant parameters in the liver and kidney of CCl4-intoxicated mice. Data areexpressed as the means ± SEM (n¼ 8). BHPF 1, 2 and 3: 100, 200 and 400 mg/kg of BHPF, respectively. Values having different superscripts aresignificantly different at p5 0.05.

of GSPE was attributed to its potent antioxidant activity, the

attenuation of renal tubular damage and the enhancement of

the regeneration response.49,50 The remarkable hepato- and

nephroprotective effect of BHPF may be related to the

presence of different proanthocyanidins, which may confer a

synergistic action. ECG, a major component in GSPE,

exhibited a protective role against renal failure, along with

the ability to reduce serum uric acid, urea, and creatinine

concentrations.51 Diverse pharmacological activities have

been attributed to ECG, including potent antioxidant, anti-

fibrogenic and hepatoprotective activity.13,52 Besides, it was

proved that ECG has more effective antioxidant activity over

vitamin C.53 All these attributes might contribute to the

hepato- and nephroprotective effect of BHPF. Based on the

results of this study, the potent hepatoprotective effect

previously reported for the total extract of B. hookeri44

could be attributed to its proanthocyanidin and ECG contents.

Furthermore, the results of this study confirmed that

B. hookeri proanthocyanidin has a potent nephroprotective

effect.

Experimental evidence indicates that oxidative stress is a

major link between liver injury and hepatic fibrosis. Persistent

hepatocellular damage disrupts the regeneration process of

damaged tissues and overwhelms the liver’s defensive mech-

anisms. The injured hepatocytes are potent sources of ROS and

lipid peroxidation products that stimulate the transformation of

hepatic stellate cells (HSCs) to fibrogenic myofibroblast-like

cells, which produce collagen.15,48 An excessive extracellular

matrix is accumulated in liver tissues, which may progress to

hepatic fibrosis or cirrhosis. Inactivation of HSCs represents a

promising approach to block the progression of hepatic

fibrosis.15 The protective effect of BHPF against infiltration

by inflammatory cells and other CCl4-induced pathological

changes in the liver, along with the protective effect against

oxidative stress, indicated that a dietary supplement of BHPF

has hepatoprotective and antifibrotic therapeutic potential.

Figure 4. Hepatoprotective effect of BHPF in CCl4-intoxicated mice. (A) Group I (normal control): showing normal histological structure of the centralvein and intact hepatocytes. (B) Group II (CCl4-treated group): showing severe loss of hepatic architecture with multiple focal necrosis, ballooningdegeneration in the hepatocytes. (C) Group VI (CCl4 + 200 mg/kg of silymarin): showing absence of histopathological alterations. (D and E) Groups Vand IV (CCl4 + 400 mg/kg and CCl4 + 200 mg/kg, respectively of BHPF): showing normal histological structure. (F) Group III (CCl4 + 100 mg/kg ofBHPF): showing diffuse Kupffer cell proliferation in between the hepatocytes (H&E,� 20).

Acknowledgments

The authors would like to thank Eng. Therese Labib, the

taxonomy specialist at the herbarium of El-Orman Botanical

Garden, Giza, Egypt for his kind help regard to identification

of B. hookeri leaves used in our study.

Declaration of interest

The authors have declared no conflicts of interest. The current

research received no fund from any organization.

References

1. Abdel-Daim MM. Synergistic protective role of ceftriaxone andascorbic acid against subacute diazinon-induced nephrotoxicity inrats. Cytotechnology. 2014. [Epub ahead of print]. doi: 10.1007/s10616-014-9779-z.

2. Abdel-Daim MM, El-Ghoneimy A. Synergistic protectiveeffects of ceftriaxone and ascorbic acid against subacute deltame-thrin-induced nephrotoxicity in rats. Renal Fail. 2015;37(2):297–304.

3. Abdel-Daim MM, Abuzead SMM, Halawa SM. Protective role ofSpirulina platensis against acute deltamethrin-induced toxicity inrats. PLoS One. 2013;8(9):e72991.

4. Al-Sayed E, Abdel-Daim MM. Protective role of cupressuflavonefrom cupressus macrocarpa against carbon tetrachloride-inducedhepato- and nephrotoxicity in mice. Planta Med. 2014;80(18):1665–1671.

5. Abdel-Daim MM, Abd Eldaim MA, Mahmoud MM.Trigonella foenum-graecum protection against deltamethrin-induced toxic effects on hematological, biochemical, and oxidativestress parameters in rats. Can J Physiol Pharmacol. 2014;92(8):679–685.

