Td

Ea

b

c

a

KASBHA

I

brapu

omhpgttcf

w

0h

Phytomedicine 19 (2012) 1059– 1067

Contents lists available at SciVerse ScienceDirect

Phytomedicine

j ourna l ho mepage: www.elsev ier .de /phymed

herapeutic effect of Acacia nilotica pods extract on streptozotocin inducediabetic nephropathy in rat

nayat A. Omaraa,∗, Somaia A. Nadab, Abdel Razik H. Farraga, Walid M. Sharafa, Sayed A. El-Toumyc

Pathology Department, National Research Center, 12622 Dokki, Cairo, EgyptPharmacology Department, National Research Center, 12622 Dokki, Cairo, EgyptChemistry of Tannins Department, National Research Center, 12622 Dokki, Cairo, Egypt

r t i c l e i n f o

eywords:cacia niloticatreptozotociniochemicalistopathologicalntioxidant activity

a b s t r a c t

The aim of the present study was to examine the effect of aqueous methanol extract (150 and300 mg/kg body weight) of Acacia nilotica pods in streptozotocin-induced diabetic rats for 60 days, andits biochemical, histopathological and histochemical study in the kidney tissues. Diabetic rats exhib-ited hyperglycemia, elevated of serum urea and creatinine. Significant increase in lipid peroxidation(LPO), superoxide dismutase (SOD) and reduced glutathione (GSH) was observed in diabetic kidney.Histopathological examination revealed infiltration of the lymphocytes in the interstitial spaces, glomeru-lar hypertrophy, basement membrane thickening and tubular necrosis with loss of their brush border insome of the proximal convoluted tubules in diabetic rats. Acacia nilotica extract lowered blood glucose lev-

els, restored serum urea and creatinine. In addition, Acacia nilotica extract attenuated the adverse effectof diabetes on LPO, SOD and GSH activity. Treatment with Acacia nilotica was found to almost restorethe normal histopathological architecture of kidney of streptozotocin-induced diabetic rats. However,glomerular size and damaged area showed ameliorative effect after treatment with the extract. In con-clusion, the antioxidant and antihyperglycemic properties of Acacia nilotica extract may offer a potentialtherapeutic source for the treatment of diabetes.

ntroduction

Diabetic nephropathy, a major long-term complication of dia-etes mellitus, is the most common cause of end-stage renal diseaseequiring dialysis worldwide (King et al. 1998) and is becoming

staggering challenge to public healthcare systems due to therohibitive costs of renal replacement therapy that could becomenaffordable even for developed countries.

Early diabetic nephropathy is characterized by hypertrophyf the glomeruli and tubular epithelial cells, thickening of base-ent membranes and enhanced renal blood flow and glomerular

yperfiltration (Hostetter 2001). This is accompanied by increasedrotein excretion and subsequent development of progressivelomerulosclerosis, with accumulation of extracellular matrix pro-eins in the glomerular mesangium thickening of glomerular andubular membranes, and tubulointerstitial fibrosis, all of themontributing to the inexorable progressive deterioration of renal

unction (Parving et al. 2000).

Free radicals have been shown to be harmful as they reactith important cellular components such as proteins, DNA and

∗ Corresponding author. Tel.: +20 2 33371362; fax: +20 2 33370931.E-mail address: eomara67@hotmail.com (E.A. Omara).

944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.phymed.2012.07.006

© 2012 Elsevier GmbH. All rights reserved.

cell membrane (Mantena et al. 2008). The body on the otherhand, requires free radicals for immune system responses. How-ever, an overload of these molecules has been linked to certainchronic diseases of heart, liver and some form of cancers (Prakashet al. 2007). All organisms contain anti-free radical defense system,which includes antioxidant enzymes such as catalase, peroxidaseand superoxide dismutase and antioxidants like ascorbic acid andtocopherol.

All over the world, people depended on herbs for the treatmentof various ailments before the advent of modern medicine. In Egypt,many plants are used today in folk medicine and are sold at herbalvendors and shops (Abdel-Azim et al. 2011). The ancient Egyptianswere familiar with many medicinal herbs and aware of their use-fulness in the treatment of various diseases. They used the plantorgans such as roots, rhizomes, flowers, leaves, fruits, seeds, andoils. They applied their medicaments in the form of powders, pills,suppositories, creams, pastes, and ointments (Shahat et al. 2001;Dagmar 2006). However, scientific evidence for the medicinal prop-erties of such plants is not always demonstrated.

The genus Acacia comprises about 1350 species (Seigler 2003),

distributed through the tropics and to some extent in the temper-ature regions. A. nilotica is a multipurpose tree of Fabaceae familythat is used extensively for the treatment of various diseases, e.g.cold, bronchitis, diarrhea, dysentery, biliousness, bleeding piles and

1 edici

lobtlMa1hgr2elr1

tdItpdnke

M

G

55aswtPAws

P

UIUSo

E

wrw(luHobar

060 E.A. Omara et al. / Phytom

eucoderma (Ambasta 1994). It is used by traditionally in treatmentf various cancer types of mouth, bone and skin. In West Africa, theark and gum are used against cancers and/or tumors (of ear, eye, oresticles) and indurations of liver and spleen, the root for tubercu-osis, the wood for smallpox and the leaves for ulcers (Kalaivani and

athew 2010). A. nilotica has been reported to have many biologicalctivities including antihypertensive, antispasmodic (Gilani et al.999), antidiabetic (Ahmad et al. 2008), hypochlesterolemic, and itad a beneficial effect on the hyperlipidemia associated with hyper-lycemia (Laakso 1995; Maciejewski et al. 2001) and decreased theisk of liver failure associated with diabetes mellitus (Ahmad et al.008). Nubians in South Egypt believe that diabetic patients mayat as much food rich in carbohydrates without risk, if they regu-arly take powdered pods of A. nilotica (Boulos 1983). A. nilotica iseported to be rich in tannins and polyphenols (Caster and Cowan988; Kumar 1983).

