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Guidelines for Authors

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In the Name of ALLAH, Most Gracious, Most Merciful

Volume (7) – NO.(2)

July 2014 – Ramadan 1435H

Scientific Publications & translation

Deposit No.: 1429/2022

EDITORIAL BOARD

Editor-in-Chief:

Prof. Hassan M. Mousa

Editorial Board:

Prof. Moustafa Zeitoun

Prof. Hassan Abdel-Rhman Abdel-Rhman

Dr.Anber M.A. Hassanein

DR.Ihab Salah Ashoush

Advisory Board:

Prof. Abdulrahman I. Al-Humaid, Saudi Arabia

Prof. Hani M. Gohar, Egypt

Prof. Abdrab Al-Rasoul Omran, Saudi Arabia

Prof. Heungshik S. Lee, Korea

Prof. Hamzah M. AboTarboush, Saudi Arabia

Prof. Hassan A. Melouk, USA

Prof. Steven D. Lukefahr, USA

Prof. Ibrahim M. Al-Shahwan, Saudi Arabia

Prof. Mohamed M. Youssef, Egypt

Prof. Jean Boyazoglu, France

Prof. Abdulmageed M. Kamara, Egypt

Prof. Maher H. Khalil, Saudi Arabia

Prof. William E. Artz, USA

Prof. Ghanem M. Al-Ghamdi, Saudi Arabia

Journal of Agricultural and Veterinary Sciences,

Qassim University, Vol. 7, No. 2, pp. 101-202 Eng, pp (July 2014/Ramadan 1435H)

Contents Page

English Section

Veterinary Medicine A Control Program for Abscess Disease of Sheep

Khaled B. Alharbi ............................................................................................................................. 101

Reproductive, Hematological and Hepato-renal Effects of Ciprofloxacin in Male

Rats. Aida E. Bayad, Ibrahim M. El-Ashmawy, Abdel Salam F. El-Sawy And Eman A. Sabbah ... 109

Androgen ablation mitigates defect of B cells to a prostate cancer and increase

survival rate of TRAMP mice. Saleh Altuwaijri ................................................................................................................................ 125

Food Science and Human Nutrition Probable Atrophied Islet Cells Revival after Treatment with Olea eurpaea and

Lepidium sativum Extracts in Alloxan Induced Diabetic Mice Amr M. Awad , Ahmed A. Tayel , Ahmed I. Ibrahim ................................................................. 137

Evaluation of different modification methods for potato starch and its applications in

salad dressing manufacture Gadallah, Mohamed G.E., Yousif, El-Sayed I and Seror, Afaf, M. ........................................... 147

Impact of Different Dietary Proteins on Blood Glucose and Lipids Profile in

Diabetic Rats El- Hofi, M.A.; A.E. Metwly; I.S. Ashoush; Safaa A. Abd El-Aziz and Marwa, M. Yousef ... 163

Anti-diabetic effect of olive leaves extract in alloxan-diabetic rats Mousa , H. M. ., Farahna M., Ismail1, M. S., Al-Hassan1 A. A. , Ammar, A. S. and Abdel-

Salam1 A. M ...................................................................................................................................... 181

Plant Production Screening of thirteen garlic (Allium sativum L.) Genotypes for characteristics of

Yield and Quality under Sohag Conditions

Hazem A. Obiadalla Ali ................................................................................................................... 193

V

Journal of Agricultural and Veterinary Sciences,

Qassim University, Vol. 7, No. 2, pp. 101-202 Eng, pp (July 2014/Ramadan 1435H)

Veterinary Medicine

Journal of Agricultural and Veterinary Sciences

Qassim University, Vol. 7, No. 2, pp. 101-108 (July 2014/Ramadan 1435H)

101

A Control Program for Abscess Disease of Sheep

K. B. Alharbi

Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, P. O. Box 6622 Buraydah, Saudi Arabia

[email protected]

( Received 24/2/2014; accepted 3/4/2014)

ABSTRACT. A program for controlling abscess disease of sheep, based on vaccination, zinc injection and antiseptic washing, was implemented on a sheep flock comprising 50 Najdi ewes and 3 breeding

rams. The animals in the flock were vaccinated with a bacterin (GlanvacTM) and washed with Dettol

antiseptic at its standard dilution (1/125) every 6 months. They are also injected subcutaneously with 5 mg/kg bodyweight zinc as zinc oxide suspended in olive oil, once annually. The ewes were then allowed

to breed freely and the born F1 generation of lambs (n=20) joined the control program at the age of 3-4

months. The ewes and the lambs were monitored for abscess development and for general health for two years. In the first year, two vaccinated ewes developed abscesses on the head with incidence of 3.8%

(2/53). Two non-vaccinated lambs developed abscesses at the age of 3 months before joining the control

program. The program was continued on the parent stock ewes and their F1 generation lambs for a second year. The incidence of abscesses was 0% for both dams and lambs in the second year. The F1 generation

reached maturity and was bred to produce F2 generation of lambs (n=8) that also joined the control

grogram at the age of 3-4 months. The F2 generation of lambs remained free of abscesses until the age of 5 months, the time when the experiment time expired. The results show that the program is highly

effective in the control of sheep abscess disease as from the second year of its implementation. Its

alternative, test and cull seropositive animals will not be accepted by sheep owners in this country especially when the number of positive animals is high and there is the added cost of running the ELISA

test for each animal in the flock.

Keywords: Najdi sheep, abscess disease, control program

Khaled B. Alharbi 102

INTRODUCTION

Abscess disease is one of the sheep diseases that are causing great concern

worldwide. It lacks effective control measures and once introduced into a flock, it is

there to remain. This is because the bacterium highly pollutes the sheep

environment, its response to vaccination is controversial and the limitations in

detecting subclinically infected animals (Ivanovic et al., 2009, Williamson, 2001).

There is no available vaccine that is known to confer solid immunity against abscess

disease. Production of more effective vaccines was suggested by targeting all known

C. pseudotuberculosis antigens (Dorella et al., 2009). In some countries vaccination

failure is blamed on improper implementation of caseous lymphadenitis (CLA)

vaccination program (Paton et al 1991). A vaccine for sheep abscesses is

commercially available in Saudi Arabia (GlanvacTM). This vaccine is made of

killed germs with reports from the manufacturer of it being effective in decreasing

the incidence and severity of the disease in sheep flocks. The vaccine will not totally

prevent the formation of abscesses in exposed animals, but has been shown to

reduce the number of new abscesses developing in a flock (Mahmoud et al., 2009).

Locally produced vaccines from indigenous bacterial strains are likely to confer

better protection effects compared to imported vaccines (Fountaine et al., 2006). The

repeated infection was attributed to the high contamination of the environment of

pens with the pathogenic bacteria.

Investment in Intensive sheep production in the Kingdom of Saudi Arabia

has resulted in the emergence of abscess disease with annual increasing frequency.

This continued increase in the incidence of abscesses prompted the search to find a

suitable control program. A survey carried on the magnitude of the disease in

Qassim region showed that abscess disease incidence was about 22.4% especially in

the Najdi breed (Al-Harbi 2011). The records of the University Veterinary Teaching

Hospital, Qassim, Saudi Arabia, showed that the number of sheep and goats brought

for treatment from abscess disease is increasing annually. Clinical abscess disease

has reached very high incidence rate in some Najdi sheep farms, (44%) (Al-Harbi

2011) and the economic threat of the disease is largely confined to lower sale value

of infected sheep on the market especially during the holly months of the year when

sales are high. Abscesses can also develop in internal organs such as the lungs, liver,

kidney, heart etc. leading to emaciated and unhealthy animals (thin ewe syndrome).

Almost all sheep farmers at Qassim region (central Saudi Arabia) believe that

the available vaccines in the Kingdom have poor protective effect against sheep

abscesses and the control of abscess disease by vaccination needs intensive

investigation.

Results of three experiments carried at the University experimental station,

Qassim, on the immunology of abscess disease showed that the disease incidence is

reduced by vaccination, zinc injection and routine washing of animals with

antiseptics (Mahmoud et al., 2009; Al-harbi 2011). For this reason, this experiment

was planned utilizing the 3 previous results together aiming at finding an effective

A Control Program for Abscess Disease of Sheep 103

control program for abscess disease in sheep farms in the Kingdom of Saudi Arabia

by increasing immunity of the animals against abscess disease by vaccination,

boosting the general immunity of sheep by zinc injection and by fighting the

bacterium in the environment by washing the sheep and pens with an antiseptic

(Dettol). The formulated program is applied on a flock of Najdi ewes as well as their

lambs for 2 years.

MATERIALS AND METHODS

Animals

Fifty Najdi ewes and 3 rams were bought from the local market and kept in pen at

the University Experimental Farm. They were fed on berseem (Medicago sativa) and

had free access to drinking water. They were treated against internal parasites by

injecting them with ivermectin (1 ml/50 kg). They were allowed to breed freely

during the experimental period. The pens are sprayed with Detoll at its

recommended dilution to control bacterial contamination.

Twenty lambs were born (first generation F1) from the original stock of ewes

during the first year of experimental period. These were marked as first generation

of lambs and joined the control program at the age of 3-4 months. When they

reached maturity, the F1 generation was bred to give the F2 generation of lambs

(n=8) that also joined the control program at the age of 3-4 months.

Method

Ewes are vaccinated with abscess disease vaccine (GlanvacTM) every 6

months and injected subcutaneously with zinc, as zinc oxide suspended in olive oil,

at a dose rate of 5 mg/kg body weight, once annually. They are then washed with an

antiseptic, Dettol, at its standard dilution rate of 1 ml in 125 water, twice annually.

Figure 1 shows the program of controlling sheep abscesses.


Fig. (1). The abscess disease control program.

All sheep are screened for development of abscesses and general health after

vaccination.

RESULTS

Development of Abscesses in the first year

Two of the vaccinated ewes developed abscesses on the head with an incidence rate

of 3.8% (2/53) on the first year of the application of the control program. Another

two non-vaccinated lambs born in the farm developed abscesses one on the head and

the other on the right pre-scapular lymph node at the age of 3 months (Fig. 2). These

lambs were eliminated and did not join the control program.

BREEDING

EWES

SRAY

SRAY


Fig. (2). Non-vaccinated lamb developing a prescapular abscess during the First year of the research.

Flock Health

The experimental flock of sheep remained free from all infectious diseases

known to be fatal for sheep such as hemorrhagic septicaemia and enterotoxaemia.

Five ewes died during the experimental period. Three died from fractured hip

being attacked by the males during the breeding season. They remained recumbent

for few days but they eventually died. One ewe died from acute peritonitis that has

been confirmed by postmortem. During winter, very severe lice infection (probably

from stray dogs) occurred and it resulted in the death of one ewe. The flock was

sprayed with an acaricide (diazinon) and the flock was freed from the infection.

Lambs Health

Five lambs were lost out of the total twenty born during the first year

(mortality: 25%). Three were rejected by their mothers and 2 died from the high heat

wave during summer. Good F2 lamb crop was obtained in the second year (n=8).

The bred F1 generation gave a healthier F2 lamb crop.

Development of Abscesses in Vaccinated Lambs

The parent stock of ewes, the first generation and second generations of

lambs remained free of abscesses during the second and final year of the project.


DISCUSSION

The main objective of developing vaccines for veterinary use is welfare and control

(or elimination) of diseases. In the veterinary profession, vaccination might be a

corner stone determining economic failure or success of an enterprise, such as sheep

production projects. In recent years the veterinary profession has been faced with

many problems worldwide in disease control. These problems included the

appearance of new diseases or emergence of disease strains that do not respond well

to vaccination such as abscess disease of sheep. Such diseases need special research

studies to formulate suitable control programs that probe vaccine development plus

the use of other immunogenic avenues that help fight disease. This is because simple

vaccination might not work and control programs need to be carefully studied and

evaluated before authentication and implementation.

Abscess disease of sheep is a worldwide problem without success in its

elimination. We have been studying abscess disease in sheep farms at Qassim for 16

years concentrating on its epidemiology, nature, pathology, immunology and finally

control. Results of vaccination alone were similar to those obtained worldwide.

There is no effective complete control of this disease by vaccination and Qassim

sheep farmers are completely unsatisfied with vaccination outcome.

The use of serological testing (ELISA) followed by culling of positive

reactors can be very effective in controlling abscess disease of sheep and goats

(Dercksen et al 2000). However, the use of such procedure lacks practicality in

many parts of the world especially when large percentage of sheep in the flock tests

positive. Clinical abscess disease has a prevalence of 44% in some sheep farms in

Saudi Arabia (Alharbi 2011) and if number of sheep with subclinical infection

(diagnosed by ELISA) added to this percentage the number will even go higher. No

sheep owner in Saudi Arabia, or in many other parts of the world, having large

number of positive reactors to sheep abscess disease, will accept the test-and-cull

policy to be implemented to his sheep farm especially when there is an added cost of

the ELISA test. Baird and Malone (2010) examined six flocks of sheep clinically

and by ELISA for abscess disease and advised owners to remove positive sheep

from the flock. Of the six tested flocks, one was dispersed after only 2 blood tests

and the recommendations were not followed in another.

Zinc injection has been used to boost the immunity of sheep and thus the

effectiveness of the control program. Zinc has been shown to have strong wound

healing effect and immune stimulating function (Underwood 1981; Hambridge et

al., 1986). All kinds of immune cells showed decreased functions after zinc

depletion (Ibs and Rink 2003). In monocytes, all functions were impaired whereas

natural killer cells cytotoxicity is decreased. Phagocytic activity of neutrophils and

macrophages was reduced in zinc deficient animals (Ibs and Rink, 2003). Addition

of zinc resulted in restored normal functions and resulted in the release of cytokines

by immune cells. Microbial growth was diminished in abscesses and it was


suggested that a protein in the abscess fluid through its binding effect with zinc

inhibited bacterial growth within an abscess (Ibs and Rink 2003; Wu and Wu, 1987).

When an abscess ruptures, it highly pollutes the sheep premises and this

necessitates strict hygienic measures to control the dispersed bacterium. This can be

achieved by cleaning the sheep pens as well as washing (dipping) the sheep with

safe antiseptics such as Dettoll. We have been dipping sheep in acricides for years to

control scab infection. It is high time now to start dipping them in antiseptics to

reduce the incidence if abscess disease.

There is no previous report on the use of a comprehensive control program

for the control of sheep abscesses that utilized vaccination, zinc injection and

antiseptic washing of animals and pens. Al-Harbi (2011) and Mahmoud et al (2009)

showed that vaccination, zinc injection and antiseptic washing controlled abscess

disease for a period of 7 months, and the disease re-emerged after that protection

period. In this experiment, a control program was formulated based on these facts

and was repeated bi-annually in a cyclic manner. The program has been found

effective in controlling abscesses in the main sheep stock as well as its first and

second generations. Abscesses disappeared completely from the stock flock, its first

and second generation of lambs in the second year of application. Such results might

lay the foundation for further investigations on probing new avenues of abscess

disease control other than routine vaccination and test and cull policies. This is to be

achieved by application of comprehensive control programs that utilize vaccination

and probe the use of other additive components that stimulate the immune system as

well as clean the environment from the causative organism. When the incidence of

the disease is lowered to a minimum then the test and cull program will more

feasible. The incidence of abscess disease is increasing worldwide and vaccination

alone has failed to eliminate it from sheep farms anywhere in the world.

Acknowledgements

This research work was financed by the Deanship for Research, Qassim University.