6. Abdel-Daim MM, Ghazy EW. Effects of Nigella sativa oiland ascorbic acid against oxytetracycline-induced hepato-renaltoxicity in rabbits. Iran J Basic Med Sci. 2015;18(3):221–227.

7. Abdou RH, Abdel-Daim MM. Alpha-lipoic acid improves acutedeltamethrin-induced toxicity in rats. Can J Physiol Pharmacol.2014;92(9):773–779.

8. Abdel-Daim MM, Ghazy EW, Fayez M. Synergistic protective roleof mirazid (Commiphora molmol) and ascorbic acid againsttilmicosin-induced cardiotoxicity in mice. Can J PhysiolPharmacol. 2014;93(1):45–51.

Figure 5. Nephroprotective effect of BHPF in CCl4-intoxicated mice. (A) Group I (normal control): showing normal histological structure of theglomeruli and tubules at the cortex with absence of histopathological alterations. (B) Group II (CCl4-treated group): showing marked inflammatory cellaggregation in between the tubules, marked degeneration in the lining epithelium of all the tubules, and blood vessel congestion. (C) Group VI(CCl4 + 200 mg/kg of silymarin): showing absence of histopathological alterations. (D) Group V (CCl4 + 400 mg/kg of BHPF): showing normalhistological structure. (E and F) Groups IV and III (CCl4 + 200 mg/kg and CCl4 + 100 mg/kg of BHPF, respectively): showing mild inflammatory cellinfiltration in between the tubules (H&E,� 20).

9. Ibrahim AE, Abdel-Daim MM. Modulating effects of Spirulinaplatensis against tilmicosin-induced cardiotoxicity in mice. Cell J.2015;17(1):137–144.

10. Abdel-Daim MM, Abdelkhalek NK, Hassan AM. Antagonisticactivity of dietary allicin against deltamethrin-induced oxidativedamage in freshwater Nile tilapia; Oreochromis niloticus.Ecotoxicol Environ Saf. 2015;111:146–152.

11. Abdelkhalek NK, Ghazy EW, Abdel-Daim MM. Pharmacodynamicinteraction of Spirulina platensis and deltamethrin in freshwaterfish Nile tilapia, Oreochromis niloticus: Impact on lipid peroxida-tion and oxidative stress. Environ Sci Pollut Res Int. 2015;22(4):3023–3031.

12. Abdel-Daim MM. Pharmacodynamic interaction of Spirulinaplatensis with erythromycin in Egyptian Baladi bucks (Caprahircus). Small Ruminant Res. 2014;120(2–3):234–241.

13. Han X, Shen T, Lou H. Dietary polyphenols and their biologicalsignificance. Int J Mol Sci. 2007;8:950–988.

14. Bagchi D, Bagchi M, Stohs SJ, et al. Free radicals and grape seedproanthocyanidin extract: Importance in human health and diseaseprevention. Toxicology. 2000;148(2–3):187–197.

15. Li J, Li J, Li S, et al. Ameliorative effect of grape seedproanthocyanidin extract on thioacetamide-induced mouse hepaticfibrosis. Toxicol Lett. 2012;213(3):353–360.

16. Filho VC. Chemical composition and biological potential of plantsfrom the genus Bauhinia. Phytother Res. 2009;23(10):1347–1354.

17. Bodakhe SH, Ram A. Hepatoprotective properties of Bauhiniavariegata bark extract. Yakugaku Zasshi. 2007;127(9):1503–1507.

18. Sosa S, Braca A, Altinier G, Della Loggia R, Morelli I, Tubaro A.Topical anti-inflammatory activity of Bauhinia tarapotensis leaves.Phytomedicine. 2002;9(7):646–653.

19. Maddigan L, Allan R, Reid R. Coastal Plants of the Burdekin DryTropics. Queensland, Australia: NQ Dry Tropics, BurdekinSolutions Ltd. & Townsville and Coastal Dry Tropics LandcareIncorporated; 2008.

20. Bruce RD. An up-and-down procedure for acute toxicity testing.Fundam Appl Toxicol. 1985;5(1):151–157.

21. Park SW, Lee CH, Kim YS, et al. Protective effect of baicalinagainst carbon tetrachloride-induced acute hepatic injury in mice.J Pharmacol Sci. 2008;106(1):136–143.

22. Reitman S, Frankel S. A colorimetric method for the determinationof serum glutamic oxalacetic and glutamic pyruvic transaminases.Am J Clin Pathol. 1957;28(1):56–63.

23. Tietz NW, Burtis CA, Duncan P, et al. A reference method formeasurement of alkaline phosphatase activity in human serum. ClinChem. 1983;29(5):751–761.