Plants have played a major role in the introduction of newherapeutic agents. A medicinal plant, Galega officinalis, led to theiscovery and synthesis of metformin (Cusi and Defronzo 1998).

t is our opinion that instead of random search of plants, a selec-ive search based on traditional knowledge would be focused androductive and certainly more economic. In the present study wasesigned to investigate the effect of aqueous methanol extract of A.ilotica pods on streptozotocin-induced diabetic in rat includingidney functions, antioxidant parameters, and histopathologicalxaminations for the renal tissue.

aterials and methods

eneral

NMR experiments were performed on a Bruker AMX 400 and00 instruments with standard pulse sequences operating at 400,00 MHz in 1H NMR and 100, 125 MHz in 13C NMR. Chemical shiftsre given in ı values (ppm) using tetramethylsilane as the internaltandard and DMSO-d6 as solvent at room temperature. HRESI-MSas taken on a Micromass Autospec (70 eV) spectrometer. UV spec-

ral data was measured on a Shimadzu 240 spectrometer in MeOH.aper chromatography Whatman 1, using solvent systems A (15%cOH) and B (n-BuOH:AcOH:H2O, 4:1:5, upper layer). Compoundsere visualized by exposure to UV light (365 nm), before and after

praying with AlCl3 and Naturestoff-polyethylene glycol reagents.

lant material

Pods of Acacia nilotica were collected in April 2009 from thepper Egypt. Identification of the plants was confirmed by Prof. Dr.

brahium El-Garf, Department of Botany, Faculty of Science, Cairo,niversity and comparison with herbarium specimens. Voucherpecimens were kept in herbarium, Department of Botany, Facultyf Science, Cairo, University (Boulos 1999).

xtraction and isolation compounds

Air-dried ground pods of A. nilotica (1.5 kg) were defattedith petroleum ether (40–60 ◦C), and extracted three times at

oom temperature with CH3OH:H2O (7:3). The combined extractsere filtered, evaporated under reduced pressure and lyophilized

180 g). Twenty grams of the dry residue was used for pharmaco-ogical studies. Weighed samples of pods of A. nilotica extract weresed to prepare the solutions, which were diluted with distilled2O to the appropriate concentration for the experiment. The rest

f the dry extract was redissolved in 2 l H2O and extracted with n-utanol (3× 2 l). After evaporation of solvents, the n-butanol extractnd the remaining H2O phase gave dark brown solids 50 and 70 g,espectively. The n-butanol extract was loaded on a polyamide 6S

ne 19 (2012) 1059– 1067

column chromatography (80 cm × 3 cm). The column was elutedwith H2O, and then H2O–CH3OH mixtures of decreasing polarityand 10 fractions (1 l, each) were collected. The major phenolicsfractions obtained were combined into five fractions after chro-matographic analysis. Fraction 1 (1.5 g) was fractionated by columnchromatography on Sephadex LH-20 with aqueous EtOH (0–70%)for elution to give compounds 1 (17 mg) and 4 (25 mg). Fraction 2(1.6 g) was subjected to column chromatography on cellulose andn-BuOH saturated with H2O as an eluent to give two major subfrac-tions, then one of them was separately fractionated on a SephadexLH-20 to yield pure samples 2 (21 mg) and the second was fraction-ated by column chromatography on Sephadex LH-20 with aqueousEtOH (50%) for elution to give compound 3 (15 mg). Using the sameprocedure fraction 3 (1.8 g) and fraction 4 (1.4 g) gave chromato-graphically pure samples 4 (15 mg) and 5 (15 mg). Fraction 5 (1.5 g)was chromatography on Sephadex LH-20 using aqueous acetone(0–25%) for elution to give pure sample 6 (20 mg) and 7 (25 mg).

Total phenolic content

The concentration of total phenolics of the plant extract wasdetermined according to the method described by Kumar et al.(2008). Gallic acid was used as standard. Briefly, a mixture of 100 �lof plant extract (100 �g ml–1), 500 �l of Folin–Ciocalteu reagentand 1.5 ml of Na2CO3 (20%) was shaken and diluted up to 10 ml withwater. After 2 h, the absorbance was measured at 765 nm using aspectrophotometer. All determinations were carried out in tripli-cate. The total phenolic content value was expressed as the gallicacid equivalents (GAE) in milligrams (mg) per 1 g weight of theextract, using the standard curve generated with the series of gallicacid standard.

Total flavonoid content

Total flavonoid concentration of plant extract was determinedaccording to the reported procedure by Kumaran and Karunakaran(2007). 100 �l of plant extract (10 mg ml−1) in methanol was mixedwith 100 �l of 20% AlCl3 in methanol and a drop of acetic acid, andthen diluted to 5 ml with methanol. The absorbance was measuredat 415 nm after 40 min against the blank. The blank consisted of allreagents and solvent without AlCl3. All determinations were carriedout in triplicate. The total flavonoid content value was expressedas the rutin equivalents (RE) in milligrams (mg) per 1 g weight ofthe extract, using the standard curve generated with the series ofrutin standard.

Analytical HPLC

Analysis was performed on an Agilent 1200 Series HPLC coupledwith a diode array detector (DAD). The analytical column was anAgilent ZORBAX Eclipse Plus C18 column (4.6 mm × 150 mm, 5 �m,PN 883952-702), was used at 30 ◦C. Two solvents were used witha constant flow rate of 1.0 ml/min. Solvent A consisted of 0.5 aceticacid/H2O, solvent B methanol. All the solvents used were of HPLCgrade. For the elution program, delivered at a flow rate of 1.0 ml permin as the following proportions of solvent B were used: 0–15 min,20% B; 15– 30 min, 40% B; 30–45 min, 60% B; 45–55 min, 80% B;55–60 min, 90% B.