The professional help of Prof. Osama Mahmoud, Mr. Ahmed Aldubeey, Mr.

Abdullah Alhawas is highly acknowledged. Also, the help of Mr. Barood Khan for

caring of experimental animals is acknowledged.

REFERENCES

Al-Harbi, K. B. (2011). "Prevalence and etiology of abscess disease in sheep and

goats at Qassim region, Saudi Arabia". Veterinary World 4(11), 495 – 499.

Baird, G. J. and Malone, F. E. (2010). "Control of caseous lymphadenitis in six

sheep flocks using clinical examination and regular ELISA testing".

Veterinary Record 166, 358 – 292.

Dercksen, D. F., Brinkhof, J. M. A., Dekker-Nooren, T., Maanen, K., Bode, C.

F., Baird, G. and Kamp, E. M. (2000). "A comparison of four serological


tests for the diagnosis of caseous lymphadenitis in sheep and goats".

Veterinary Microbiology 75, 167 – 175.

Dorella, F. A., Pacheco, L. G., Seyffert, N., Portela, R. W., Meyer, R., Miyoshi,

A. and Azevedo, V. (2009) "Antigens of Corynebacterium

psuedotuberculosis and prospectus for vaccine development". Expert Rev.

Vaccines 8, 205 – 213.

Fontaine, M. C., Baird, G., Conner, K. M., Rudge, K., Sales, J. and Donachie,

W. (2006). "Vaccination confers significant protection of sheep against

infection with a virulent United Kingdom strain of C. psuedotuberculosis".

Vaccine 14, 33 – 34.

Hambridge, K. M., Casey , C. E. and Krebs, J. (1986). "Zinc. In: Trace Elements

in Man and animals Nutrition", 5th

edition, volume 2, pp 1-37. Academic

Press, Orlando.

Ibs, K. H. and Rink, L. (2003). "Zinc-altered immune function". Journal of

Nutrition 133, 1452S – 1456S.

Ivanovic, S, Zutic, I., Pavlovic, I and Zujovic, M. (2009). Caseous lymphadenitis

in goats. Biotechnology in Animals, 25, 999 – 1007.

Mahmoud, O.M., E.M. Haroun and O.H. Omer, (2009) "Effect of zinc injection

on the immunity of sheep vaccinated against abscess disease", R. J. Sci., 2,

10-13.

Paton, M. W., Mercy, A. R., Sutherland, S. S., Ellis, T. M. and Duta, S. R.

(1991)."The effect of antibody to caseous lymphadenitis in ewes on the

efficacy of vaccination in lambs". Australian Veterinary Journal 68 (4),

143 – 146.

Underwood, E. J. (1981). "Mineral Nutrition of Farm Animals". Common

Agricultural Bureaux, UK.

Williamson, L. H., (2001). "Caseous lymphadenitis in small ruminants. Veterinary

Clinics of North America". Food Animal Practice 17, 359 – 371.

Wu, F. Y. H. and Wu, C. H. (1987). "Zinc in DNA replication and transcription".

Annual Review of Nutrition 7, 251 – 272.



101

Reproductive, Hematological and Hepato-renal Effects of Ciprofloxacin in

Male Rats.

Aida E. Bayad1, Ibrahim M. El-Ashmawy2,3*, Abdel Salam F. El-Sawy2

And Eman A. Sabbah2

1 Univ. Fellow ,Veterinary Services Center, and 2Pharmacology Department, Faculty of Veterinary Medicine, Alexandria University, Egypt

3 Department of Veterinary Medicine,Faculty of Agricultural and Veterinary Medicine, Qassim

University,P.O.Box 1482,Buraydah,Al-Qassim, Saudi Arabia Corresponding Auther : Ibrahim M. El-Ashmawy, [email protected]

(Received 10/10/2013; accepted 22/2/2014)

Abstract. The present study was designed to investigate the effects of the antimicrobial agent

ciprofloxacin on male fertility as well as some liver and kidney function tests in male rats. In addition,

some hematological and histopathological investigations were also studied. The animals were divided

into three equal groups, each of 15 rats. The first and second groups given 30 and 60 mg/ kg B.wt.

ciprofloxacin orally for ten days using stomach tubes. The third group was given saline and kept as a

control. ciprofloxacin induced mild to moderate toxic effects on the reproductive parameters especially reproductive organs weight and semen characteristics , as well as liver and kidney functions in male rats.

Attention should be paid on using higher doses of ciprofloxacin. That's due to the resultant fertility

problems due to abnormal semen characters in addition to a significant toxic effect of the drug on liver and kidneys .

Keywords: ciprofloxacin, sperm count,and motility, liver, kidney, testis, prostate.

Aida E. Bayad et al. 110

INTRODUCTION

The fluoroquinolones differ significantly from the earlier agents (quinolones) in

having a broader spectrum of antibacterial activity, increased potency, decreased

potential for emergence of resistance and exhibit rapid and extensive tissue

distribution because of its low protein binding ability(Walker et al., 2007).

Fluoroquinolones that are marketed for use in veterinary medicine today are

typically well-absorbed orally, have a large volume of distribution, penetrate nearly

every tissue and cell in the body and have extended elimination half-life, allowing

for every 24-48 hour dosing (Chu and Fernandes,1989) and (Walker et al., 2007).

Ciprofloxacin is a bactericidal agent. This agent has good activity against

many gram-negative bacilli and cocci, including most species and strains of

Pseudomonas aeruginosa, Klebesiella spp., E. coli, Enterobacter, Campylobacter,

Shigella, Salmonella, Aeromonas, Haemophilis, Proteus, Yersinia, Serratia and

Vibrio spp. They are also active against Bordetella bronchisepta, Brucella spp.,

Chlamydia spp. and Mycoplasma spp. (Plumb, 2005).

The quinolone antimicrobial target bacterial DNA gyrase and topoisomerase

IV (Drlica and Zhao, 1997). For many Gram positive bacteria (such as

Staphylococcus aureus), topoisomerase IV is the primary activity inhibited by the

quinolones (Ng et al., 1996). In contrast, for many Gram –negative bacteria (such as

E. coli), DNA gyrase is the primary quinolone target (Hooper, 2000 and Alovero et

al., 2000).The two strands DNA must be separated to permit DNA replication and

transcription.

Studies on the safety of fluoroquinolones are relatively scarce, but there are

some reports on toxicity of some fluoroquinolones ( Dollery ,1999, Abd-Allaah et

al. ,2000 , Baykal et al. ,2005, Demir et al. ,2007, Pfister et al. ,2007 ,and Khaki et

al. ,2008). These studies need further investigations. So, this study was designed to

investigate the adverse effect of ciprofloxacin on male reproductive system of albino

rats. This was assessed by examination of histology of reproductive organs (prostate

gland, testis and epididymis) and examination of semen samples. This study was

extended to investigate the effect of ciprofloxacin on some hematological

parameters, liver and kidney functions.


Drugs: Ciprofloxacin (ciproxin ® 10% solution, Alexandria Pharmaceutical

company). Its chemical name is 1- cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-

piperazinyl)-3-quinolone carboxylic acid. Its molecular weight is 367.9. Its

solubility in water is 1 in 25 (Dollery , 1999).

Animals: Male albino rats weighing from 160-190 g B. Each was used to study

some of the adverse effect of ciprofloxacin on male fertility, some hematological

parameters as well as hepatic and renal function tests.

Reproductive, Hematological and Hepato-renal Effects… 111

Experimental Design: Fourty-five mature male albino rats were used to study the

effects of two dose levels of ciprofloxacin on blood picture, semen characteristics,

weight and histology of the reproductive organs as well as renal and hepatic

functions. Rats were fed on bread, barley, corn, green leaves of vegetables and

watered ad-libitum. The animals were divided into 3 equal groups, each of 15 rats.

The first group was given 30 mg/ kg B.wt. ciprofloxacin orally using stomach tube.

The second group was given 60 mg/kg B.wt.ciprofloxacin orally. These doses were

calculated according to Paget and Barnes (1964). The third group was given saline

and kept as a control. All treated groups were administrated ciprofloxacin orally

while control group was administrated saline orally for 10 consecutive days. Five

rats from each group were sacrificed after two weeks, one month, and two months

post treatment. Blood, tissues and semen were obtained from them as follow.

1- Blood sampling: Two blood samples from each control and treated rats

were taken before sacrifying them from the orbital plexus under light ether

anesthesia using hematocrit tubes.

One sample was taken with EDTA for making blood picture while the other

sample was collected on clean dry centrifuge tubes for making sera samples.

2- Preparation of serum: Blood samples were left to clot at room temperature

then centrifuged for 15 minutes at 3000 r.p.m to obtain clear serum. The sera were

identified and stored in deep freezer at -20 Co till used for biochemical analysis.

3- Fertility studies: After sacrificing of rats, the epididymal content of each

rat was taken by sharp cutting of the tail of epididymes and squeezed gently on

sterile glass slide to estimate the progressive motility, sperm cell count and sperm

abnormalities according to the method described by Bearden and Fuquay (1980).

4- Weight of Internal Body Organs: After collection of blood samples and

epidydimal sperm examination, the testis, epidydimis and acessory genital gland

were dissected out, grossly examined and weighted. The index weight (I.W) of each

organ was calculated as described by (Matousek, 1969).

Index weight (I.W) = Organ weight X 100 Body Weight

5- Hematological studies: Red blood cells, white blood cells count,

hemoglobin and packed cell volume were measured ( Schalm, 1986).

6-Biochemical studies: The collected sera were used to investigate the effect

of ciprofloxacin on serum alanine and aspartate aminotransferases, albumin, total

protein , creatinine and urea, using commercial available kits and calculation of

globulin, by subtracting albumin from total protein values to obtain globulin value.

7- Histopathological studies: Five rats from each group of the control and

treated rats were decapitated. The testis, epididymis and prostate gland were dissected

out, grossly examined, dried by filter paper and weighed separately. Also, tissues from


the livers and kidneys of killed control and treated rats were collected. All specimens

were preserved in 10% neutral buffer formalin solution (Culling, 1974).

8-Statistical analysis: The obtained data were statistically analyzed for variation

among groups using the GLM procedure of the SAS computer program (SAS, 1987).

RESULTS

I-Effect of ciprofloxacin on fertility in male rats:

1. Effect of ciprofloxacin on male reproductive organs weight:

a- Rats of 1st group (30 mg/kg B. wt orally): Rats treated with ciprofloxacin

showed significant decrease in weight of testes, accessory sex glands after one and

two months from onset of drug administration. While there was significant decrease

in epididymes weight after two months from drug administration (Tables 1-3).

b- Rats of 2nd

group (60 mg/kg B. wt orally): There were significant

decrease in testes and epididymis weight after one and two months from drug

administration, while there were significant decrease in accessory sex glands weight

after one month from onset of drug administration but returned back to normal level

after two months from drug administration (Tables 1-3).

Table (1). Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on the index

weight of testes, at different periods in adult male rats. Values are expressed as mean ±

standard error. (n=5)

Treatment Testes index weight

2nd week 4th week 8th week

Control 1.51 ±0.005 A 1.77±0.03A 1.35± 0.04 A

Cipro. (30 mg/kg b.wt) 1.57±0.05 A 1.43±0.03 B 1.24±0.007 AB

Cipro (60 mg/kg B. wt) 1.63±0.03A 1.28±0.01 C 1.14± 0.04 B

Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. =

ciprofloxacin.

Table (2). Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on the index

weight of epididymis at different periods in adult male rats. Values are expressed as

mean ± standard error. (n=5)

Treatment Epididymes index weight


Control 0.56±0.01 A 0.62± 0.01 A 0.52±0.03 A

Cipro. (30 mg/kg B. wt) 0.64± 0.02 A 0.60±0.03 A 0.44± 0.01 B

Cipro.(60 mg/kg B. wt) 0.59±0.06 A 0.45± 0.01 B 0.42±0.01 B


ciprofloxacin.


Table (3): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on the index

weight of accessory sex glands (prostate and seminal vesicle) at different periods in adult

male rats. Values are expressed as mean ± standard error. (n=5)

Treatment Accessory sex glands index weight


Control 0.68± 0.02 A 0.88±0.05 A 0.83±0.10 A

Cipro.(30 mg/kg B. wt) 0.70± 0.05A 0.74±0.03 B 0.80±0.13 A

Cipro.(60 mg/kg B. wt) 0.71± 0.03 A 0.70±0.01 B 0.66±0.01 A


ciprofloxacin.

2. Effect of ciprofloxacin on semen characters:

a- Rats of 1st group (30 mg/kg B. wt orally): Rats administrated 30 mg

ciprofloxacin / kg B.wt showed no significant change on semen characters (motility,

abnormalities, sperm cell count) throughout the period of experiment (Tables 4-6).

b- Rats of the 2nd group (60 mg/kg B. wt orally). There was significant

decrease on sperm motility after two weeks and one month from drug administration

but returned to normal level after two months from drug administration. The sperm

cell count of treated rats with ciprofloxacin showed significant decrease after two

months from drug administration. Sperm abnormalities showed increase after one

month from onset of drug administration (Tables 4-6).

Table (4). Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on sperm

progressive motility% at different periods in adult male rats. Values are expressed as

mean ± standard error (n=5).

Treatment Sperm motility %


Control 90.2±2.13 A 87.4±2.78 A 85.00±2.23 A

Cipro.(30 mg/kg B. wt) 89.6±2.03 A 66.0± 11.44AB 72.60±7.15 A

Cipro.(60 mg/kg B. wt) 59.6±9.22 B 63.80±4.24 B 78.00± 8.30 A

Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = ciprofloxacin.

Table (5): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on sperm cell

count X106/ml at different periods in adult male rats.Values are expressed as mean ±

standard error (n=5).

Treatment sperm count (X106/ml)


Control 119.00±8.71A 130.00± 8.51 AB 120.00±9.35 A

Cipro.(30 mg/kg B. wt) 114.00±10.41 A 152.00± 9.56 A 91.00± 16.76 AB

Cipro.(60 mg/kg B. wt) 112.00±12.90 A 116.00± 12.98 B 86.00±5.78 B

Values on the same column carrying the same letter are not significantly different (p< 0.05). Cipro. = ciprofloxacin.


Table (6): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on sperm

abnormalities at different periods in adult male rats. Values are expressed as mean ±


Treatment Sperm abnormalities %


Control 7.40±0.50 A 8.60±1.6 B 8.00±0.70 A

Cipro.(30 mg/kg B. wt) 8.00±0.70 A 15.60±2.87 AB 6.80±1.46 A

Cipro.(60 mg/kg B. wt) 8.80±0.58 A 21.00±6.34 A 9.60±2.99 A


ciprofloxacin.

3. Effect of ciprofloxacin on hematological pictures:

a- Rats of 1st group (30 mg/kg B. wt orally): Rats treated with ciprofloxacin

showed significant decrease in hemoglobin after two weeks from onset of drug

administration. There was significant decrease in PCV after one month from onset

of drug administration but returned back to normal level after two months from drug

administration (Tables 7-10).

b- Rats of 2nd group (60 mg/kg B. wt orally): Rats treated with

ciprofloxacin showed significant decrease in RBCs count and hemoglobin after two

weeks from onset of drug administration. There was significant decrease in PCV

after two weeks and one month from onset of drug administration but returned to

normal level after two months (Tables 7-10).

Table (7): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on RBCs count at

different periods in adult male rats. Values are expressed as mean ± standard error (n=5).