24. Babson SR, Babson AL. An improved amylase assay using dyedamylopectin. Clin Chim Acta. 1973;44(2):193–197.

25. Schattmann K. A spectrophotometric quantitative caffein-freemethod for determination of the serum bilirubin index withthe Lange universal colorimeter. Arztl Wochensch. 1952;7(49):1154–1156.

26. Richmond W. Preparation and properties of a cholesterol oxidasefrom Nocardia sp. and its application to the enzymatic assay of totalcholesterol in serum. Clin Chem. 1973;19(12):1350–1356.

27. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymaticdetermination of total serum cholesterol. Clin Chem. 1974;20(4):470–475.

28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Proteinmeasurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265–275.

29. Larsen K. Creatinine assay in the presence of protein with LKB8600 Reaction Rate Analyser. Clin Chim Acta. 1972;38(2):475–476.

30. Coulombe JJ, Favreau L. A new simple semimicro method forcolorimetric determination of urea. Clin Chem. 1963;9:102–108.

31. Whitehead TP, Bevan EA, Miano L, Leonardi A. Defects indiagnostic kits for determination of urate in serum. Clin Chem.1991;37(6):879–881.

32. Mihara M, Uchiyama M. Determination of malonaldehyde precur-sor in tissues by thiobarbituric acid test. Anal Biochem. 1978;86(1):271–278.

33. Nishikimi M, Appaji N, Yagi K. The occurrence of superoxideanion in the reaction of reduced phenazine methosulfate andmolecular oxygen. Biochem Biophys Res Commun. 1972;46(2):849–854.

34. Beutler E, Duron O, Kelly BM. Improved method for thedetermination of blood glutathione. J Lab Clin Med. 1963;61:882–888.

35. Hellstrom J, Sinkkonen J, Karonen M, Mattila P. Isolation andstructure elucidation of procyanidin oligomers from Saskatoonberries (Amelanchier alnifolia). J Agric Food Chem. 2007;55(1):157–164.

36. Khallouki F, Haubner R, Hull WE, et al. Isolation, purification andidentification of ellagic acid derivatives, catechins, and procyani-dins from the root bark of Anisophyllea dichostyla R. Br. FoodChem Toxicol 2007;45(3):472–485.

37. de Souza LM, Cipriani TR, Iacomini M, Gorin PA, Sassaki GL.HPLC/ESI-MS and NMR analysis of flavonoids and tannins inbioactive extract from leaves of Maytenus ilicifolia. J PharmBiomed Anal. 2008;47(1):59–67.

38. Hartisch C, Kolodziej H. Galloylhamameloses and proanthocyani-dins from Hamamelis virginiana. Phytochemistry. 1996;42(1):191–198.

39. Mabry TJ, Markham KR, Thomas MB. The SystematicIdentification of Flavonoids. New York, Heidelberg, Berlin:Springer-Verlag; 1970.

40. Verardo V, Arraez-Roman D, Segura-Carretero A, Marconi E,Fernandez-Gutierrez A, Caboni MF. Identification of buckwheatphenolic compounds by reverse phase high performance liquidchromatography–electrospray ionization-time of flight-mass spec-trometry (RP-HPLC–ESI-TOF-MS). J Cereal Sci. 2010;52(2):170–176.

41. Reed JD, Krueger CG, Vestling MM. MALDI-TOF mass spec-trometry of oligomeric food polyphenols. Phytochemistry. 2005;66(18):2248–2263.

42. Jaiswal R, Jayasinghe L, Kuhnert N. Identification and character-ization of proanthocyanidins of 16 members of the Rhododendrongenus (Ericaceae) by tandem LC–MS. J Mass Spectrom. 2012;47(4):502–515.

43. Manna P, Sinha M, Sil P. Aqueous extract of Terminalia arjunaprevents carbon tetrachloride induced hepatic and renal disorders.BMC Complementary Altern Med. 2006;6(1):33.

44. Al-Sayed E, Martiskainen O, Seif el-Din SH, et al.Hepatoprotective and antioxidant effect of Bauhinia hookeri extractagainst carbon tetrachloride-induced hepatotoxicity in mice andcharacterization of Its bioactive compounds by HPLC-PDA-ESI-MS/MS. BioMed Res Int. 2014;2014:1–9.

45. Ogeturk M, Kus I, Colakoglu N, Zararsiz I, Ilhan N, Sarsilmaz M.Caffeic acid phenethyl ester protects kidneys against carbontetrachloride toxicity in rats. J Ethnopharmacol. 2005;97(2):273–280.