Drugs and chemicals

Streptozotocin and diagnostic kits were purchased from Sigma(St. Louis, MO, USA). Glibenclamide was obtained from Sanofi-aventis Deutchland GmbH, Germany. The powder was dissolved indistilled water and orally administrated at dose 0.03 mg/kg b.wt. for

edici

6(

A

H1e5oaaRwo1

E

giDopR

F(

E.A. Omara et al. / Phytom

0 days. This dose equals the therapeutic dose for human (4 mg/kg)Abd El-Rahim et al., 2010).

nimals

Male mature Sprague Dawley rats purchased from Animalouse Colony, National Research Centre, Cairo, Egypt (weighing:20–150 g). Animals were divided into seven equal groups (six ratsach) housed under standard environmental conditions (23 ± 1 ◦C,5 ± 5% humidity and a 12-h light:12-h dark cycle) and maintainedn a standard laboratory diet ad libitum with free access to waternd they were housed in polycarbonated clean cages. Animal carend the experimental protocols were approved by the Nationalesearch Centre Animal Care and Use Committee was in accordanceith the guidelines of the International Association for the Study

f Pain Committee for Research and Ethical Issues (Zimmermann983).

xperimental design

Rats were divided into two main groups: normal non-diabeticroups; group 1: was normal control, groups 2 and 3 were admin-stered A. nilotica extract 150 mg and 300 mg/kg b.wt., respectively.

iabetic groups were injected intraperitoneally with single dosef streptozotocin (60 mg/kg b.wt. dissolved in 10 mM citrate bufferH 4.5), 7 days post injection, fasting blood sugar was determined.ats with blood glucose level above 200 mg/dl were included in the

ig. 1. (A) Chemical structures of the isolated compounds. (B) HPLC chromatogram of A. n2); catechin-4′-O-gallate (3); catechin-3′-O-gallate (4); gallic acid (5); quercetin 3-O-gluc

ne 19 (2012) 1059– 1067 1061

experiment. The diabetic groups: group 4, was control diabetic andreceived equivalent volume of saline; groups 5, and 6 were orallyadministered 150 and 300 mg/kg b.wt., respectively. While group 7,was given the reference drug glibenclamide (25 mg/kg). Treatmentschedule continued for 60 days.

Biochemical analysis

Blood samples were drawn after overnight fast from retro-orbital Venus plexus for determination of serum levels of glucose,creatinine (Slot 1965), and urea (Fawcett and Scott 1960). At the endof the experiment, all animals were sacrificed, and then kidneyswere removed and a part from the kidney was homogenate andused for determination of: (a) lipid peroxidation (LPO) was deter-mined by estimation of malondialdhyde (MDA) content accordingto Uchiyama and Mihara (1978); (b) the reduced glutathione (GSH)according to the method of Moron et al. (1979); and (c) nitric oxide(NO) according to Miranda et al. (2001) and superoxide dismutase(SOD) activity using Suttle (1986) method.

Histopathological and histochemical studies

A part from the rat kidney from all groups was removed and

immediately fixed in 10% neutral buffered formalin, dehydratedin gradual ethanol (50–100%), cleared in xylene and embedded inparaffin. 4–5 �m thick sections were prepared and stained withHematoxylin and Eosin (H&E) for photomicroscopic observation

ilotica pods extract. Identification of compounds: catechin (1); catechin-7-O-gallateoside (6) and quercetin (7).

1 edicine 19 (2012) 1059– 1067

(((

M

iPIamsds

S

l

R

C

topapce

B

i(etwgt

(((c0n

tasaeo3

sbt(t

Srum glucose

Con

trol

A. n -1

50

A.n-3

00

Dia

betic

Dia

b.+

A.n 1

50

Dia

b+ A

.n- 3

00

Diab+

Glib

. 25

mg

0

50

100

150

200

250

A B B

C

DE A

Crea tinine

Con

trol

A. n

-150

A.n-3

00

Dia

betic

Diab.

+ A.n

150

Dia

b+ A

.n- 3

00

Dia

b+Glib

. 25

mg

0.0

0.2

0.4

0.6

0.8

1.0

AC AC A

B

AC ACC

mg

/d

lm

g /d

l

Urea

Con

trol

A. n

-150

A.n-3

00

Dia

betic

Diab.

+ A.n

150

Diab+

A.n

- 300

Dia

b+Glib

. 25

mg

10

20

30

40

50

ADA

A

B

CD

D

AC

mg

/ d

l

Fig. 2. Effect of A. nilotica at doses (150 mg and 300 mg/kg) on kidney in control anddiabetic rat on blood glucose, creatinin and urea. ANOVA – one way, at p < 0.05. The

062 E.A. Omara et al. / Phytom

Drury and Wallington 1980). The periodic acid Scfiff’s techniquePAS) was used to demonstrate the presence of polysaccharidesglycogen) in the kidney (Mac-Manus and Cason 1950).

orphometry measurement

Morphometry measurement was achieved using computerizedmage analyzer (Leica Qwin 500 image) in Image Analyzer Unit,athology Department, National Research Center, and Cairo, Egypt.mage processing and analysis system was used for interactiveutomatic measurement of Renal morphometry was studied byeasuring: diameter of glomeruli and total area damage on slides

tained with H&E by 15 random fields per slide. Mean score ofiameter of glomeruli and total area damage was calculated in eachtudy group and compared to control.

tatistical analysis

Data were statistically analyzed using ANOVA two-way test andeast significant difference of the means (LSD) at p < 0.05.

esults

hemistry

Fractionation of the extract resulted in isolation and identifica-ion of seven phenolic compounds (1–7) (Fig. 1A). The structuref the isolated compounds was established through chromatogra-hy as well as conventional chemical and spectroscopic methods ofnalysis (UV, 1/2D NMR). The HPLC constituent profile of A. niloticaods recorded at 280 nm and 340 nm can be observed in Fig. 1B. Theontent of total phenolic compounds was (504.99 ± 1.35 mg GAE/Gxtract) and flavonoids (205.43 ± 3.58 mg RE/G extract).

iochemical studies

Blood glucose level of diabetic rat (220.5 ± 2.30 mg/dl) wasncreased significantly (p < 0.05) as compared to the control group67.8 ± 1.2 mg/dl). Treatment with A. nilotica aqueous alcoholicxtract at dose (150 and 300 mg/kg b.wt.) significantly decreasedhe glucose level of the diabetic groups in dose dependant mannerith mean values of 98.3 ± 0.94 and 83.4 ± 2.20 mg/dl in two-dose

roups (150 and 300 mg/kg). Glibenclamide-treatment normalizedhe glucose level (72.3 ± 1.5 mg/dl) as illustrated in Fig. 2.