Treatment RBCs count (X106/cmm)


Control 5.90 ±0.35 A 5.81±1.02 A 6.29±0.20 A

Cipro. (30 mg/kg B. wt) 5.52±0.23 AB 4.82±0.46 A 6.60±0.24 A

Cipro.(60 mg/kg B. wt) 4.86±0.10 B 3.76±0.84 A 6.07±0.41 A

Values on the same column carrying the same letters are not significantly different (p< 0.05).

Table (8): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on Hb at different

periods in adult male rats. Values are expressed as mean ± standard error (n=5).

Treatment Hb (g/dl)


Control 11.70±0.25 A 9.77±0.85 A 11.42±0.60 A


Cipro.(60 mg/kg B. wt) 6.90±0.40 C 7.42±1.62 A 10.16±0.46 A


ciprofloxacin.


Table (9): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B.wt) on WBCs count at

different periods in adult male rats. Values are expressed as mean ± standard error (n=5).

Treatment WBCs count (X103//cmm)


Control 8.48 ±0.47 AB 7.24±0.29 A 7.94±0.32 A




ciprofloxacin.

Table (10): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on PCV at different

periods in adult male rats. Values are expressed as mean ± standard error (n=5).

Treatment PCV %


Control 34.80±2.17 A 36.60±0.05 A 34.78±1.22AB

Cipro.(30 mg/kg B.wt) 33.00±1.48 A 27.58±2.26 B 36.72±0.71 A

Cipro.(60 mg/kg B. wt) 27.80±0.80 B 27.34±2.13 B 32.50±0.98 B


ciprofloxacin.

4. Effect of ciprofloxacin on biochemical findings :

a- Rats of 1st group (30mg/kg B wt orally): Rats treated with ciprofloxacin

at 30 mg/kg B.wt orally showed significant increase in serum urea and creatinine

level after two weeks from onset of drug administration. There was significant

decrease in albumin level after two weeks and one month from onset of drug

administration . Serum ALT level increased throughout the periods of the

experiment, while serum AST increased after two weeks and one month from onset

of drug administration but returned to normal level after two months from drug


b- Rats of 2nd

group (60 mg/kg B. wt orally): Rats treated with ciprofloxacin

at 60 mg/kg B.wt orally showed significant increase in creatinine level after two

weeks from onset of drug administration. Serum urea level increased throughout the

period of the experiment . Rats treated with ciprofloxacin showed significant

decrease in albumin level after two weeks and one month from onset of drug

administration. Serum ALT level increased throughout the experimental period,

while serum AST level increased after two weeks and one month from onset of drug

administration and returned back to normal level after two months following drug



Table (11): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on serum

creatinine level at different periods in adult male rats. Values are expressed as mean ±


Treatment Creatinine (mg%)


Control 0.44 ±0.02 B 0.50±0.04B 0.50±0.04 A

Cipro.(30 mg/kg B. wt) 0.60±0.50 A 0.56±0.04 AB 0.66±0.06 A



ciprofloxacin.


urea level at different periods in adult male rats. Values are expressed as mean ±


Treatment Urea (mg%)


Control 20.80±0.37 C 22.80±0.73B 21.40±0.60 B

Cipro.(30 mg/kg B. wt) 31.20±0.37 B 22.40± 0.81B 20.60±3.42 B

Cipro. (60 mg/kg B. wt) 34.40±1.50 B 25.60± 1.02 A 35.60±1.96 A


ciprofloxacin.


albumin level at different periods in adult male rats. Values are expressed as mean ±


Treatment Albumin (g/dl)


Control 4.47±0.05 A 4.4±0.07 A 3.54±0.31A

Cipro. (30 mg/kg B. wt) 4.05±0.08 B 3.32±0.12 C 3.38±0.13 A

Cipro. (60 mg/kg B. wt) 3.53±0.06 B 3.86±0.05 B 4.12± 0.26 A

Values at same column carrying the same letters are not significantly different (p< 0.05). Cipro. = ciprofloxacin.


globulin level at different periods in adult male rats. Values are expressed as mean ±


Treatment Globulin (g/dl)


Control 1.53±0.05 AB 1.20±0.35 A 1.46±0.31A


Cipro. (60 mg/kg B. wt) 2.07±0.24 A 1.54±0.25 A 1.26±0.40 A




total protein level at different periods in adult male rats. Values are expressed as mean

± standard error (n=5).

Treatment Total protein (g/dl)


Control 6.00 ± 0.00 A 5.60±0.40 A 5.00±0.00 A

Cipro.(30 mg/kg B. wt) 5.40±0.24 A 5.40±0.24 A 5.00± 0.00 A

Cipro.(60 mg/kg B. wt) 5.60± 0.24 A 5.40±0.24 A 5.38±0.24 A


ciprofloxacin.


ALT level at different periods in adult male rats. Values are expressed as mean ±


Treatment ALT (IU/L)


Control 23.40±1.31 B 24.00±0.70 B 20.76±1.30 B





AST level at different periods in adult male rats. Values are expressed as mean ±


Treatment AST (IU/L)


Control 80.80±3.15 B 74.20±3.81 C 75.80±1.65 A

Cipro.(30 mg/kg B. wt) 134.40±7.13 A 101.80±0.37 B 83.20±0.91 A



ciprofloxacin.

5 - Effect of ciprofloxacin on histopathological findings:

a- Rats of 1st group (30 mg/kg B. wt orally): The testes of the treated rats

showed changes of the lining epithelium of some seminiferous tubules. Some of

epididymal tubules revealed absence of sperm accompanied by degeneration and

necrosis of spermatozoa. The prostate gland showed congested blood vessels.

Congestion of portal blood vessels of liver with diffuse leucocytic


infiltration.Interstitial lymphocytic cellular infiltration were seen in many cases in

between the renal tubules( Fig. 1,5).

b- Rats of 2nd

group (60 mg/kg B. wt orally): The histopathological changes

of the male genital organs and accessory genital glands showed congested blood

vessels. Some of the seminiferous tubules were lined by few layers of

spermatogonia and showed incomplete spermatogenesis. Some of epididymal

tubules revealed absence of sperms. The prostate gland showed congested blood

vessels and leukocytic cellular infiltration. Severe congestion of the portal blood

vessels of liver and leucocytic infiltration in the portal areas. Congestion of the renal

blood vessels, vacuolar and hydropic degeneration of the lining epithelial cells of

some renal tubules (Fig.2-4).

c- Rats of 3rd group (control): Rats of this group showed no histopathological

alterations.

Fig.1. Testes of rats administered with ciprofloxacin (30 mg/kg B.W. for 10 days) and killed 2 weeks

post administration showed pyknosis in the nuclei of primary spermatogonial cells as well as

karyorrhexis in the other spermatogonial cells of the seminiferous tubules. H&E stain x 400.

Fig.2. Testes of rat administered with ciprofloxacin (60 mg/kg B.W. for 10 days) and killed 2 weeks

post administration showing necrobiotic changes of the lining epithelium of some

seminiferous tubules evidenced by pyknosis and karyorrhexis in the nuclei of spermatogonial

cells. H&E stain x 400.


Fig.3. Prostate gland of rat administered with ciprofloxacin (60 mg/kg B.W. for 10 days) and killed

8 weeks post administration showing eosinophilic cellular debris in the lumen of some

glandular acini. H&E stain x 200

Fig.4. Liver of rat administered with ciprofloxacin (60 mg/kg B.W. for 10 days) and killed 2 weeks

post administration showing sever congestion of the portal blood vessels and leucocytic

cellular infiltration in the portal areas particularly mononuclear cells. H&E stain x 200.

Fig.5. Kidney of rat administered with ciprofloxacin (30 mg/kg B.W. for 10 days) and killed 2 weeks

post administration showing homogenous eosinophilic casts in the lumen of some renal

convoluted tubules. H&E stain x 200.


DISCUSSION

The present study investigated the possible adverse effects of the antimicrobial

agent ciprofloxacin on male fertility as well as some liver and kidney function tests

in male rats. In addition, some hematological and histopathological investigations

were also studied.

Measurements were made at different periods from onset of drug

administration to follow up the induced effects of the drug on the reproductive,

hepatic and renal functions. The duration of the present study was two months to

cover complete spermatogenic cycle in rats which ranges from 48-52 days

(Clermont and Harvey, 1965).

The obtained results revealed that different doses of ciprofloxacin caused a

significant decrease in the weight of the testes, epididymis and accessory genital glands.

The present results are in agreement with those reported by Khaki et al. (2008)

who found that, oral administration of 12.5 mg ciprofloxacin/ kg B.wt daily for 60

days caused significant decrease in weights of testes, epididymis and seminal vesicles.

Concerning the effect of ciprofloxacin on epididymal sperm characteristics,

the obtained data revealed that higher doses of ciprofloxacin have deleterious

effects on male reproductive function.. There was a significant decrease in

progressive sperm motility percentage, and sperm count, while sperm abnormalities

were significantly increased .

These results are in agreement with Demir et al. (2007) who showed that

ciprofloxacin treatment for ten days in rats resulted in a marked reduction in sperm

count and motility. Also, Abd-Allaah et al. (2000) reported that ciprofloxacin

treatment for 15 days in rats caused marked reduction in sperm count and motility.

Grabe et al. (1986) found that ciprofloxacin was detected in the prostatic tissue and

seminal fluid in high concentrations. This ensure a persistent harmful effect on the

reproductive function.

Moreover, Bedford (1975) and Orgebin-Crist et al. (1976) reported that the

composition of epididymal fluid play a role in sperm maturation and storage. In the

same direction, the present study revealed some alterations in the male reproductive

organs represented by necrosis, congestion and degeneration. These changes can affect

the environment and medium necessary for sperm formation, maturation and storage.

Ciprofloxacin like other chemical agents may directly interfere in the process

of spermatogenesis. These deleterious effects induced by ciprofloxacin on male

reproductive function could be attributed to the induced histopathological changes

in male organs caused by increased free radical generation in the testes following

ciprofloxacin treatment (Weyers et al., 2002).

The obtained results demonstrated that, oral administration of ciprofloxacin

in male rats at both doses (30 mg and 60 mg/kg B.wt) induced insignificant changes

on white blood cells, while it induced significant decrease in RBCs count,


hemoglobin level and packed cell volume especially with dose of 60 mg

ciprofloxacin/ kg B. wt. These effects of ciprofloxacin might be due to its direct

effect on the hemopoietic system. Gilbersto and Jones,(1972) investigated the effect

of nalidixic acid on blood. They admitted the relationship between nalidixic acid

(mother nucleus of enrofloxacin) and incidence of anemia and leukopenia.

The present study demonstrated the effect of ciprofloxacin on liver enzymes.

It is apparent from the present study that, ciprofloxacin induced a significant

increase in alanine aminotransferase (ALT) activity following administration of

both doses of ciprofloxacin throughout the period of experiment, while aspartate

aminotransferase (AST) showed increase in its activity on administration of both

doses of ciprofloxacin after four weeks from drug administration, but returned to

normal level after eight weeks from onset of drug administration. Transaminases

including AST and ALT are found in most tissues but in unequal proportions, ALT

occurs in the liver, but only in the cytoplasm of parenchymal cells, in contrast to

AST which is equally distributed between the cytoplasm and mitochondria. When

liver damage occurs, the cell membranes become permeable or the cell walls may

rupture, so that these enzymes diffuse into the blood stream and increased levels are

found in circulatory blood (Hoe and Wilkinson, 1973).

Accordingly, the reported findings in the present study may be attributed to

damage of the hepatic cells by the direct effect of drug and its metabolites resulting in

escape of the liver enzymes in blood stream due to the harmful effects on the hepatic

cells. This conclusion is supported by the findings reported by Coles (1974), who

mentioned that increased serum transaminase activities suggest hepatocellular damage.

The obtained results are further confirmed by Basaran et al. (1993), they

found that hepatic function disorders in those receiving fluoroquinolones manifested

mainly with increase in hepatic enzymes in blood serum. Matysiak et al. (2008)

found that administration of ciprofloxacin to white female rats in dose of 4 mg /kg

B.wt twice daily for 7 days induced liver damage. These findings were supported by

the histopathological findings of the present work.

The biochemical studies were carried out also to investigate the degree of

renal damage following oral administration of ciprofloxacin in male rats. The

present study demonstrated that, the level of blood urea and creatinine were

significantly increased especially at dose level of 60 mg ciprofloxacin /kg B. wt.

The elevated level of urea and creatinine in serum is known to reflect the state of

glomerular filtration and indicates kidney disease (Coles, 1974). Fluoroquinolones

were found to be more concentrated in the renal tissues (Lambert and Jaupitre,

1984, Bergeron et al., 1985 and Alesting 1990) this suggestion is supported by the

reported histopathological findings which revealed congestion of renal blood

vessels, hyaline casts in the lumen of some renal convoluted tubules and hydropic

degeneration of the lining epithelial cells of some renal tubules. Also, this

suggestion is further confirmed by the findings reported by Wolfarm (1988).He

reported that, the potential changes caused by fluoroquinolones in the kidneys of


rats include mild interstitial nephritis, crystaluria, occult blood in the urine and

decreased renal function. Also, Walker et al. (2007) found occasionally mild

interstitial inflammation of kidney tubular walls being associated with precipitation

of fluoroquinolones complexes.

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125

Androgen ablation mitigates defect of B cells to a prostate cancer and increase

survival rate of TRAMP mice.

Saleh Altuwaijri1, 2, 3

1Veterinarian Medicine Department, Qassim University. 2Tianjin Institute of Urological Surgery, Tianjin

Medical University, Tianjin, China.3 Clinical Research Laboratory, Saad Research and Development

Center (SRDC) , SAAD Specialist Hospital, Al Khobar 31952, Saudi Arabia

[email protected]


Abstract. Androgen ablation is the most commonly used therapy for prostate cancer. However, the

effects of androgen ablation on immune system, especially B cell distribution are not well understood. We have used transgenic adenocarcinoma mouse prostate (TRAMP) mice to characterize the B cell

distribution in periphery blood and spleen upon androgen ablation. We found a decrease of the B cell

population in wild type TRAMP mice compared to normal wild type mice. Furthermore, the B cells increased when in castrated TRAMP mice compared to the wild type TRAMP mice. Interestingly, we

found an increase in immature B cells in the spleen of castrated TRAMP mice, which might be due to the

resistance to apoptosis during B cell maturation. Our data suggest that androgen ablation might play an important role in the regulation of B cell tolerance.

mailto:[email protected]

Saleh Altuwaijri 126

INTRODUCTION

Prostate cancer is routinely screened for and is frequently diagnosed early in the

course of the disease when the patient has only small, non-invasive tumors or even

precancerous prostatic intraepithelial neoplastic (PIN) lesions. In these cases, the

(pre)cancerous prostate lesions generally cause symptoms in the patients that are

less severe than the serious side-effects of the standard prostate cancer treatments.

Therefore, the standard of care in these patients is a period of active surveillance that

can last for years before the prostate tumor begins to pose a more serious risk to the

health of the patient(Gray, et al., 2013). Interactions between immune and cancer

cells occur throughout the development of tumors (Dalgleishand O'Byrne, 2006).

The immunosurveillance effects of the immune system allow for the initial

recognition and successful elimination of the neoplastic cells at the early stage.