46. Wagner H, Ulrich-Merzenich G. Synergy research: Approaching anew generation of phytopharmaceuticals. Phytomedicine. 2009;16(2–3):97–110.

47. Prochazkova D, Bousova I, Wilhelmova N. Antioxidant andprooxidant properties of flavonoids. Fitoterapia. 2011;82(4):513–523.

48. Shin MO, Yoon S, Moon JO. The proanthocyanidins inhibitdimethylnitrosamine-induced liver damage in rats. Arch Pharm Res.2010;33(1):167–173.

49. Ulusoy S, Ozkan G, Yucesan FB, et al. Anti-apoptotic and anti-oxidant effects of grape seed proanthocyanidin extract in preventingcyclosporine A-induced nephropathy. Nephrology. 2012;17(4):372–379.

50. Saad AA, Youssef MI, El-Shennawy LK. Cisplatin induced damagein kidney genomic DNA and nephrotoxicity in male rats: Theprotective effect of grape seed proanthocyanidin extract. FoodChem Toxicol. 2009;47(7):1499–1506.

51. El-Adawi H, El-Azhary D, Abd El-Wahab A, El-Shafeey M, Abdel-Mohsen M. Protective effect of milk thistle and grape seed extractson fumonisin B1 induced hepato-and nephro-toxicity in rats. J MedPlant Res. 2011;5(27):6316–6327.

52. Tipoe GL, Leung TM, Liong EC, Lau TYH, Fung ML, Nanji AA.Epigallocatechin-3-gallate (EGCG) reduces liver inflammation,oxidative stress and fibrosis in carbon tetrachloride (CCl4)-inducedliver injury in mice. Toxicology. 2010;273(1–3):45–52.

53. Sajilata MG, Bajaj PR, Singhal RS. Tea polyphenols as nutraceut-icals. Compr Rev Food Sci Food Safe. 2008;7(3):229–254.

Protective role of polyphenols from Bauhinia hookeri against carbon tetrachloride-induced hepato-and...

Documents

Hepato-protective Efficiency of Ethanol Leaf Extract of Moringa

Cyclooxygenase-1 serves a vital hepato-protective function in chemically induced acute liver injury

EVALUATION OF ACETYLCHOLINESTERASE INHIBITION BY BAUHINIA ALBA AERIAL PARTS METHANOL EXTRACT AND BIO-ACTIVE CONSTITUENTS

Isolation and Biochemical Characterization of a Galactoside Binding Lectin from Bauhinia variegata Candida (BvcL) Seeds

Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata

Antiobesity Activity of Bauhinia purpurea Extract: Effect on Hormones and Lipid Profile in High Calorie Diet Induced Obese Rats

Recognition profile of Bauhinia purpurea agglutinin (BPA

New Zealand sea lions Phocarctos hookeri and squid trawl fisheries: bycatch problems and management options

The Inhibition of aluminium corrosion in hydrochloric acid solution by exudate gum from Raphia hookeri

Bioactivity-Guided Isolation of Anticancer Agents from Bauhinia Kockiana Korth

BUL: A novel lectin from Bauhinia ungulata L. seeds with fungistatic and antiproliferative activities

Costs of hepato-pancreato-biliary surgery and readmissions in privately insured US patients

SEEDLING CHARACTERISTICS OF JHINJERA (BAUHINIA RACEMOSA LAMK.)

The influence of parasitoidism on the anatomical and histochemical profiles of the host leaves in a galling Lepidoptera -Bauhinia ungulata system

Hepatoprotective and antioxidant effect of bauhinia hookeri extract against carbon tetrachloride-induced hepatotoxicity in mice and characterization of its bioactive compounds by HPLC-PDA-ESI-MS/MS

REVIEW JURNAL KIMIA BAHAN ALAM \" Evaluation of Anti-Inflammatory, Anti-diabetic activity of Indian Bauhinia vahlii (stem bark) \"

NEONATAL MORTALITY IN NEW ZEALAND SEA LIONS (PHOCARCTOS HOOKERI ) AT SANDY BAY, ENDERBY ISLAND, AUCKLAND ISLANDS FROM 1998 TO 2005

The involvement of AMPK/GSK3-beta signals in the control of metastasis and proliferation in hepato-carcinoma cells treated with anthocyanins extracted from Korea wild berry Meoru

Mixed monolayers of Bauhinia monandra and Concanavalin A lectins with phospholipids, part II

Purification and molecular cloning of a new galactose-specific lectin from Bauhinia variegata seeds