Serum creatinine level significantly elevated in diabetic group0.78 ± 0.032 mg/dl) when compared with the control values0.49 ± 0.02 mg/dl). Moreover, groups received A. nilotica extract150 and 300 mg/kg) or those treated with glibenclamide theirreatinine values were within the normal range (0.54 ± 0.014,.50 ± 0.025 and 0.56 ± 0.022 mg/dl) whereas these three groupson-significantly different from each other Fig. 2.

Urea values also were elevated significantly in the non-reated diabetic group (43.2 ± 1.10 mg/dl) than normal controlnimals. A. nilotica extract-treatment groups (150 and 300 mg/kg)howed significant decrease in urea serum level to 32.5 ± 1.20nd 28.4 ± 1.60 mg/dl, respectively, dose dependent effect. How-ver, glibenclamide had similar effect to that of the lower dosef A. nilotica (150 mg/kg) to reduce urea concentration to be5.6 ± 1.30 mg/dl (Fig. 2).

Malondialdhyde (MDA) concentration in the renal homogenateignificantly elevated (807.33 ± 3.23 nmol/g tissues) in the dia-

etic group comparing to the control rat (259.17 ± 2.33 nmol/gissue). A. nilotica extract caused significant reduction in MDA level369.67 ± 4.5; 352.78 ± 3.16 nmol/g tissue in 150 and 300 mg/kg –reated groups) as shown in Fig. 3.

different capital letters above columns are significantly different.

Reduced glutathione (GSH) content significantly decreased inthe renal tissue of diabetic rat (8.43 ± 1.00 mmol/g tissue) com-pared with control group (17.19 ± 1.10 mmol/g tissue). Whiletreatment either with A. nilotica extract or glibenclamide resultedsignificant increase in GSH-renal content (A. nilotica 150 mg/kg:13.60 ± 1.10; A. nilotica 300 mg/kg: 14.37 ± 0.87; and gliben-

clamide: 16.00 ± 0.09 mmol/g tissue); GSH value non-significantlydifferent than normal control group (Fig. 3).

E.A. Omara et al. / Phytomedicine 19 (2012) 1059– 1067 1063

MDA

Con

trol

A. n

-150

A.n

-300

Diabe

tic

Diab.

+ A.n

150

Diab+

A.n

- 300

Diab+

Glib

. 25

mg

0

200

400

600

800

1000

A B AA

C

D D

nM

ol/g

tis

su

e

GSH

Con

trol

A. n

-150

A.n

-300

Diabe

tic

Diab.

+ A.n

150

Diab+

A.n

- 300

Diab+

Glib

. 25

mg

0

5

10

15

20

25

ACAD A

B

CD DAD

mm

ol /g

tis

su

e

SOD

Con

trol

A. n

-150

A.n

-300

Dia

betic

Dia

b.+

A.n

150

Diab+

A.n

- 300

Dia

b+G

lib. 2

5 m

g

0

20

40

60

80

100

A

B C

D

EF

G

µg

/ g

tis

su

e

NO

Con

trol

A. n

-150

A.n-3

00

Dia

betic

Dia

b.+

A.n 1

50

Dia

b+ A

.n- 3

00

Diab+

Glib

. 25

mg

0

20

40

60

80

A AA

C

AA A

uM

/mg

pro

tein

F l and dc

ncpg6tt2

wbt

H

ntc

As

ig. 3. Effect of Acacia nilotica at doses (150 mg and 300 mg/kg) on kidney in controapital letters above columns are significantly different.

Superoxide dismutase (SOD) in tissue homogenate was sig-ificantly inhibited in diabetic rats (27.3 ± 0.39 �g/g tissue)omparing with the control group (69.61 ± 0.18 �g/g tissue) at

< 0.05. All tested treatments (A. nilotica: 150 and 300 mg/kg andlibenclamide) significantly increased SOD activity (64.33 ± 0.43;1.17 ± 0.47 and 46.24 ± 0.87 �g/g tissue, respectively) comparingo the diabetic non-treated group. Furthermore, this result showedhat A. nilotica 150 mg/kg < A. nilotica 300 mg/kg < glibenclamide5 mg/kg on their effect to increase SOD activity (Fig. 3).

Nitric oxide (NO) value significantly reduced in diabetic grouphen compared with the control and any other treated groups (dia-

etic: 44.39 ± 2.80; control: 62.40 ± 2.50 �M/g tissue). Whereas, allreated diabetic groups had normal NO value as shown in (Fig. 3).

istopathological and histochemical studies

The sections from control showed normal histology of the kid-ey of rats. The proximal convoluted tubules, the distal convolutedubule and the renal corpuscles with glomerulus and glomerular

apsule are very clear and prominent (Fig. 4A).

Kidney tissue studies in the groups receiving the extract of. niotica at dose (150 and 300 mg/kg body weight) in compari-on to the control group did not show any abnormal microscopic

iabetic rat on MDA, GSH, SOD, and NO. ANOVA – one way, at p < 0.05. The different

findings. The extract did not induce mortality in any of thesequentially treated rats. After 60 days of streptozotocin adminis-tration severe changes were observed in kidneys. Histopathologicalchanges in kidney of diabetic rat showed signs of tubular necrosiswith loss of their brush border, vacuolar degeneration of prox-imal tubules and thickened basement membrane. The epitheliallining cells were disrupted with pyknotic nuclei. Hemorrhage andlymphocytic infiltrate in the interstitial area and periglomerularlymphocytic infiltration were noticed. Many tubules containedprotein casts (Fig. 4B and C). Most of the glomeruli showed atro-phy while remaining glomeruli also showed segmental sclerosiswith basement membrane thickening with and spread interstitialfibrosis. Bowman’s capsules were considerable increased in sizeoccupying the whole glomeruli spaces (Fig. 4B). These changes werefound to be significantly reduced in kidneys of the experimentalgroup treated with A. nilotica extract. The group of rat’s receivedlow dose of extract (150 mg/kg) showed moderate improvementin the tubular and glomerular morphology (Fig. 4D).