(Wang, 2006). In the meantime, this constant immunological selective pressure

leads to the development of neoplastic cells, which are able to escape the immune

system by mimicking immunosuppressive processes associated with the induction of

tolerance and the prevention of auto-immune disorders via modulation of B cell

function (Maloney, 2005). Activated lymphocytes subsequently migrate to the

primary tumor and exert their newly acquired tumoricidal effects (Neeson, 2006). It

is now evident that tumor environment contributes to the suppression of the anti-

tumor immune functions of tumor, thereby promoting cancer metastasis (Yi, et al.,

2007). The mechanisms involved in the defective immune response and the humoral

immune response associated with tumor proliferation are not well understood.

Androgen ablation is the most commonly used therapy for prostate cancer

that cannot be treated with surgery or radiation (Pope, et al. 2002). However,

virtually all prostate cancer patients treated with androgen ablation respond but

eventually develop resistance to therapy, an ominous clinical state for which no

consistently effective therapy exists. (Hiroshi, et al. 2007).Prostate cancer in

TRAMP mice closely mimics the course of the human disease, from the

development of precancerous PIN lesions to invasive prostate adenocarcinoma or

neuroendocrine tumors and then to metastatic disease (Greenberg, et al. 1995).

Therefore this model is an ideal system in which to investigate the effects of

therapeutic vaccination at different stages of prostate cancer progression. Transgenic

mouse models have been developed to study the initiation and progression of human

prostate cancer. Among those, the TRAMP model mimics human prostate cancer in

both its histopathologic and lethal features (Greenberg, et. 1995). In mice without

prostate cancer, it has been found that androgen ablation in castrated normal mice

leads to a transient increase in B cells (Olsen and Kovacs, 2001). In mice with

prostate cancer, a progressive decrease in T cell recognition of the prostate gland

and B cell distribution and function have not been defined. Our data suggest that

immunotherapy approaches for prostate cancer may prove most effective when

applied after androgen ablation.

To address the effect of androgen ablation on B distribution in prostate cancer,

we used wild type and castrated TRAMP mice to characterize the B cell distribution in

Androgen ablation mitigates defect … 127

periphery blood and spleen. We found a decrease in B cells in TRAMP mice

compared with normal mice. Furthermore, the total number of B cells increases in

castrated TRAMP mice compared with the intact TRAMP mice. Interestingly, we

observed an increase in immature B cell population in the spleens of castrated

TRAMP mice, which might be due to the resistance to apoptosis. Our data suggest that

androgen ablation might play an important role in the regulation of B cell tolerance.

MATERIALS AND METHODS.

Animal. We obtained TRAMP (C57BL/6-TRAMPxFVB) mice from Jackson

Laboratory and 4-week old male nude mice from Charles River Laboratories. Mice

were anesthetized with 40 mg/kg sodium pentobarbital intraperitoneally (Nembutal,

Abbot Laboratories; Chicago, IL). Surgical castration was performed via a midline

scrotal incision allowing bilateral access to the hemiscrotal contents. After exposing

each testicle, a 3-0 Vicryl suture was used to ligate the spermatic cord and then

remove the testicle.

ELISA. Plasma DHT were determined using the DHT ELISA Kit (Alpha

Diagnostic International, San Antonio, Texas) according to the manufacturer’s

instructions.Cytology and automated hematology analysis. We measured

hematologic parameters from blood samples obtained from 8 to 12-week-old mice

using an Abbott CELL-DYN 4000 system.

Cell staining and isolation using FACS.We performed immunofluorescence

analysis using fluorochrome-conjugated antibodies purchased from eBiosciences,

including APC-anti-B220, and PE–anti-IgM., as well as FITC–anti-CD45 from

Pharmingen (San Diego, CA). We incubated 1–5 X 105 cells on ice for 30 min with

saturating amounts of antibody and isolated B cells from bone marrow, spleen, and

peripheral blood using the EasySep FITC selection kit (Stem Cell Technologies).

Statistics.Two-sided Student’s t test were used for statistical analysis (p<0.05). Data

were presented as the mean ± standard deviation (SD), (Altuwaijri, et al. 2003).

RESULT

Decreased whit blood and lymphocyte cells in peripheral blood in TRAMP mice.

Recent studies indicated infiltration of immune cells into the lumen of prostate with

cancer in mice (Zhang, et al. 2006), which suggested the influence of tumor cells on

the immune system. However, it is unclear about the effects of hormonal

chemoprevention on B cells and prostate tumorigenesis via early androgen

deprivation (preceding the expected onset of prostate cancer). . To address this

question, we first analyzed the immune cells in peripheral blood. Automated

analysis of hematological parameters showed no significant difference in red blood

cells, platelets, hemoglobin, hematocrit between TRAMP and control mice (data not

shown). However, the total number of white blood cells (WBC) (Figure 1A) as well


as the total number of lymphocytes (Figure1B) were decreased in TRAMP mice

compared with control mice.

Decreased B cells in the spleen of TRAMP mice.

Association between tumor and autoimmune and immunodeficiency

conditions provided a strongly support for the involvement of immune system in

tumorigenesis (Gilardoni, et al. 1999). The effects of immune system on prostate

cancer has not been understood. To investigate the association between prostate

cancer and B cell distribution and function, we analyzed the spencocytes in TRAMP

mice (at the age of 24 weeks). We use the flow Cytometry technique to analyze the

distribution of lymphocytes from the spleen of TRAMP and control mice (Figure

2A). We then stained splenocytes in these mice with B220+, a marker for B cells

(Figure 2B-C). B cells in TRAMP mice were significantly decreased (55%) than that

in control mice (81%). These results are in agreement with the previous findings that

tumors repress immune system by inhibiting the expansion of immune cells. (Zhang,

et al. 2006).

Figure (1). A) The total white blood cells, and B) the total number of lymphocytes were significantly

decreased in TRAMP mice compared to Wt mice.

0

1

2

WBC

Wt TRAMP

Wt

10

3 c

ells

/µl

Lymphocytes

0

1

2

Wt

10

3 c

ells

/µl

*

TRAMP

Wt

A) B)

0

1

2

WBC

Wt TRAMP

Wt

10

3 c

ells

/µl

Lymphocytes

0

1

2

Wt

10

3 c

ells

/µl

*

TRAMP

Wt

A) B)


Figure (2) Flow cytometer technique used to analyze. A) The distribution of lymphocyte from the

spleen of wild type and TRAMP. B) Wild type and TRAMP splenocyte with B220+

which is a marker for B cells. The positive B cells in TRAMP mice were 54% in

comparison to the Wild type mice which were 80%.

Castration significantly improved the survival rate of TRAMP mice.

Androgen ablation therapy with suppression of androgen functions is an

effective treatment for most prostate cancer patients in the initial stage of disease

(Best, et al. 2005). To investigate whether the TRAMP mice mimic this clinical

process in human, we castrated the TRAMP mice at age of 12 weeks. At age of 30

week, 100% un castrated TRAMP mice had overt prostate cancer. However, only

52% of the castrated TRAMP mice had prostate cancer (pl 0.05). Two castrated

mice did have overt tumors that later led to death (data not shown). Although

androgen deprivation before the development of prostate cancer can decrease the

occurrence of this disease in male mice, the androgen-independent prostate cancer in

some cases was observed. Accordingly, the castrated TRAMP mice have a higher

survival rate and lower androgen level in serum compared with the non-castrated

TRAMP mice (Figure 3A-B). Therefore, using TRAMP mice model, we were able

to recapitulate the clinical situation that androgen deprivation therapy offers benefit

for prostate cancer patients.

Immature B cells are increased in castrated TRAMP mice.

To further explore the role of immune cells, in particular B cells, in prolonging

the survival in castrated TRAMP mice, we analyzed the distribution of B cell

subpopulations within splenocytes in the castrated TRAMP mice using flow cytometry.

We found that mature B cells (B220high

) were increased in castrated TRAMP mice

compared with non-castrated TRAMP mice (Figure 4A-B). This suggested that the

17.85

80.89

45.31

54.96

TRAMP Wild TypeWild Type

Spleen

B) C)

A)

17.85

80.89

45.31

54.96

TRAMP Wild TypeWild Type

Spleen

B) C)

A)


increased number of B cells in peripheral blood (data not shown) and spleen of castrated

TARMP mice might be due to expansion of pre-B-cell population in the bone marrow.

Surprisingly, the immature B cells (B220low

) were also elevated in castrated TRAMP

mice compared with non-castrated TRAMP mice (Figure 4A-B). These results suggested

that androgen ablation may have intrinsic, direct effects on the B cell development in the

bone marrow, which might influence the function of B cells.

Figure (3) A) TRAMP mice have a lower survival rate and B) androgen level is low in serum of

castrated TRAMP mice than wt-TRAMP mice.

Figure (4). A-B) Flow Cytometry analysis of the distribution of B cells subpopulations within

spenocyte showed that mature B cells (B220high) were increased in castrated TRAMP

mice compared to Wt-TRAMP mice. A-B) The immature B cells (B220low ) were

elevated in castrated TRAMP mice compared to Wt-TRAMP mice

Androgen level

0

1

4

TRAMP

WtTRAMP

Castration

*

Su

rviv

ing

0.0

1.0

0.8

0.6

0.4

0.2

20 30 40 5010

TRAMP

Wt

TRAMP

Castration

Age ( weeks)

A) B)

Androgen level

0

1

4

TRAMP

WtTRAMP

Castration

*

Su

rviv

ing

0.0

1.0

0.8

0.6

0.4

0.2

20 30 40 5010

TRAMP

Wt

TRAMP

Castration

Age ( weeks)

Su

rviv

ing

0.0

1.0

0.8

0.6

0.4

0.2

20 30 40 5010

TRAMP

Wt

TRAMP

Castration

Age ( weeks)

A) B)

A)

45.31

4.53

53.96

TRAMP Wild Type TRAMP Castration

18.92

17.60

64.16

B)A)

45.31

4.53

53.96


18.92

17.60

64.16

B)

45.31

4.53

53.96


18.92

17.60

64.16

TRAMP Castration

18.92

17.60

64.16

B)


DISCUSSIONS

It is well known that many autoimmune diseases have a strong association with

female. Sex hormones, including androgen, have been shown to be able to regulate

the immune systems (Olsen,et al. 2001, Li, et al. 2006). The causal relationship

between sex hormones and susceptibility to autoimmunity is consistent with the

observation that male rheumatoid arthritis patients display low levels of androgens

(Smithson, et. 1998)and that prostate cancer patients treated with androgen-ablation

therapy were reported to have an increased incidence of rheumatoid arthritis

(Cutolo, et al. 1984,Cutolo, et a. 1991). Furthermore, the increase of mature and

immature B cells has been linked to a variety of tumors and autoimmune diseases, in

this study; we demonstrated B cells were increased in castrated TRAMP mice

compared with non-castrated TRAMP mice, Androgen ablation therapy increases

the survival rate of TRAMP mice. More over, loss of androgen resulted in expansion

of the mature and immature B cells in the castrated TRAMP mice compared with the

non-castrated TRAMP mice. It would be interesting to investigate whether the

castrated TRAMP mice have a higher chance of developing any autoimmune

diseases, such as rheumatoid arthritis.

In mice, expansion of the pre-B-cell population in the bone marrow is

observed after castration (Viselli, et al. 1995, Wilson, et al. 1995) and DHT

supplementation may suppress B-cell precursors (Medina and Kincade, 1994). In

normal male mice, castration leads to a dramatic increase in IgM+ naïve splenic B

cells (Wilson, et al. 1995, Viselli, et al. 1997). Furthermore, this was reversed with

testosterone supplementation, implicating androgens in naïve B-cell production and

export (Medina andKincade, 1994). It would be interesting to furtherinvestigate the

role of androgen in the development B cell in bone marrow, in particular which

stage of B cell development in the bone marrow is affected by androgen. In the

meantime, it is worthwhile to pursue whether there is a link between the effect of

androgen on B cell development and autoimmune diseases and tumors.

The abbreviations used are: AR, androgen receptor; TRAMP, transgenic

adenocarcinoma mouse prostate; DHT, 5-alpha-dihydrotestosterone.

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Food science and human nutrition



137

Probable Atrophied Islet Cells Revival after Treatment with Olea eurpaea and

Lepidium sativum Extracts in Alloxan Induced Diabetic Mice

Amr M. Awad 1*, Ahmed A. Tayel 1.2, Ahmed I. Ibrahim 1

1 Genetic Engineering and Biotechnology Research Institute, Univ. of Sadat City, El-Sadat City, Egypt

2 College of Agriculture and Veterinary Medicine, Qassim Univ., Saudi Arabia.


ABSTRACT. The simulation of atrophied islet cells of the pancreas, by extracts of Olea eurpaea leaves and Lepidium sativum seeds, in alloxan-induced diabetic mice was evaluated. 35 mice were divided into

four groups; Group 1 was given only feed and distilled water throughout the period of the experiment. In

Group 3, mice were pretreated with the extract of O. eurpaea leaves for 15 days, whereas Group 4 mice were pretreated with the extract of L. sativum seeds for 15 days. Diabetes was induced in mice of Groups

2, 3, and 4 with 150 mg/kg of body weight of alloxan monohydrate. Group 2 mice were used as the

experimental positive control. The results showed a significantly high blood glucose level in Group 2 mice indicating the diabetic state. The blood glucose level of mice in Group 3 and 4 reduced when

compared with the value of Group 2 mice. The histopathological examination revealed atrophied islet of

Langerhans in Group 2 mice. Groups 3 and 4 showed remarkable reviving islet cells. The results suggest that the crude extract of O. eurpaea leaves and L. sativum seeds has a protective effect against alloxan

induced pancreatic damage .This study indicates worth anti-diabetic activity of this plants and

recommends the use of herbal drugs for diabetic mellitus treatment .

Keywords: Diabetes mellitus, Plant extracts, Glucose level; Histopathology, β- cell, Lesions

Amr M. Awad et al 138

INTRODUCTION

Diabetes mellitus is a widely spread disease and one of the major causes of death

around the world. Diabetes is a conditional disease characterized by high level of

blood glucose which is caused by an absence or decrease of insulin secretion. The

causes of diabetes mellitus include poor diet, obesity, environmental factors,

hereditary factors and drug usage (Sunday et al., 2012). Alloxan is a chemical

compound used to induce experimental diabetes by leading the beta cells of the

Langerhans islets to swell and finally degenerate. Alloxan diabetic mice have been

reported to have increased vascular permeability, with no recorded fiber loss

(Ragavan and Krishnakumari, 2006). Medicinal plants have always been considered

a healthy source of life for all people. The properties of medicinal plants are very

useful in treatment of various diseases. Many herbals in our environment are known

to possess biological activities that are beneficial in managing diseases (Cowan,

1999). For example plant parts such as Olea eurpaea leaves and Lepidium sativum

seeds, which are used in Arab and Islamic countries for treating many

gastrointestinal disorders, were reported to have potential curative and protective

abilities on damaged tissues (Farag et al., 2003; Orlovskaya and Chelombit'ko,

2007). So that these plants were chosen as candidates for treating alloxan-induced

diabetes mice The importance of using herbal medicine in treating various diseases, like

diabetes mellitus, is to eliminate the possible side and accumulative effects of

synthetic and chemical drugs (Devi et al., 2003; Budhwani et al., 2010). This work

has been done to investigate the effect of O.eurpaea leaves and L. sativum seeds

extracts on diabetes mellitus and histopathological features of diseased mice.

Material and Methods

This experiment was performed to evaluate the, efficacy of O.eurpaea leaves and L.

sativum seeds extract on Alloxan induced diabetic mice.