Post treatment with the plant extract at 300 mg/kg dose showed

considerable improvement in glomeruli and tubules. Bowman’scapsule showed comparatively less hypertrophy. Glomeruli werecompact with wide spacing in the Bowman’s capsule. Tubuleswere organized but debris was seen. Lumens were maintained.

1064 E.A. Omara et al. / Phytomedicine 19 (2012) 1059– 1067

Fig. 4. Effect of A. nilotica extract (150 and 300 mg/kg/day) for 60 days treatment on kidneys structures of rats with streptozotocin-induced diabetes. (A) Section of thekidney of control rat showing normal histological structure glomerulus (G) and renal tubules (T) (H&E 400×). (B) Section of the kidney of diabetic rat showing vacuolardegeneration of the epithelial cells lining the renal tubules(arrow head), shrinked glomeruli, widened urinary space of the Bowman’s capsule(long arrow) and areas ofhemorrhage in interstitial tissue (star) (H&E 400×). (C) Higher magnification of section of the kidney of diabetic rat showing marked degeneration of the epithelial cells liningthe renal tubules (long arrow) and pyknotic cells (arrow head) (H&E 1000×). (D) Section of the kidney of diabetic rat treated with A. nilotica at dose (150 mg/kg body weight)showing architecture similar to control, however some areas presented mild vascular degeneration of renal tubules (T), some glomeruli showed lobulation (G), hemorrhagein interstitial tissue (long arrow)and few and pyknotic cells (arrow head) (H&E 400×). (E) Section of the kidney of diabetic rat treated with A. nilotica at dose (300 mg/kgbody weight) showing normal glomeruli (G) with normal tubule (T), only few pyknotic cells (H&E 400×). (F) Section of the kidney of diabetic rat treated with Glibenclamidea with sa

EtdEat

swb6

t dose (25 mg/kg body weight) showing tubules more or less normal tubules (T),

rrow) and few pyknotic cells (arrow head) (H&E 400×).

pithelium was intact in most of the tubules hence improved his-oarchitecture of kidney (Fig. 4E). The thickening of the walls wasecreased by the A. nilotica (150 and 300 mg/kg) treatment (Fig. 4E).xperimental group of the animals injected with streptozotocinnd glibenclamide showed mild improvement in of glomeruli andubules (Fig. 4F).

The periodic acid Schiff’s (PAS) technique was used to demon-

trate the presence of glycogen in the kidney. The PAS +ve materialsere mainly distributed at the brush border and basement mem-

rane of the tubules (Fig. 5A). Treating rats with streptozotocin for0 days caused reduction of glycogen content in the kidney cells

light shrinked glomeruli (G), hemorrhagic area in between the renal tubules (long

(Fig. 5B), and absent in the brush borders of proximal convolutedtubules. Also, the glomeruli were less positive than those of the con-trol group. Strong reaction in the basement membrane was alsoseen (Fig. 5C). Glycogen content in kidney sections of the grouptreated with A. nilotica (150 and 300 mg/kg) and glibenclamideappeared similar to control group (Fig. 5D–F). However, glomeruliof the diabetic with (150 mg/kg) of A. nilotica group revealed posi-

tive PAS reaction and moderate dense positive brush border of theproximal convoluted tubules. The diabetic with (300 mg/kg) of A.nilotica group kidney sections revealed positive PAS reaction andintense brush border of the proximal convoluted tubules.

E.A. Omara et al. / Phytomedicine 19 (2012) 1059– 1067 1065

Fig. 5. Effect of A. nilotica extracts (150 and 300 mg/kg/day) for 60 days treatment on glycogen content in kidneys tissue of rats with streptozotocin-induced diabetes. (A)Photomicrograph of the kidney of control rats section with PAS positive material in the cytoplasm and brush borders of the proximal tubules and glomeruli were also positiveto PAS reaction. (B) Streptozotocin-diabetic rat showing a decreased amount of PAS positive material with strong reaction in the basement membrane and absent in the brushborders (arrow head). (C) Streptozotocin-diabetic rat showing decrease stainability of PAS materials with marked increase in the thick of basement membrane of tubules andglomeruli. Absent in the brush borders were observed (arrow head). (D) Section of the kidney of diabetic rat treated with A. nilotica at dose (150 mg/kg body weight) showingincrease stainability of PAS materials of tubules and glomeruli. Moderate preservation of brush borders was observed. (E) Section of the kidney of diabetic rat treated with A.nilotica at dose (300 mg/kg body weight) showing marked increase of amount of PAS positive material in tubules and glomeruli. Preservation of brush borders was observed(arrow). (F) Section of the kidney of diabetic rat treated with glibenclamide at dose (25 mg/kg body weight) showing moderate increase of PAS +ve materials in tubules andglomeruli. Preservation of brush borders was observed (PAS reaction 400×).

M

atapai

orphometry studies

In the morphometric studies, we observed that the quantitativenalysis of kidney tissue damage by image analyzer showed thathe group treated with A. nilotica at doses (150 and 300 mg/kg/day)

nd glibenclamide achieved a significantly ameliorated when com-ared to the diabetic group (p < 0.05). Quantitative analysis of therea of damage showed a dose-relationship in the area of damagen diabetic with A. nilotica at doses (150 and 300 mg/kg/day) and

glibenclamide. The data are showed in (Fig. 6). Also, we observedan atrophy of glomerular size with Bowman’s space dilated instreptozotocin-untreated diabetic rats. Diabetic rats of 60 daysduration, exhibited a significant (p < 0.05) atrophy of total areaoccupied by glomerular capillaries. Thus, mean of the glomeruli

size was significantly decreased in the diabetic rats compared tocontrols. A. nilotica at doses (150 and 300 mg/kg/day) and gliben-clamide ameliorated the decreased in mean glomeruli size ascompared with diabetic group (Fig. 7).