Plant materials extraction

Dried plant parts (O.eurpaea leaves and L. sativum seeds) were powdered to

get ~ 60-mesh size using a mixing grinder. 50 g from plant powder were mixed with

200 ml of 70% ethanol and agitated using a rotary shaker (150 rpm) for 6 h then

filtered through Buchner funnel using Whatman no. 41 filter paper for removing of

plant particles. Plant residues were re-extracted with 100 ml of solvent, filtered and

the total extracts were pooled and evaporated under reduced pressure using a flash

evaporator (Büchi, Flavil, Switzerland) at 45 °C to omit almost 90% of solvent.

Extracts were further dried in a desiccator under vacuum till constant weight. The

final weights of dry extract were recorded, and then dry matters were suspended in

sterilized water with few drops of Tween 80 to have a concentration of 20% (w/v).

Probable Atrophied Islet Cells Revival …

139

Collation and Acclimatization of Mice The current study was conducted on a total of 35 male inbred C57BL/6J mice

aging 10-15 weeks and weighing 15-25 grams each. Mice were purchased from the

animal house of the Medical Technology Center, Medical Research Institute,

Alexandria University. Mice were grouped in 4 groups.each group of mice was

housed in serene bottomed wire cages arranged in rows and fed on standard pellet

diet (Hindustan Lever Ltd. India).

Housing conditions

Animals were housed in groups of 5 mice into polycarbonate cages and

maintained at controlled temperature (22ο C) with light/dark cycles of 12 hours each

where mice had free access to water and a commercial pellet diet.

Induction of Diabetes

Diabetes was induced by administration of Alloxan (Alloxan monohydrate,

Sigma, St. Louis, MO.) intra-peritoneal, at a dose of 150 mg/kg body weight

(Katsumata et al., 1999).

Treatment

The extract was administered orally at a dose of 1000 mg/kg body weight

from O.eurpaea leaves and at a dose of 1500 mg/kg body weight from L. sativum

seeds for 15 days daily.

Statistical evaluation

Statistical evaluation was done using Minitab program Quality Tools

(capability six pack). Statistical significance was set at (P<0.05).

Experimental Design

The experimental animals were randomized into four groups (groups 1, 2,

3and4). 5 animals on Group 1and 10 animals for other groups and treated as follows:

Group I: Consisted of 5 mice which served as normal control and were

given distilled water and feed only.

Group II: Consisted of 10 Alloxan induced diabetic mice served as control

and having free access to distilled water and feed only.

Group III: Consisted of 10 mice Alloxan induced diabetic mice and were

treated with crude extract of O.eurpaea leaves at dose of 1 g/kg body weight daily

for 15 days once a day.

Group IV: Consisted of 10 mice Alloxan induced diabetic mice and were

treated with crude extract of L. sativum, at dose of 1.5 g/kg body weight daily for 15

days once a day.

Animals were administered with the extract daily. Alloxan was administered

on the specified days after which the experimental animals were fasted overnight


prior to blood glucose determination. Fasting blood glucose of mice in all the groups

were measured before and 72 hours after induction of diabetes. The mice were

sacrificed at the end of the experiment and their pancreas harvested for

histopathological studies. Blood glucose level was determined using the Optima FP-

300 Spectrophotometer.

Histopathological examination of pancreas and liver

Immediately following animal scarification, liver & pancreatic organs were

excised from all animals under study and processed routinely for histopathological

study using conventional Hematoxylin and Eosin stain. This was done as described

by Bopanna et al. (1997) with minor modifications.

1-Fixation: The pancreatic tissues and liver tissues were fixed in neutral

buffered formalin (10% formaldehyde in phosphate buffered saline PBS); a process

that stabilizes the tissues to prevent decay.

2-Embedding: The fixed pancreatic specimens and liver specimens were

consecutively immersed in ascending concentration gradient of ethanol (70, 80, 90

and 100%) to dehydrate the tissue, followed by a clearing agent such as xylene and

finally hot molten paraffin wax (impregnation).

3-Sectioning: The embedded pancreatic tissues and liver tissues were then

sectioned into very thin (2-8 µm) sections using a microtome. These slices were then

placed on a glass slide for staining.

4-Staining : To facilitate microscopic examination, the pancreatic tissue

sections and liver tissues were stained with one or more stain(s) to give contrast to

the tissue; as without staining, it would be very difficult to identify differences in

cell morphology. Hematoxlyin and Eosin (H&E) are the most commonly used stains

in histology and histopathology. Hematoxlyin stains nuclei blue while Eosin stains

the cytoplasm pink.

RESULT AND DISCUSSION

The present study was carried out on 35 male inbred C57BL/6J mice that were used

as a model for experimental possible revival of atrophied islet cells of the pancreas

by O. eurpaea and L. sativum extracts in Alloxan induced diabetic mice. Animal

sacrification was performed following the end of each induction protocol at 15 days

after Alloxan injection and daily treatment by O.eurpaea leaves and L. sativum.

Alloxan induced selective cytotoxicity in the β-Cell of pancreas through the reactive

oxygen species generation, and this resulted in the reduction of synthesis and release

of insulin. The induced hyperglycemia by Alloxan was associated with polydipsia

and loss in body weight (Dunn et al., 1943; Roy et al., 2005). Sera were collected for

monitoring glucose level; pancreas was subjected to a detailed histopathological

study for monitoring islet cell histological changes as well as inflammatory

infiltrates and treatment effect on the body weight.


141

Monitoring Diabetic Status

Effect on body weight: In positive control and experimental diabetic mice a

weight losses were observed in this study .The reduction in weight in diabetic group

was significant when compared with normal control group. The effects of

administration of extracts of O.eurpaea leaves and L. sativum on the body weight in

"Normal control, Diabetic, and Diabetic treated mice” is recorded in Table (1) after

15 days of administration to plant extract .The body weight increased significantly

(p<0.005) to extent of 3.514 to 10.700 %, compared to pretreatment period, whereas

in diabetic control group, the body weight decreased by 9.901%. As far, the relative

efficacy in increasing or maintaining body weight (4.195%) was recorded in

O.eurpaea extract treatment, whereas weight of diabetic group treated with L.

sativum extract increased with 3.514%.

Table (1). Effect of treatment with O. eurpaea and L. sativum extracts on the body weight gain in

normal and Alloxan induced diabetic mice.

Groups

of mice

Treatment mg/kg body

wt. oral

Body weight (gm)

Pretreatment 0 day Post treatment day 15 (Mean + SD)

Mean + SD Percentage deviation

1 Normal control 21.7±3.5 24.3±3.6 10.700

2 Diabetic control 22.2±2.9 20.2±2.7 9.901-

3 1000 O.eurpaea leaves 27.4±3.1 28.6±2.9 4.195

4 1500 Lepidium Sativum 30.2±4.6 31.3±4.3 3.514

Observations of the study support the results of some researches who

reported significant increase in the body weight after treatment with herbal

preparation in hypoglycemic animals (Bopanna et al., 1997; Pari and Saravanan;

2000; Devi et al., 2003).

Effect on blood glucose level in normal and experimentally diabetic mice:

Alloxan caused specific destruction of cells of islet of pancreas and resulted in the

increase in blood glucose levels. It was evident that Alloxan administration of

150mg/kg body causes diabtogenic response in mice, the effect of administration to

the extracts of O.eurpaea and L. sativum on blood glucose in normal control,

diabetic control and diabetic treated mice is shown in Table 2 .


Table (2). Effect of O. eurpaea and L. sativum extracts on the blood glucose levels in normal and

Alloxan treated diabetic mice

Groups

of rat

Treatment mg/kg body

wt. oral

Glucose levels mg/dl

Pretreatment 0 day Post treatment day 15 (Mean + SD)

Mean + SD Percentage deviation

1 Normal control 89.7±15.1 73.3±13 -18.28

2 Diabetic control 123.4±16 129.4±9.9 4.8622

3 Olea Eurpaea 117.1±16.2 74.1±7.5 -36.72

4 Lepidium Sativum 123.3±9.7 75.5±8.4 -38.8433

After 15 days of treatment with of O.eurpaea and L. sativum extracts, mean

levels of blood glucose decreased significantly to 74.1and 75.5 mg/dl, as compared

to diabetic positive control (129.4 mg/dl).

The extract mechanism in reducing blood glucose level is not well

understood. The probable cause of reduction of blood glucose might be to increase

glucose uptake peripherally and increase sensitivity of insulin receptor in case of L.

sativum extract. Blood glucose reduction following administration of insulin is well

documented (Yeap et al., 2010).

Fig (1). Microscopic examination of normal

control showing normal pancreatic

tissues formed of normal exocrine

glands and interlobar septa .(H&E×40)

Fig (2). Normal Mice Pancreas showing exocrine

glands A, interlobar septa B and Islets of

Langerhans C (H & E x 40).

A

B C


143

Fig (3). Mice Pancreas treated with Alloxan after 7

days showing atrophied islets A and

severe vascular congestion B (H & E x40)

Fig (4). Pancreatic islet showing moderate

reduction in size and cellularity

(arrow), mild degenerative changes

(H&E x40).

Fig (5). Microscopic examination of pancreatic

tissues by treated Olea e-urpaea leaves

showing mildly regenerating islets A and

decrease interlobar septa (H & E x 40)

Fig (6). Mice pancreas given Olea eurpaea

leaves showing moderately

regenerating islets A and mild vascular

congestion B and There is still mild

recovery of the islets as well as mild

inflammation (H &E x 40).

B

A

A

A

B


Fig(7). Microscopic examination of pancreatic

tissues by treated Lepidium Sativum

showing mildly regenerating islets A and

decrease interlobar septa (H & E x 40)

Fig (8). Mice pancreas given L. sativum showing

moderate vascular congestion A and

mild infiltrates of lymphocytes B (H &

E x 40). Sections showed mild islet

recovery, mild vascular congestion and

mild peri-islet infiltrates of lymphocytes

and There is still mild recovery of the

islets as well as mild inflammation

The microscopic examination of histopathological samples from treated

animals is illustrated in Figures (1-8). Pancreas tissue examination following

histopathology showed that, comparing to normal group (Fig 1 and 2), the pancreas

of Alloxan diabetic group reduced in size which could alter the release of insulin and

thus, hinder glucose uptake (Fig 3 and 4). Fig (2, 3) showed the reviving of islet

cells which increases insulin release. They also showed increase in lymphocytes as a

means of self-regulation and protection. Inflammation or necrosis of cells could

result into increase in lymphoid follicles and neutrophils in response to tissue

damage (Fig 5 and 6). The incidence of diabetes surged over the years, with no

defined measure of treatments, requires approaches for prevention, to revive and

increase the intergrity of pancreatic cells. The extract of O.eurpaea leaves as

depicted from the results has the ability to maintain the integrity of pancreatic cells.

The extract of L. sativum lead treated pancreas cells to be gathered together and

small preserved islets similar to the normal on Fig (7, 8)

Our findings reveal that the significant decrease of serum glucose level in

extract treated diabetic mice. Alloxan does not only destroy the pancreatic β-cells

but causes kidney damage, which is however reversible, while streptozotocin

selectively destroys pancreatic insulin secreting β-cells (Gilman, 1990).The present

study revealed that the immediate action of Alloxan induced diabetes by destroying

β-cells even at a single dose of 150 mg/kg of body weight. The ultra-structure of

Alloxan diabetic pancreas showed considerable reduction in the Langerhans islets

and depleted islets. The diabetic mice showed pancreatic islet regeneration

(Yamamoto et al., 1981). The regenerative effect of the pancreatic cells by

A

B

A


145

O.eurpaea and L. sativum extracts showed that exocrine cells of pancreas may

enlighten the positive effects of these agents on the production of insulin.

CONCLUSION

The crude extract of Olea Eurpaea leaves and L. sativum seeds showed protective

property against Alloxan induced diabetes. However, this study gives a strong hit for

further toxicity and mechanistic study of these extracts for protecting and reviving

the atrophied islet cells. This will increase the acceptability and control of the use of

the plant extracts.

REFERENCES:

Bopanna K.N., Kannan J., Sushma G., Balaraman R., Rathod S.P. (1997). Antidiabetic and antihyperlipaemic effects of neem seed kernel powder on

alloxan diabetic rabbits, Indian Journal of Pharmacology, 29:162-167.

Budhwani K., Shrivastava B., Singhai A. K., Chautrvedi L., Budhwani G.

(2010). Exploring Herbal Solution For Diabetes. Pharmacia,1 (1): 29-32.

Cowan, M.M. (1999). Plant products as antimicrobial agents. Clinical Microbiology

Reviews, 12: 564–82.

Devi B.A., Kamalakannan N., Prince P.S.M. (2003). Supplementation of

Fenugreek leaves to diabetic rats: Effect on carbohydrate metabolic enzymes

in diabetic lives and kidney. Phytotherapy Research,17:1231-1233.

Dunn D., Sheehan H., Letchin N.M. (1943). Necrosis of islets of langerhans

produced experimentally. Lancet, 1: 484-487.

Farag, R.S., El-Baroty, G.S., Basuny, A.M., (2003). Safety evaluation of olive

phenolic compounds as natural antioxidants. International Journal of Food

Sciences and Nutrition, 54, 159–174.

Gilman A.G. (1990). Goodman and Gilman’s the pharmacological basis of

therapeutics. Pergamon Press, New York, 8th edn, pp. 1317- 1322.

Katsumata K., Kastsumata Y.,Ozawa T., Katsumata K. Jr.(1999). Potentiating

effects of combined usage of three Sulfonylurea drugs on the occurrence of

Alloxan diabetic rats. Hormone and Metabolic Research, 25:125-126.

Orlovskaya T. V., Chelombit'ko V. A. (2007). Phenolic compounds from

Lepidium sativum. Chemistry of Natural Compounds, 43 (3): 323.

Pari L., Saravanan G. (2000). Antidiabetic effect of cogent db, a herbal drug in

Alloxan induced Diabetic mellitus .Comparative Biochemistry and

Physiology, 131:19-25.


Ragavan, B., Krishnakumari.S. (2006). Effect of T. Arjuna Stem Bark Extract on

Histopathology of Liver, Kidney and Pancreas of Alloxan-Induced Diabetic

Rats. African Journal of Biomedical Research, 9: 189 - 197.

Roy S., Sehgal R., Padhy B. M. and Kumar V. L. (2005). Antioxidant and

protective effect of Calotropis procera against alloxan diabetes in rats.

Journal of Ethnopharmacology, 102:470-73.

Sunday J. J., Spencer N. C. O., Kingsley O., Akintola A. A., Binyelum N.,

Favour A. O. (2012). Possible Revival of Atrophied Islet Cells of the

Pancreas by Vernonia amygdalina in Alloxan Induced Diabetic Rats. Journal

of Applied Pharmaceutical Science, 2 (9):127-131.

Yamamoto H., Uchigata Y., Okamoto H. (1981). STZ and alloxan induces DNA

strand breaks and poly (ADP ribose) synthetase in pancreatic islet. Nature,

294: 284-286.

Yeap S.K., HO W.Y., Beh B. K., Liang W. S., Ky H., Yousr A.N., Alitheen N. B.

(2010). Vernonia amygdalina, an ethnoverterinary and ethnomedical use of

green vegetables with multiple bioactivities. Journal of medicinal plants

research, 4(25): 2787-2812.



147

Evaluation of different modification methods for potato starch and its

applications in salad dressing manufacture

Gadallah, Mohamed G.E.1*, Yousif, El-Sayed I.2 and Seror, Afaf, M.3

1Food Science and Human Nutrition Department, College of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia

2Food Science Dept., Fac. of Agric., Ain Shams Univ., Shoubra El-Kheima, Cairo, Egypt.

3Starch and Glucose Company, Cairo, Egypt

*Corresponding author e-mail ( [email protected] )


ABSTRACT. The effect of pregelatinization, acid-thinning and dextrinization methods on physicochemical

and rheological properties of potato starch was evaluated. The obtained modified potato starches were used at

5% to replace fat in salad dressing processing. No significant difference was noticed in moisture content between native and pregelatinized potato starches. The nitrogen free extract (99.20 - 99.91%) suggest that

pure potato starch was obtained by the applied isolation method. A gradual decrease in water binding

capacity was recorded for dextrinized and acid-thinned potato starches compared to native potato starch. Similar observation was recorded for oil binding capacity for acid-thinned and pregelatinized potato starches

in compared to native potato starch. Data also indicated that higher solubility and lower swelling power at 90 oC were recorded by dextrinized potato starch. The large granules probably had slightly lower pasting temperature and higher peak viscosity at 95 oC for acid-thinned and native potato starch as compared to that

from the pregelatinized potato starch which recorded slightly lower peak viscosity. Emulsion stability was higher for control salad dressing, followed by samples containing pregelatinized and acid-thinned potato

starches. It could be concluded that salad dressing with 5% of native, pregelatinized and acid-thinned potato

starches had acceptable sensory attributes.

Keywords: modified potato starch, physicochemical properties, salad dressing, sensory evaluation

Gadallah, Mohamed G.E et al 148

1. INTRODUCTION

Starch is necessary to impart functionally desirable attributes to foods. Native

starches provide viscous, cohesive and sticky pastes when they are heated and gels

when these pastes cool. (Adebowale et al., 2005). In general, native starch presents

low shear stress resistance and thermal decomposition, in addition to high

retrogradation and syneresis. Starch can be modified to obtain pastes with specific

attributes that can resist extreme food processing requirements like heat, stirring and

low pH conditions.

Physical modification of starch is made using heat and moisture

(pregelatinization); while chemical treatments involve the introduction of functional

groups into the starch molecule using reactions of derivatization (etherification,

esterification and cross linking) or decomposition (acid or enzymatic hydrolysis and

oxidation) (Singh et al., 2007). Knowledge about physical and chemical

modification effects on starch granules structure is necessary to understand their

functional properties and allow development of starches with desired properties

enhancing their uses especially in food industry. Modification treatment, reaction

conditions and starch source are critical factors that govern the pasting behavior of

starch pastes (Reddy & Seib, 1999; Gonzalez & Perez, 2002 and Singh et al., 2004).

The physical and chemical properties are related to the structural and

molecular features of the starch granules. The ratio of amylose to amylopectin,

length of amylopectin chains, starch granule size distribution, and phosphate content

are among the parameters known to influence the granule properties. (Vasanthan &

Bhatty, 1995). Potato starch granules show a unimodal size distribution. Several

properties are correlated to the granule sizes as e.g. the viscosity and phosphate

content with corresponding influence on quality and properties of starch blends and

food products (Wang et al., 2003).

Salad dressing is an oil-in-water emulsion, where oil is the discontinuous

phase and water is the continuous phase. According to United States Food and Drug

Administration (FDA), Salad dressing is defined as a semisolid emulsified food with

the same, ingredient and optional ingredients as salad dressing with the exception of

cooked starch paste. There are two types of salad dressing; pour-able and spoon-able

which vary in flavor, chemical and physical properties (especially viscosity). The

example of spoon-able is salad dressing and the pour-able one is salad cream

Babajide and Olatunde (2010).

Therefore, the aim of this study was to investigate the effects of different

modification methods on physicochemical properties of potato starch and its

application in salad dressing.

Evaluation of different modification methods …

149

2. MATERIALS AND METHODS

2.1. Materials:

Fresh tubers of potatoes (medium size), corn oil, eggs, salt (sodium chloride), sugar

(sucrose), fresh mustard flour, vinegar 5% and white pepper were purchased from

the local market, Cairo, Egypt.

2.2. Potato starch isolation:

The starch was isolated from potato tubers according to the method of

Jimenez–Hernandez et al., (2007). The extracted starch was packed in glass

container and stored at room temperature (23 ± 1 oC) till further analysis.

2.3. Preparation of modified potato starches:

2.3.1. Pregelatinized starch:

Potato starch solution 1:1 (750 g starch + 750 ml deionized water) was

incubated at 63oC for 5 min. Pregelatinized starch was produced by drying at room

temperature (23 ± 1oC) for 24 h according to Knight, (1969).

2.3.2. Acid thinned starch:

Potato starch (750 g) was mixed with 375 ml of 0.1N HCL solution and 375

ml deionized water for 30 min. Then, the pH was adjusted to 7.0 with 1N NaOH.

Neutralized starch was dried at room temperature (23 ± 1oC) for 24 h, following

washing three times with distilled water and filtration with filter paper No.1. Acid-

thinned starch was produced by drying at room temperature (23 ± 1oC) for 24 h

according to Caglarirmak and Cakmakli, (1993).

2.3.3. Dextrinized starch:

Potato starch (750 g) mixed thoroughly with 600 ml of 0.1N HCL and then

dried at 50oC for 32h to 5% moisture content. The dried starch was dissolved in 750

ml deionized water and pH was adjusted to 7.0 by adding 0.1N NaOH solution. The

starch was dried at room temperature (23 ± 1oC) for 24 h according to Caglarirmak

and Cakmakli, (1993).

2.4. Chemical composition:

Native and modified potato starches and salad dressing were analyzed for their

moisture, ash, lipids (ether extract) and crude protein contents (N x 6.25) according to

the methods described in AOAC (2000). The nitrogen free extract (NFE) was

calculated by difference. Caloric values of salad dressing were calculated with

following equation, caloric values = (crude protein x 4) + (lipids x 9) + (NFE x 4).

2.5. Physical properties of potato starches:

Bulk density, water binding capacity, oil binding capacity and pH of native

and modified potato starches was determined according to the methods described by

Adeleke and Odedeji (2010).


2.6. Swelling power and solubility:

Swelling power and solubility were determined at 50, 70 and 90 o

C using the

method of Leach et al., (1959). A 1% aqueous suspension of starch (100 ml) was

heated in a water bath at 90oC for 1 h with constant stirring. The suspension was

cooled for half an hour at 30oC. Samples were then poured into preweighed centrifuge

tubes, centrifuged at 3000 xg for 10 min and weight of sediments was determined. For

the measurement of solubility, the supernatants were poured into aluminium dishes

and evaporated at 110 ± 2 oC for 12 h and weight of dry solids was determined.

2.7. Visco-amylograph parameters:

Rheological properties of native and modified potato starch pastes were

measured by using Barabender amylograph (OHG Duisburg kulturstra Be 51-55, D-

4100 Duisburg, Germany) according to Merco and Juliano (1981). The parameters

calculated from the obtained amylograms were gelatinization temperature oC.,

maximum viscosity, viscosity at 95 o

C (B.U), viscosity after 15 min at 95 o

C (B.U),

viscosity at 50 o

C (B.U), viscosity after 15 min at 50 o

C (B.U), break-down (B.U)

and setback (B.U).

2.8. Processing of salad dressing:

Salad dressing was prepared according to procedure described by Singh and

kulkarni (1990) as follows: the ingredients of the salad dressing were: oil 40%, fluid

egg yolk 4%, salt 2%, sugar 11%, mustard flour 0.5%, starch 5%, vinegar 14%,

white pepper 2% and water to make 100%. Starch paste was prepared in water using

85 ± 5 o

C for 5 min; the vinegar was then added on composition of cooking. This

was then mixed with egg yolk, other dry ingredients and salad oil with moderate

agitation and then passed three times through a colloid mill. Native and modified

potato starches were used at 5% to replace fat in salad dressing; the product was

packed in glass containers and stored at 4 to 5oC until further analysis. The same

ingredients without modified starch were used to prepare the control salad dressing.

2.9. Emulsion stability of salad dressing:

Emulsion stability (%) of salad dressing samples was determined according to

the method of Sathivel et al., (2005) as follows: Salad dressing sample (25 g) was

placed into a 10 ml centrifugal tube and stored at –20 oC for 2 days and then allowed to

thaw at room temperature (23 ± 1oC) for 1h. The thawed samples were centrifuged at

1500 xg for 40 min, and the amount of oil was measured. Oil recovery percentage was

calculated as weight of oil recovered / 25 g of salad dressing sample x 100.

2.10. Sensory evaluation of salad dressing:

Salad dressing samples prepared with native and modified potato starches

and control sample were subjected to sensory evaluation as described by Babajide

and Olatunde (2010). Sensory characteristics like appearance (10), color (10),

flavour (10), consistency (10) and overall acceptability (10) were evaluated by 10


151

trained panellists of the staff numbers of Food Science Department, Faculty of

Agriculture, Ain Shams University.

2.11. Statistical analysis:

Statistical analysis was carried out using the PROC ANOVA followed by

Duncan’s multiple range test for comparison between means (from 3 replicates)

using Statistical Analysis System program (SAS, 1996). Different alphabetical

letters in the column are statistically differed at 5% level of significant. (Snedecor

and Cochran, 1980).

3. RESULTS AND DISCUSSION

3.1. Proximate composition of potato starches:

The data of the proximate analysis of the native and modified potato starches are

shown in Table (1). No statistical (P ≤ 0.05) difference was observed between the

moisture content of native and pregelatinized potato starches, they had an ordinary

value of 14.05% being significantly less than that of acid-thinned starch and more

than that of dextrinized one. In this respect, Jimenez – Hernandez et al., (2007)

indicated that commercially up to 20% of moisture is allowed in starch as raw

material. The contents of crude protein ranged between 0.33% in acid thinned starch

to 0.55% in native potato starch, meanwhile the lipid contents of potato starch

samples ranged between 0.11% for pregelatinized starch to 0.18% in native starch.

No statistical (P ≤ 0.05) differences were found in the ash content between the

pregelatinized and acid-thinned potato starches (0.20%) being less than those of

dextrinized (0.30%) and native starch (0.22%). The starch ashes are mainly

composed of phosphorus, sodium, potassium, magnesium and calcium (Beynum and

Roels, 1985). The data obtained for calculated NFE which ranged from 99.20% to

99.91% suggest that pure starch was obtained by the isolation methods employed in

this work. These results are in agreement with Jimenez–Hernandez et al., (2007).

Table (1). Proximate composition of native and modified potato starches (% on dry weight basis).

Starch samples Moisture

Crude

protein

(N x 6.25)

Lipids Ash

Nitrogen

free extract

(NFE)*

Native potato starch 14.05b 0.55a 0.18a 0.22b 99.91d

Modified potato starch

Pregelatinized 14.05b 0.42b 0.11d 0.20c 99.27b

Acid-thinned 14.45a 0.33d 0.14c 0.20c 99.33a

Dextrinized 13.70c 0.35c 0.16b 0.30a 99.20c

Values followed by the same letters in the same column are not significantly different (p ≤ 0.05).

NFE* calculated by difference. No. of replicates: 3.


3.2. Physical properties of potato starches:

The bulk density (BD), water binding capacity (WBC) and oil binding

capacity (OBC) of native and modified potato starches by pregelatinization, acid-

thinning and dextrinization are presented in Table (2). Significant (P ≤ 0.05)

differences in bulk density and pH values of native, pregelatinized, acid-thinned and

dextrinized potato starches could be observed. Bulk density value for native potato

starch was 0.866 g/cm3, while dextrinized potato starch recorded 0.904 g/cm

3. Bulk

density is generally affected by the particle size and density of the flour and it is

very important in determining the packaging requirement, material handling and

application in wet processing in the food industry (Karuna et al., 1996).

A gradual decrease in the WBC of the potato starch were recorded for

dextrinized and acid-thinned potato starch samples being 1.37 and 1.36 g/g,

respectively in compared to 1.42 g/g for the native potato starch. Similar observation

was recorded for OBC for acid-thinned and pregelatinized potato starch in compared

to the native potato starch being 1.84, 1.52 and 1.98 g/g, respectively. Hence, both

hydrophilic and hydrophobic capacities of the native potato starch were impaired

after dextrinization. The low WBC of starch samples may be due to the reduction of

the amorphous region in the starch granules. This reduces the number of available

binding sites for water in the starch granules, Lawal (2004). Contrarily, WBC

increased after pregelatinization of potato starch, which may be attributed to the fact

that the hydrophilic tendency of starch increases after heat-moisture treatment

(Singh et al., 2009).

Table (2). Physical properties of native and modified potato starches.

Starch samples Bulk density

(g/cm3) WBC (g/g) OBC (g/g) pH

Native potato starch 0.866d 1.42b 1.98a 6.48d


Pregelatinized 0.877c 1.53a 1.84a 6.63c

Acid-thinned 0.889b 1.36c 1.93a 7.08a

Dextrinized 0.904a 1.37c 1. 52b 6.97b


WBC: water binding capacity, OBC: oil binding capacity. No. of replicates: 3.

3.3. Swelling power and solubility of potato starches:

The swelling power and solubility of native and modified potato starches

differed significantly (P ≤ 0.05), except, solubility of pregelatinized and acid-

thinned potato starches at 90oC as shown by Figure (1). The high swelling power of

the potato starches might be due to the weak internal organization resulting from


153

negatively charged phosphate ester groups within the granule, Kim et al., (1996).

The high swelling and water binding capacity of native, pregelatinized and acid

thinning potato starches make it a potential additive in some types of food products,

as these properties are essential for proper texture in these foods.

All modified potato starches, except dextrinized potato starch exhibited lower

solubilities at 90oC, less disintegration take place during gelatinization and caused

these lowering of solubility Figure (2). The solubility of the native, pregelatinized

and acid-thinned potato starches at 90oC was 13.87, 12.49 and 12.52 %,

respectively. A higher solubility (52.38 %) and lower of swelling power 8.13 g/g

were noticed for dextrinized potato starch at 90oC. The behavior of dextrinized

potato starch may be as a result of depolymerization and structural weakening of the

starch granules. Similar observations are obtained by Singh et al., (2004) and Ferrini

et al., (2008).

Fig. (1). The swelling power as a function of heating treatment of native and modified potato

starches.

0.00

5.00

10.00

15.00

20.00

25.00

30.00

50 70 90

Sw

ell

ing

po

wer

(g/g

)

Temperature oC

Native potato starch Pregelatinized potato starch

Acid-thinned potato starch Dextrinized potato starch


Fig. (2): The solubility as a function of heating treatment of native and modified potato starches.

3.4. Visco-amylograph parameters of potato starches:

The characteristics of native and modified potato starch pastes are given in

Table 3. The gelatinization (pasting) temperature of potato starch samples ranged

between 61.75oC for acid-thinned to 64.30

oC for pregelatinized potato starch. The

large granules probably had slightly lower pasting temperature and higher peak

viscosity at 95oC which being 1045 and 1030 B.U for acid-thinned and native potato

starch, respectively, as compared with that from the pregelatinized potato starch

which recorded slightly lower hot peak viscosity (685 B.U). The decrease in pasting

temperature was attributed to depolymerization of the starch molecules, resulting in

a weakened granule organization (Chavez-Murillo et al., 2008). In addition, higher

amylose content lower initial pasting temperature, or probably due to the absence of

free fatty acids and lysophospholipids in tuber and root starches (Gujska et al., 1994,

Ferrini et al., 2008).