1066 E.A. Omara et al. / Phytomedici

Fig. 6. Damaged areas in the renal tissue of control, diabetic and treated groups.× = zero value; no damaged area were found in normal treated groups (mean ± SEof mean, n = 10 fields/slid/rat), ANOVA – one way; the different capital letters aresignificantly different at p < 0.05.

Glomerular area s

Contr

ol

A. n

-150

A.n

-300

Dia

betic

Dia

b.+ A

.n15

0

Dia

b+ A.n

- 300

Dia

b+ Glib

. 25

mg

0

200

400

600

800

A AA

B

C

AA

Glo

meru

lar

are

a (

µ2)

Fig. 7. Glomerular damaged areas in the renal tissue of control, diabetic and treatedgc

D

msahit((tivXstcv

It can be concluded that the aqueous methanol extract of A.

roups (mean ± SE of mean, n = 10 fields/slid/rat), ANOVA – one way; the differentapital letters are significantly different at p < 0.05.

iscussion

The present study revealed that diabetic rats in the no treat-ent group developed sever hyperglycemia, elevated values of

erum urea and creatinine. As well as, significant increase in LPOnd reduction in SOD, GSH and NO contents in the renal tissueomogenate was detected. Oxidative stress was incriminated as an

mportant mediator in the pathophysiology of diabetic nephropa-hy (Lee et al. 2003; Kowluru et al. 2004). Both hyperglycemiaAllen et al. 2003) and activation of the renin–angiotensin systemAnjaneyulu and Chopra 2004) play a role in the generation of reac-ive oxygen species (ROS). Moreover, the oxidative stress is evidentn enhanced products of mitochondrial oxidative stress in diabeticascular complications (Suzuki et al. 1999; Kanauchi et al. 2002;u et al. 2004). Also, the increased ROS in the kidney, especially theuperoxide radicals; react with NO to form peroxynitrite, which in

urn binds to tyrosine and other protein residues, yielding highlyytotoxic compounds such as nitrotyrosine in the renal and otherascular tissues (Pacher et al. 2005; Prabhakar 2007).

ne 19 (2012) 1059– 1067

Histopathological examinations supported these biochemicalresults and kidney sections showed progressive damage. Mostkidney sections showed lesions similar to human glomerulosclero-sis, glomerular membrane thickening, arteriolar hyalinization andwidespread tubular necrosis. Progressive glomerulosclerosis andfibrosis associated with decreased kidney function, resulting in endstage renal failure is the major finding in diabetic nephropathy(Striker et al. 1996). O’Donnell et al. (1988) and Harvey et al. (1992)proposed that the glomerular damage in diabetic kidney was due tothe increased production of Kallikrein and prostaglandin E2 whichcaused hyperfiltration and vasodilatation in diabetes.

In the present study, tubular necrosis, infiltration of lympho-cytes in the interstitial spaces with loss of brush border wasobserved in most kidney sections. These findings are in agreementwith the findings of Ramesh et al. (2007), Kim et al. (2008) andRenno et al. (2008) who showed tubular epithelial changes andenlargement of lining cells of tubules.

In the present study, we observed a hypertrophy of glomeru-lar size with Bowman’s space dilated in streptozotocin-untreateddiabetic rats. Diabetic glomerular hypertrophy constitutes an earlyevent in the progression of glomerular pathology which occurs inthe absence of mesengial expansion. In hyperglycemia there is anincrease in the entry of glucosein renal tissue (Belfiore et al. 1986).This has been postulated to cause increased intra-renal glycogendeposition, which leads to glycosylation of basement membranecollagen in the kidney (Anderson and Stowring 1973; Kim et al.2008).

The present study showed that diabetic rats receiving A. niloticapods extract had reduction of their blood glucose in comparison todiabetic control rats. Our results are agreement with Ahmad et al.(2008) and Asad et al. (2011) who reported that A. nilotica extractleaves decreases the elevated blood glucose levels in diabetic rab-bits. Such an effect may be accounted by a decrease in the rateof intestinal glucose absorption, achieved by an extra pancreaticaction including the stimulation of peripheral glucose utilizationor enhancing glycolytic and glycogenic process with concomitantdecrease in glycogenolysis and glyconeogenesis (Luzi and Pozza1997). However the effect was more significant when compared tostandard drug glibenclamide.

Our study showed that the treatment with A. nilotica extracthad antihyperglycemic effect in dose response manner. A. nilot-ica ameliorated the increased level of serum urea and creatininemore than glibenclamide treatment group. In renal homogenate,the antioxidant enzyme system (SOD and GSH), NO level and LPOshowed better improvement by the studied treatments with A.nilotica extract or glibenclamide, We investigated that A. nilot-ica pods extract is rich in tannins and polyphenols. Polyphenolsdecrease the blood glucose levels (Sabu et al. 2002; Tsuneki et al.2004) and have anti-oxidant properties (Kumar 1983). The mecha-nism of underlying A. nilotica pods extract effects involved to inhibitthe oxidative stress and enhancing the antioxidant enzyme sys-tem due to its scavenging property as evidenced by Kalaivani andMathew (2010) and Singh et al. (2009) in vitro and in vivo studies.

In this study, the general morphology of glomeruli and tubularlesions of the diabetic rats with the extract of A. nilotica pods wasmuch improved and seemed quite normal in appearance comparedwith that of diabetic rats. On the basis of the above evidences it ispossible that the presence of flavonoids and tannins are responsiblefor the observed antidiabetic activity.

Conclusion

niolitica pods showed an antidiabetic effect and diabetic nephropa-thy complications due to the presence of tannins and polyphenolstherein in the extract.

edici

R

A

A

A

A

A

A

A

A

B

B

BC

C

D

D

F

G

H

H

K

K

K

K

K

K

K

K

L

E.A. Omara et al. / Phytom

eferences

bdel-Azim, N.S., Shams, K.A., Shahat, A.A., El Missiry, M.M., Ismail, S.I., Hammouda,F.M., 2011. Egyptian herbal drug industry: challenges and future prospects.Research Journal of Medicinal Plant 5, 136–144.

bd El-Rahim, A.H., Radwan, H.A., Abd El-Moneim, O.M., Farag, I.M., Nada, S.A.,2010. The influence of amaryl on genetic alterations and sperm abnormali-ties of rats with alloxan-induced hyperglycemia. Journal of American Science6, 1739–1748.

hmad, M., Zaman, F., Sharif, T., Zabta, M.C., 2008. Antidiabetic and hypolipidemiceffects of aqueous methanolic extract of Acacia nilotica pods in alloxan-induceddiabetic rabbits. Scandinavian Journal of Laboratory Animal Science 35, 29–34.

llen, D.A., Harwood, S., Varagunam, M., Raftery, M.J., Yaqoob, M.M., 2003. Highglucose-induced oxidative stress causes apoptosis in proximal tubular epithelialcells and is mediated by multiple caspases. FASEB Journal 17, 908–910.

njaneyulu, M., Chopra, K., 2004. Effect of irbesartan on the antioxidant defensesystem and nitric oxide release in diabetic rat kidney. American Journal ofNephrology 24, 488–496.

mbasta, S.P., 1994. The Useful Plats of India, Publication and Information. Direc-torate Council of Scientific and Industrial Research, New Delhi, p. 4.

nderson, J.W., Stowring, I., 1973. Glycolytic and gluconeogenic enzyme activi-ties in renal cortex of diabetic rats. American Journal of Physiology 224, 930–936.

sad, M., Munir, T.A., Afzal, N., 2011. Acacia nlotica leave extract and glyburide:comparison of fasting blood glucose, serum insulin, �-thromboglubulin levelsand platelet aggregation in Streptozotocin induced diabetic rats. The Journal ofthe Pakistan Medical Association 61, 251–274.

elfiore, F., Rabuazzo, A.M., Iannello, S., 1986. Anabolic response of some tissues todiabetes. Biochemical Medicine and Metabolic Biology 35, 149–155.

oulos, L., 1983. Medicinal Plants of North Africa. Reference Publication, Alonac, MI,pp. 115–117.

oulos, L., 1999. Flora of Egypt. Al Hadara Publishing, Cairo, Egypt, p. 368.aster, J., Cowan, D.C., 1988. Phenology of Acacia nilotica subsp. indica Brenan. In:

Proceedings 5th Biennial Conference, Australia Ranelands Society, Longreach,Queensland, pp. 9–12.

usi, K., Defronzo, R.A., 1998. Metformin: a review of its metabolic effect. DiabetesReviews 6, 89–131.

agmar, L., 2006. International trade in medicinal and aromatic plants, actors, vol-umes and commodities, plants. In: Bogers, R.J., Craker, L.E., Lange, D. (Eds.),Medicinal and Aromatic Plants. Springer, Berlin/Heidelberg.

rury, R.A., Wallington, E.A., 1980. Coreleton’s Histological Technique, 4th ed. OxfordUniversity Press, Oxford.

awcett, J.K., Scott, J.E., 1960. A rapid and precise method for the determination ofurea. Journal of Clinical Pathology 13, 156–159.

ilani, A.H., Shaheen, F., Zaman, M., Janbaz, K.H., Shah, B.H., Akhtar, M.S., 1999.Studies on antihypertensive and antispasmodic activities of methanol extractof Acacia nilotica pods. Phytotherapy Research 8, 556–669.

arvey, J.N., Edmundson, A.W., Jaffa, A.A., Martin, L.L., Mayfiekd, R.K., 1992. Renalexcretion of Kallikrein and eicosanoids in patients with type-1 (insulin depen-dent) diabetes mellitus. Relationship to glomerular and tubular function.Diabetologia 35, 857–862.

ostetter, T., 2001. Prevention of end-stage renal disease due to type 2 diabetes.New England Journal of Medicine 345, 910–912.

alaivani, T., Mathew, L., 2010. Free radical scavenging activity from leaves of Aca-cia nilotica (L.) Wild. ex Delile, an Indian medicinal tree. Food and ChemicalToxicology 48, 298–305.

anauchi, M., Nishioka, H., Hashimoto, T., 2002. Oxidative DNA damage and tubu-lointerstitial injury in diabetic nephropathy. Nephron 91, 327–329.

im, H.J., Kong, M.K., Kim, Y.C., 2008. Beneficial effects of Phellodendri cortex extracton hyperglycemia and diabetic nephropathy in streptozotocin-induced diabeticrats. BMB Reports 41, 710–715.

ing, H., Aubert, R., Herman, W., 1998. Global burden of diabetes, 1995–2025: preva-lence, numerical estimates, and projection. Diabetes Care 21, 1414–1431.

owluru, R., Abbas, S., Odenbach, S., 2004. Reversal of hyperglycemia and diabeticnephropathy: effect of reinstitution of good metabolic control on oxidativestress in the kidney of diabetic rats. Journal of Diabetes and its Complications18, 282–288.

umaran, A., Karunakaran, J., 2007. In vitro antioxidant activities of methanolextracts of five Phyllanthus species from India. LWT – Food Science and Technol-ogy 40, 344–352.

umar, K.S., Ganesan, K., Rao, P.V., 2008. Antioxidant potential of solvent extractsof Kappaphycus alverezii (Doty) Doty – an edible seaweed. Food Chemistry 107,

289–295.

umar, R., 1983. Chemical and biochemical nature of fodder tree tannins. Journal ofAgricultural and Food Chemistry 31, 1364–1366.

aakso, M., 1995. Epidemiology of diabetic dyslipidemia. Diabetes Reviews 3,408–422.

ne 19 (2012) 1059– 1067 1067

Lee, H., Yu, M., Yang, Y., Jiang, Z., Ha, H., 2003. Reactive oxygen species regulatedsignaling pathways in diabetic nephropathy. Journal of the American Society ofNephrology 14, S241–S245.