The acid-thinned and native potato starches showed an increase in the paste

viscosity at 95oC (Maximum viscosity and viscosity at 95

oC, respectively). However,

a drastic fall in viscosity was recorded from 1045 to 670 B.U. and from 1030 to 670

B.U, when the acid-thinned and native potato starch pastes were holed for 15 min at

95oC. The structure of starch probably became more fragile by stirring, resulting in

the decrease of viscosity and the increase of breakdown (Ohishi et al., 2007). On the

other side, during the holding period for 15 min at either 95 or 50oC, gelatinized

potato starch showed gradual increases in viscosity from 610 to 670 B.U, thus

improved breakdown of starch and from 670 to 805 B.U up to the end of cooling

time. Gelatinized potato starch exhibited a slightly larger degree of setback or

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

50 70 90

So

lub

ilit

y (

%)

Temperature oC

Native potato starchPregelatinized potato starchAcid-thinned potato starch

Dextrinized potato starch


155

retrogradation as compared to native and acid-thinned potato starch. The differences

in the set-values after cooling to 50oC had been attributed to differences in amylose

content of the starch samples (Paredez-Lopez, 1988). The results of potato starch

pasting properties suggest that, the samples with high paste viscosities are desirable

in used as thickeners, whereas low peak viscosities are desirable for high-calorie

food formulations such as weaning food and specially foods. Similar observations

are in accordance with Weissenborn et al., 1994, Jimenez-Hernandez 2007 and

Yadav et al., 2007.

Table (3): Visco-amylograph parameters of native and modified potato starches.

Starch samples Gelatiniza-

tion temp. oC

Maximum

Viscosity Viscosity

at 95 oC

(B.U.)

Viscosity

after 15

min at 95

oC (B.U.)

Viscosity

at 50 oC

(B.U.)

Viscosity

after 15

min at 50

oC (B.U.)

Break-

down

(B.U.)

Setback

(B.U.)

oC B.U*

(1) (2) (1) (2)

Native 63.25 95.00 1030 1030 675 660 700 0 355 -370 -15


Pregelatinized 64.30 93.00 685 610 670 760 805 75 15 150 -90

Acid-thinned 61.75 95.00 1045 1045 670 650 680 0 375 -395 -20

Dextrinized --- --- --- --- --- --- --- --- --- --- ---

*B.U.: Brabender Unit., ---: Not recorded. No. of replicates: 3.

Breakdown (1) = maximum viscosity – viscosity at 95 oC

Breakdown (2) = maximum viscosity – viscosity after 15 min at 95 oC

Setback (1) = viscosity at 50 oC - viscosity at 95 oC

Setback (2) = viscosity at 50 oC – viscosity after 15 min at 95 oC

3.5. Proximate composition and caloric values of salad dressing:

The proximate composition and caloric values of salad dressing incorporated

with native and modified potato starches are listed in Table (4). The moisture

content of the salad dressing samples ranged between 43.50 % for the sample

containing native potato starch to 47.69 % for the sample containing dextrinized

potato starch. Protein content in salad dressing containing dextrinized and acid-

thinned potato starch showed a lower values being 1.06 and 1.07%, respectively

than that of the control sample (1.16%) and those containing native and

pregelatinized potato starch (1.15%). Salad dressing containing dextrinized potato

starch had lower lipid content (73.99%), without significant (P ≤ 0.05) difference

between the samples containing native, pregelatinized and acid-thinned potato

starches. while the control sample exhibited significantly the highest lipid content

being statistically similar to that containing native potato starch. Salad dressing


containing acid-thinned potato starch exhibited higher ash content (2.20 %) than

other samples, the ash content ranged between 2.03% in salad dressing containing

dextrinized potato starch to 2.07% in the sample containing pregelatinized potato

starch. The caloric values of salad dressing samples containing potato starches

recorded the values ranged between 761.80 k. calories for that containing

dextrinized potato starch to 771.69 k. calories for the control sample. These results

are in accordance with Liu et al., (2007).

Table (4): Proximate composition of salad dressing incorporated with native and modified potato

starches.

Samples Moisture

Crude

protein

(N x 6.25)

Lipids Ash

Nitrogen

free extract

(NFE)*

Caloric

values

Control

(without

starch)

47.40ab 1.16a 75.99a 2.06b 20.79b 771.69

Salad dressing containing 5 % potato starch

Native 43.50c 1.12ab 75.16ab 2.05b 21.67ab 767.58

Pregelatinized 45.37bc 1.15a 74.12b 2.07b 22.66a 762.30

Acid-thinned 47.14ab 1.07bc 74.24b 2.20a 22.48a 762.40

Dextrinized 47.69a 1.06c 73.99b 2.03b 22.93a 761.80


No. of replicates: 3.

3.6. Emulsion stability and pH of salad dressing:

Emulsion stability of salad dressing samples containing potato starches were

discerningly arranged in the following order: salad dressing samples containing

native potato starches > samples prepared with dextrinized potato starch > samples

containing acid-thinned potato starch and finally that containing pregelatinized potato

starch (Table 5). Oil recovery was higher for control sample (100 %), followed by

samples containing pregelatinized and acid-thinned potato starches. Oil separation

indicates a coalescence destabilizing mechanism (Girard et al., 2002 and Diftis et al.,

2005). The destabilization velocity of oil-in-water emulsions is strongly influenced

by the droplet size and concentration (Chanamal & McClements 2000 and Perrechil

et al., 2010). All salad dressings showed a pH value between 3.63 and 3.74.


157

Table (5). Quality attributes of salad dressing incorporated with native and modified potato

starches.

Samples Emulsion stability

(Oil recovered %) pH

Control (without starch) 100.00a 3.68b

Salad dressing containing 5 % potato starch

Native 97.60d 3.74a

Pregelatinized 99.80b 3.63c

Acid-thinned 99.75b 3.69b

Dextrinized 98.20c 3.69b


No. of replicates: 3.

3.7. Sensory characteristics of salad dressing containing potato starches:

The mean score values of sensory characteristics (appearance, color, flavor,

consistency and acceptability) of the salad dressing samples containing native and

modified potato starches compared to that of control samples are illustrated by

Figure (3). No significant (P ≤ 0.05) differences could be found in all sensory

characteristics between control and samples containing native, pregelatinized, acid-

thinned and dextrinized potato starches. Therefore, preparation of salad dressing

with native, pregelatinized and acid-thinned potato starches will not negatively

affect the sensory attributes of salad dressing. Similar findings are observed by

Adebayo et al., (2010) and Babajide & Olatunde (2010).


Fig. (3). Sensory characteristics of salad dressing containing native and modified potato starches.

CONCLUSIONS

In this work the effect of modification method on physicochemical properties of

potato starches was investigated. The high swelling and water binding capacity of

native, pregelatinized and acid thinning potato starches make it a potential additive

in some types of food products. The results of potato starch pasting properties

suggest that, the samples with high paste viscosities are desirable in used as

thickeners, whereas low peak viscosities are desirable for high-calorie food

formulations such as weaning food and specially foods. It could be concluded that

the preparation of salad dressing with native, pregelatinized and acid-thinned potato

starches will not negatively affect the sensory attributes of salad dressing.

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163

Impact of Different Dietary Proteins on Blood Glucose and Lipids Profile in

Diabetic Rats

El- Hofi, M.A.*; A.E. Metwly *; I.S. Ashoush**; Safaa A. Abd El-Aziz***

and Marwa, M. Yousef ****

*Food Science Dept., Fac. Agric., Ain Shams Univ., Shoubra El-kheima Cairo, Egypt

**Food Science and Human Nutrition Dept., Fac. Agric. & Vet. Medicine, Qassim Univ., P.O. Box 6622, Buraidah 51452, Kingdom of Saudi Arabia, E-mail: [email protected]

***Medicinal Food, National Organization for Drug Control and Research (NODCAR), Giza, Egypt

**** Egyptian Society for Diabetes Care- Giza, Egypt


Abstract. In the current study the effect of whey protein concentrate (WPC), whey protein hydrolysate (WPH), casein and soy protein (SP) on blood glucose, lipid profile and protein pattern in alloxan-induced

diabetes in rats was investigated. Male albino rats were divided into six groups each of 7 rats. Two groups

served as control (normal and diabetic rats) and the other four groups were induced diabetes then given the diet supplemented with 20 % of casein, WPC, WPH and SP for 28 days.

The results revealed increases in serum biochemical parameters including glucose, triglycerides,

total cholesterol and LDL-cholesterol, in addition to decreases in HDL-cholesterol and body weight. While, at the end of the feeding period, all tested groups showed increase in their body weight in different

values, also the mentioned biochemical parameters turned to their initial values. On the other hand non-

significant changes were found in protein pattern parameters.

Therefore it can be concluded that the highest recovery of hypoglycemic effect was observed in

the groups which received whey protein hydrolsate, soy protein came in the second place followed by

whey protein concentrate while, casein came in the last place.

Keywords: Hyperglycemia; Glucose; Lipid profile; Protein pattern; Rats

El- Hofi, M.A.; et al 164

INTRODUCTION

Milk as food has thousands of years of history. It is considered to be the ideal food for the new born of all species. Isolated milk protein products, as food ingredients, have become commercially available only in this century. In bovine milk 80% of the proteins are caseins and 20% whey proteins. Caseins are well known for their good nutritive value and excellent functional properties for food formulation (Freidman, 1996). Whey proteins are increasingly being used for nutritional purposes because they consistently score high in traditional tests of protein quality (Potter et al. 1993). Protein of animal origin, such as casein, are generally hypercholesterolemic and atherogenic, when compared with vegetable proteins, as soy bean protein, in both humans and experimental animals (Carrol and Kurowska 1995).

Hyperglycemia is one of the major risk factors in the development of vascular complications in diabetes, which is treated with diet and insulin administration, intensive blood glucose control dramatically reduces the complications that result from poorly controlled diabetes. However, for many patients, achieving tight glucose control is difficult with current regimens. Thus, development of a novel adjuvant therapy is needed to achieve better control of glycemia in diabetic patients (Shah et al. 2007).

Whey proteins are particularly insulinotrophic, compared with casein in cheese and the other proteins of animal or vegetable origin (Nilsson et al. 2004). Previously; skim milk was reported to have insulinotrophic effects in untreated type 2 diabetic subjects (Gannon et al. 1986), it is known that proteins vary with respect to their effects on glucose metabolism in type 2 diabetic subjects and may stimulate insulin release and attenuate blood glucose response (Gannon et al. 1988). Food proteins are also capable of stimulating insulin response in the absence of carbohydrates and congestion of dietary protein and glucose may have synergistic effects on insulin response (Saeed et al. 2002).

The present study was designed to assess the antidiabetic effects of nutraceutical milk proteins and vegetable proteins, beside, their effect on the body weight gain, glucose level, protein pattern and lipid profile in diabetic rats.


Basal diet composition:

Basal diet consists of casein (15%), corn oil (10%), salt mixture (4%), vitamins

mixture (1%), cellulose (5%) and starch (65%) according to AOAC (2005).

Biological experimental design:

Forty two albino rats, with an initial body weight between (100-120) g were

used. The animals were housed at temperature (25 ± 1°C) in humidity controlled

room and a 12 hr light- dark cycle. Rats were allowed free access to tap water and

standard pellet diet. The animals were kept under normal healthy conditions and fed

basal diet for one week.

Impact of Different Dietary Proteins …

165

After the adaptation period one group (7 rats) served as negative control, the

rest (35 rats) were induced for hyperglycemia by intraperitoneally injected of freshly

prepared solution of alloxan monohydrate at a dose of 150mg/kg body weight (Buko

et al. 1996), after 72 h of alloxan injection the rats were fasted for 6h and their

plasma glucose levels were estimated. Rats, having plasma glucose levels above 200

mg dl−1

were considered diabetic. The diabetic rats were randomly divided into the

following five groups each of seven rats:

Control+: Fed on basal diet (positive control)

Group I: Fed on basal diet in which protein was replaced with buffalo’s

casein (100%).

Group II: Fed on basal diet in which protein was replaced with buffalo’s

whey protein (100%).

Group III: Fed on basal diet in which protein was replaced with buffalo’s

whey protein hydrolysate (100%).

Group IV: Fed on basal diet in which protein was replaced with soy protein

(100%).

The Feeding period was four weeks .Rats were kept during the whole

experiment in metal cages under hygienic conditions and ad-libitum water. The

changes in body weight were recorded weekly. Blood samples were withdrawn from

the retro-orbital plexus of the eyes from each rat according to the procedure of

Shermer (1967) at time intervals 0, 7, 14, 21, and 28 days of the experiment; serum

was separated by centrifugation at 1500 rpm for 15 min at ambient temperature to

estimate the biochemical parameters.

BIOCHEMICAL ANALYSES:

Glucose concentration:

Serum glucose was determined using enzymatic colorimetric method according to

the method of Trinder (1969).

Lipid profile parameters:

Enzymatic determination of total cholesterol (TC) in serum was carried out

according to Allain et al. (1974) Fully enzymatic determination of total triglycerides

(TG) in serum was measured colorimetrically at 546 nm, according to Fossati and

Principe (1982); high density lipoproteins- cholesterol (HDL-C) was determined

according to the method of Lopez-Virella, (1977).While, the low density

lipoproteins-cholesterol (LDL-C) was obtained by equation.

Blood protein pattern:

Enzymatic determination of total protein in serum was carried out according

to Henry (1964). Fully enzymatic determination of Albumin in serum was carried


out according to Doumas et al. (1971). While, the Globulin was calculated as: Total

globulin = Total protein - total albumin

Statistical analysis

The results were statistically analyzed according to statistical analysis system

SAS (1999). Duncan's at 5% level of significance was used to compare between

means according to Snedecor and Cochran (1980).

RESULTS AND DISCUSSION In this study, four types of protein (casein, whey protein concentrate, whey protein

hydrolysate and soy protein) were used. Hyperglycemic rat's model which was

inducted by the subcutaneous injection of alloxan is resistant to the behavioral

effects of d-amphetamine,the diminished response of diabetic rats to amphetamine

cannot be attributed to decreased levels of drug in plasma or brain, and therefore

may be due to diminished central action of this compound.

Alloxan diabetic rats have great difficulty using carbohydrate due to the low

circulating insulin levels when offered a diet rich in other substrate ,diabetic rats

show preferences for the no carbohydrate diet (John., 1978).

Effect of casein, WPC, WPH and soy protein on body weight

Results in Table (1) showed that after 28 days of feeding different sources of

protein; body weights increases were significantly higher in group of rats fed on

WPH diet compared to other groups. Also soy protein showed a higher increase in

body weight compared to group receiving casein diet. On the other hand the control

positive group (control+) which was fed on standard diet containing 15% Casein

showed a decrease in body weight while higher increases in body weight as a

function of WPH intake was observed which can be explained by the following: The

speed of amino acids absorption after protein meal showed that casein was slowly

absorbed and promoted postprandial protein deposition by inhibition of protein

break down without excessive increase in blood amino acid concentration. On the

other hand whey protein was absorbed Very fast, and rapidly stimulated protein

synthesis, also amino acids oxidation. Boirie et al. (1997), Fruhbeck (1998), Sautier

et al. (1993) and Nagaoka et al. (1992) reported that a difference was not only in

protein concentrations but also in the nature and proportions of other dietary

components which might have influenced diet intake by the rats.