Luzi, L., Pozza, G., 1997. Glibenclamide: an old drug with novel mechanism of action.Acta Diabetologica 34, 239–244.

Maciejewski, R., Rucinski, P., Burski, K., Figura, T., 2001. Changes in glucose,cholesterol and serum lipid fraction levels in experimental diabetes. AnnalesUniversitatis Mariae Curie-Sklodowska. Sectio D: Medicina 56, 363–368.

Mac-Manus, J.P.A., Cason, J.E., 1950. Carbohydrate histochemistry studies by a cety-lation technique. Periodic acid method. Journal of Experimental Medicine 91,651–654.

Mantena, R.K., Wijburg, O.L., Vindurampulle, C., Bennett-Wood, V.R., Walduck,A., Drummond, G.R., Davies, J.K., Robins-Browne, R.M., Strugnell, R.A., 2008.Reactive oxygen species are the major antibacterials against Salmonellatyphimurium purine auxotrophs in the phagosome of RAW 264.7 cells. CellularMicrobiology 10, 1058–1073.

Miranda, K.M., Espey, M.G., Wink, D.A., 2001. A rapid simple spectrophotometricmethod for simultaneous detection of nitrate and nitrite. Nitric Oxide: Biologyand Chemistry/Official Journal of the Nitric Oxide Society 5, 62–71.

Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathionereductase and glutathione S-transferase activities in rat lung and liver. Biochim-ica et Biophysica Acta 582, 67–78.

O’Donnell, M.P., Kasiske, B.L., Keane, W.F., 1988. Glomerular hemodynamic andstructural alterations in experimental diabetes mellitus. FASEB Journal 2,2339–2347.

Pacher, P., Obrosova, I., Mabley, J., Szabo, C., 2005. Role of nitrosative stress andperoxynitrite in the pathogenesis of diabetic complications: emerging new ther-apeutical strategies. Current Medicinal Chemistry 12, 267–275.

Parving, H.H., Osterby, R., Ritz, E., 2000. Diabetic nephropathy. In: Brenner, B.M. (Ed.),The Kidney. WB Saunders, Philadelphia, p. 1731.

Prakash, D., Upadhyay, G., Singh, B.N., Singh, H.B., 2007. Antioxidant and free radical-scavenging activities of seeds and agri-wastes of some varieties of soybean(glycine max). Food Chemistry 104, 783–790.

Prabhakar, N.R., 2007. Novel role for reactive oxygen species as amplifiers ofintermittent hypoxia. Focus on “reactive oxygen species mediate central car-diorespiratory network responses to acute intermittent hypoxia”. Journal ofNeurophysiology 97, 1877.

Ramesh, B., Viswanathan, P., Pugalendi, K.V., 2007. Protective effect of Umbelliferoneon membranous fatty acid composition in streptozotocin-induced diabetic rats.European Journal of Pharmacology 566, 231–239.

Renno, W.M., Abdeen, S., Alkhalaf, M., Asfar, S., 2008. Effect of green tea on kidneytubules of diabetic rats. British Journal of Nutrition 100, 652–659.

Sabu, M., Smitha, K., Kuttan, R., 2002. Antidiabetic activity of green tree phenolsand their role in reducing oxidative stress in experimental diabetes. Journal ofEthnopharmacology 83, 109–116.

Seigler, D.S., 2003. Phytochemistry of Acacia-sensu Lato. Biochemical Systematicsand Ecology 31, 845–873.

Shahat, A.A., Pieters, L., Apers, S., Nazeif, N.M., Abdel-Azim, N.S., Berghe, D.V., Vliet-inck, A.J., 2001. Chemical and biological investigations on Zizyphu Spina-christiL. Phytotherapy Research 15, 593–597.

Singh, R., Singh, B., Singh, S., Kumar, N., Kumar, S., Arora, S., 2009. Investigation ofethyl acetate extract/fractions of Acacia nilotica willd. Ex Del as potent antioxi-dant. Records of Natural Products 3, 131–138.

Slot, C., 1965. Plasma creatinine determination. A new and specific Jaffe reac-tion method. Scandinavian Journal of Clinical and Laboratory Investigation 17,381–387.

Striker, G.E., Eastman, R.D., Striker, L.J., 1996. Diabetic nephropathy: molecular anal-ysis of extracellular matrix and clinical studies update. Nephrology DialysisTransplantation 11 (Suppl. 5), 58–61.

Suttle, N.F., 1986. Copper deficiency in ruminants, recent developments. The Veteri-nary Record 119, 519–522.

Suzuki, S., Hinokio, Y., Komatu, K., Ohtomo, M., Onoda, M., Hirai, S., Hirai, M., Hirai,A., Chiba, M., Kasuga, S., Akai, H., Toyota, T., 1999. Oxidative damage to mito-chondrial DNA and its relationship to diabetic complications. Diabetes Researchand Clinical Practice 45, 161–168.

Tsuneki, H., Ishizuka, M., Terasawa, M., Wu, J.-B., Sasaoka, T., Kimura, I., 2004. Effectof green tea on blood glucose levels and serum proteomic patterns in diabetic(db/db) mice and on glucose metabolism in healthy humans. BMC Pharmacology4, 18–30.

Uchiyama, M., Mihara, M., 1978. Determination of malonaldehyde precursor in tis-sues by thiobarbituric acid test. Analytical Biochemistry 86, 271–278.

Xu, G., Yao, Q., Weng, Q., Su, B.L., Zhang, X., Xiong, J.H., 2004. Study of urinary 8-

hydroxydeoxyguanosine as a biomarker of oxidative DNA damage in diabeticnephropathy patients. Journal of Pharmaceutical and Biomedical Analysis 36,101–104.

Zimmermann, M., 1983. Ethical guidelines for investigations of experimental painin conscious animals. Pain 16, 109–110.