167

Table (1). Average body weight and weight gain (g) of Hyperglycemic rats fed on supplemented

diets containing different protein sources for 28 days. (n = 7 rats).

Data are expressed as means ± SE. Mean values in the same column within each parameter bearing the

same superscript do not differ significantly (P ≤ 0.05).

Control - : Normal rats fed on basal diet Control +: Diabetic rats fed on basal diet

I: Diabetic rats fed on basal diet supplemented 20% casein

II: Diabetic rats fed on basal diet supplemented 20% whey protein concentrate

III: Diabetic rats fed on basal diet supplemented 20% whey protein hydrolysis

IV: Diabetic rats fed on basal diet supplemented 20% soy protein

Effect of casein, WPC, WPH and soy protein on serum glucose

Results in table 2 showed that after 28days of feeding rats on different

protein sources, it could be noticed that feeding rats on diets containing WPH (group

III) exhibited the lowest level of serum glucose, followed by serum glucose level in

rats fed on SP, then those fed on casein being higher than that in negative control

rat's serum. It was found that highly significant changes within groups were found

by Boirie et al. (1997) who found that whey is considered a rapidly digested protein,

which thus promotes higher concentration of amino acids in postprandial plasma.

Several amino acids may act as direct insulin secretagogues Krebs et al. (2003).

Protein hydrolysate such as casein hydrolysate can enhance the postprandial insulin

response and reduce postprandial serum glucose levels Claessens et al. (2007).

Also, Layman (2003) and Rocha et al. (2004) found that the different effects

of different amino acids on postprandial insulin levels is well known, it is therefore

likely that protein mixes with different amino acids profiles will elicit different

postprandial insulin response. Whey protein is particularly high in branch-chain

amino acids, in particular Lucien. These amino acids are insulinogenic, meaning that

they have a higher capacity to increase an insulin response. Another possibility is the

effect of whey protein on cretin hormones released in the gut. In cretin hormones

such as glucagon- like-peptide-1 (GLP-1) released from the gut after food

consumption is involved in many digestive roles including glycemic control

(Manders et al. ;2005). The effect of protein hydrolysate (with or without

Groups Initial body weight

(g)

Final body weight

(g)

Body weight gain

(%)

Control - 107± 5.19 a 136.79±3.83c 27.75%

Control + 107± 5.19 a 88.20± 4.15e -17.62%

I 107± 5.19 a 149.5± 4.05b 39.62%

II 107± 5.19 a 121.31± 7.15d 13.29%

III 107± 5.19 a 154.30± 4.88a 44.11%

IV 107± 5.19 a 150.20± 5.14b 39.43%


combination of free amino acids) on the insulin responses in both type 2 diabetes

patients and healthy persons was studied different mechanisms of action by which

protein hydrolysis stimulate insulin secretion have been proposed. One of the

possible mechanisms is that the intracellular oxidation of amino acids increases the

ATP content in the cell, leading to closure of the ATP- sensitive K

+ channels. By the

resulting depolarization of the plasma membrane, Ca+2

channels are activated,

subsequently leading to the exocytose of insulin. (Anderson et al.,1999)

Table (2). Serum Glucose (mg/dl) of Hyperglycemic rats fed on supplemented diets containing

different protein sources for 28 days. (n = 7 rats).

Groups Initial Feeding period (days)

7 14 21 28

Control- 87.13± 11.1a 88.06 ±9.1 e 89.00± 8.1f 86.97± 11.1 f 91.03±3.1e

Control+ 87.13 ±11.1a 563.20±9.1a 492.86± 14.1a 412.42±8.1b 331.98±4.1a

I 87.13± 11.1a 485.70± 9.1b 325.40±13.1b 220.55±8.1b 177.75 ±4.1b

II 87.13±11.1a 484.70± 9.1c 320.46± 13.1d 229.55±8.1d 161.63±4.1c

III 87.13± 11.1a 383.97± 9.1d 274.54 ±11.2e 177.73± 5.1e 99.03± 3.1 e

IV 87.13± 11.1a 470.22 ±9.1c 374.41±11.1c 278.60±8.1 c 140.47± 4.5d

Data are expressed as means ± SE. Mean values in the same column within each parameter bearing the same superscript do not differ significantly (P ≤ 0.05).


I: Diabetic rats fed on basal diet supplemented 20% casein II: Diabetic rats fed on basal diet supplemented 20% whey protein concentrate



Effect of casein, WPC, WPH and soy protein on lipid profile:

Triglycerides:

Concentrations of serum triglycerides are shown in table (3). No significant

differences were observed between the tested groups before treatments. Significant

decreases in triglycerides were observed in all tested groups receiving casein, WPC,

WPH and soy protein. The reduction rate in triglycerides was significantly higher in

groups receiving whey protein hydrolysate group III compared with group receiving

casein diet, followed by group (IV) which was fed on diet containing 20% soy

protein compared with control group, these results were in agreement with Kawase

et al. (2000) who found that fermented milk supplement with whey protein

concentrate affected serum lipids through lowering triglycerides.


169

Table (3). Serum Triglyceride (mg/dl) of Hyperglycemic rats fed on supplemented diets containing



7 14 21 28

Control- 86.48±2.1a 84.24±2.3e 85.63±2.8 e 85.23±2.8d 85.66±2.8c

Control+ 86.48±2.1a 138.34±2.8a 121.79±2.8a 115.65±2.8a 109.52±2.8a

I 86.48±2.1a 117.39±2.7d 111.65±2.8b 115.65±2.8b 95.12±2.8b

II 86.48±2.1a 127.82±3.1ab 103.54±2.8cd 90.75±2.8c 77.95±2.8d

III 86.48±2.1a 123.34±2.8bc 107.00±2.8c 86.85±2.8d 69.29±2.8e

IV 86.48±2.1a 120.26±2.5c 101.67± 2.8 d 88.21±2.8cd 74.75±2.8d

Data are expressed as means ± SE. Mean values in the same column within each parameter bearing the same superscript do not differ significantly (P ≤ 0.05).






Total cholesterol:

The results on table (4) show the decreases in serum cholesterol compared

with the initial value which can be noticed for all tested groups. After feeding for 7

days a sharp decreases in total serum cholesterol can be observed for all dietary

treatments. At the end of feeding period the rate of reduction in total serum

cholesterol was relatively higher for rats fed on WPH diets compared with casein

group. Cholesterol level became the lowest value for all treatments. The mentioned

results are in agreement with Nagaoka et al. (1992) who had arrived essentially to

the same conclusions. Those authors demonstrated the cholesterol lowering effect of

WPC and the cholesterol raising effect of both casein and soy protein. Also Thomas

et al. (2007) confirmed these results. he showed that substitution of soy bean protein

for casein lowers plasma cholesterol, when casein is replaced by soy bean protein

the hypolipidemic effects by soy protein intake increases, including the

improvement of insulin/ glucagon ratio, which is involved in lower fatty acid,

biosynthesis in liver through reducing the gene expression of sterol regulatory

element binding protein (SREBP), (Torres et al. 2006). In contrast, for previous soy

intervention studies in adults with type 2 diabetes did not revealed a significant

reduction in total cholesterol following 7-12 week consumption of soy protein.

Elizabeth et al. (2012); Norton et al. (1987) and Jacobucci et al. (2001) reported

lowering effect of serum cholesterol by whey protein, the plasma cholesterol

lowering effect was more marked and due to peptide than the protein. The primary

sequence of beta lacto globulin has been described as cholesterol- lowering agent


which inhibited cholesterol absorption by changing micellar cholesterol solubility in

the intestine Nagaoka (1996). On the other hand, lovali et al. (1990) found an

elevation of blood serum cholesterol by feeding whey protein to rabbit.

Table (4). Serum cholesterol (mg/dl) of Hyperglycemia rats fed on supplemented diets containing









HDL cholesterol:

Results in table (5) describes the gradual HDL- cholesterol increases observed

during the feeding period on casein, whey protein, whey protein hydrolysates and soy

protein. HDL- cholesterol was consistently at the highest value higher for the rats fed

on WPH and consistently at the lowest value for the rats fed on casein .A significant

differences were found between the two mentioned values whereas, non-significant

differences were found between rats fed on WPC and soy protein. Chapman (1980)

reported no differences in rats fed soy bean protein and casein diets with or without

0.1% cholesterol. The present findings are inconsistent with those Park et al., (1987)

who found a cholesterol-lowering effect and a decreased HDL-cholesterol level

associated with greater lecithin cholesterol acyltransferase (LCAT) activity in rats fed

soy bean isolate versus casein, Teixeira (2000).

A recent study showed reduction of 2-3% in total and HDL cholesterol

concentration in men who consumed ≥20g soy protein/d. These results are in

agreement with Nagaoka et al. (1992) who demonstrated the ratio of total

cholesterol to HDL cholesterol decreased in rats fed on casein and whey protein

containing diets and increased in serum of rats fed on soy protein diet.


7 14 21 28

Control- 79.53+1.4a 80.27+ 2.4 c 81.01+ 6.6c 86.60+ 4.4e 88.41+ 3.4d

Control+ 79.53+1.4a 154.25 +9.8a 142.22+ 9.9 a 129.18+ 8.4a 117.39+ 5.5a

I 79.53+1.4a 152.59+ 7.5a 142.28+ 11.1a 128.14+ 9.9 a 104.66 +7.4b

II 79.53+1.4a 139.85+ 8.8b 124.61+8.8 b 111.83+ 8.6b 98.38 + 5.1c

III 79.53+1.4a 138.97+ 9.7b 126.15+ 11.4b 99.13+ 9.1d 85.11+ 7.1e

IV 79.53+1.4a 139.70+ 7.5 b 124.19+ 11.2 b 107.54+ 8.1 c 90.90+4.4 d


171

Table (5). Serum HDL (mg/dl) of Hyperglycemic rats fed on supplemented diets containing

different protein sources for 28 days. (n = 7 rats(.


7 14 21 28

Control- 49.07+ 4.8 a 48.13 +3.8 a 46.745+2.a 50.75+ 4.8 b 54.75+ 2.8 b

Control+ 49.07+ 4.8 a 22.83+ 1.8 d 29.39+2.0 d 33.47+ 2.8 e 37.55+4.8 d

I 49.07+ 4.8 a 24.87+ 1.8 d 30.15 +2.2 d 37.60 +1.1 d 41.02+3.5 c

II 49.07+ 4.8 a 29.34+ 1.7 c 38.13+ 1.8 c 45.98+ 2.3c 53.83+ 2.8 b

III 49.07+ 4.8 a 36.23+1.2 b 43.42 +2.2b 56.38 +1.8 a 66.00 +2.4 a

IV 49.07+ 4.8 a 32.12+1.4 c 41.92+2.8 b 48.82+1.8b 55.71+3.1b








LDL cholesterol:

Results in table (6) showed that, feeding on casein, WPC, WPH and soy

protein diet leads to gradual decreases in LDL cholesterol. The net results for the

arithmetic means showed a very highly significant (p<0.001) decreases for both

groups compared to the control group. Rats in group III which were fed on

supplemented diet containing whey protein hydrolysate showed significantly the

lowest serum LDL cholesterol compared to all other groups at every feeding period.

Group IV which was fed on supplemented diet containing soy protein was in the

second order; while the control rats group showed significantly the least value all

time intervals. Other studies have suggested that casein feeding decreases bile acids

production and down- regulation of the hepatic LDL-receptor Cynthia and Kristin

(1991). Similar results were reported previously by Elizabeth et al. (2012).

The ratio of LDL-cholesterol: HDL-Cholesterol was also significantly

reduced; following consumption. Nelson et al. (2002) proved a decrease in LDL

cholesterol by 20% for the whey protein against a drop of 10% for soy protein.


Table (6). Serum LDL (mg/dl) of Hyperglycemic rats fed on supplemented diets containing



7 14 21 28

Control - 30.46±2.8 a 30.47± 4.8 d 34.27 ± 4.8 c 35.85 ± 4.8 c 33.65±4.8 d

Control + 30.46 ±2.8 a 131.42± 4.8 a 112.82± 2.4a 95.71 ± 0.8 a 79.83 ± 1.8 a

I 30.46 ±2.8 a 127.72 ± 4.8a 112.46 ±1.8a 90.70± 1.8 a 63.64± 2.5 b

II 30.46± 2.8 a 110.50± 4.8 b 86.48 ± 1.2 b 65.86± 0.1 b 44.55± 5.8 c

III 30.46± 2.8 a 102.74 ±4.8 c 82.72± 2.8 b 42.74 ±1.1 c 19.11±1.8 e

IV 30.46±2.8 a 107.58± 4.8b 82.26± 4.1 b 63.22 ±1.8 b 35.19 ±5.4 d








Protein pattern:

Total protein:

The data in table (7) revealed that feeding the experimental animals on

supplemented diet containing different sources of protein; casein, WPC, WPH and

soy protein for 28 days lead to gradually increases in serum protein in groups II and

III which were fed on whey protein and whey protein hydrolysates. While, groups I

and IV which were fed on casein and soy protein supplemented diets showed no

significant differences in serum total protein levels among these who groups. The

mentioned results are in agreement with Chaan et al. (1988) reported that renal

plasma flow increased 10% in healthy human subjects and 33% in subjects with

chronic renal disease after a high-protein meal providing 1.5g protein/kg. Leila and

Ahmad (2009) reported that the substitution of soy protein for animal protein was

recommended to decrease hyper filtration, but this has not been documented very

well. It is not clear whether protein restriction can delay the progression of diabetic

nephropathy. Such an effect could be achieved by a substitution of soy protein for

animal protein in the diet.


173

Table (7). Serum Total protein (mg/dl) of Hyperglycemic rats fed on supplemented diets containing



7 14 21 28

Control- 6.166±0.13a 6.600±0.33a 6.773±0.19 a b 6.978±0.13 a b 7.145±0.23b

Control+ 6.166± 0.13a 6.498± 0.33a 6.670±0.18 b 6.863± 0.16 b 6.998± 0.23c

I 6.166±0.13a 5.850±0.23b 6.340±0.23c 6.530±0.13 c 6.848± 0.14d

II 6.166±0.13a 6.708±0.13a 6.925± 0.19a 7.121 ±0.15a 7.338± 0.16a

III 6.166±0.13a 6.641±0.17a 6.901±0.23a 7.110± 0.18a 7.483±0.16a

IV 6.166±0.13a 6.020±0.13b 6.285±0.23 c 6.635±0.13 c 6.895±0.14 d





II: Diabetic rats fed on basal diet supplemented 20%whey protein concentrate

III: Diabetic rats fed on basal diet supplemented 20%whey protein hydrolysis


Serum albumin:

The results in table (8) showed the serum albumin in male albino rats fed on

supplemented diets containing the four different sources of protein for 28 days.

Rarely gradual increases in serum albumin concentration in all rat groups were

noticed during feeding period. These increases were not significant among all the

tested rat groups.

Meanwhile, after 28 days, the concentration of plasma albumin was

statistically equal in all rat groups. Sandra et al. (2004) indicated that the

consumption of vegetable protein, including soy protein reduces urinary albumin

excretion in diabetic patients. Williams and Walls (1987) showed that consumption

of soy protein prevented the progression of renal disease in sub totally

nephroectomized rats much more effectively than consumption of casein.


Table (8). Serum Albumin (mg/dl) of Hyperglycemic rats fed on supplemented diets containing



7 14 21 28

Control- 3.665+0.19a 3.875+ 0.33bc 4.228+ 0.23ab 4.575+0.33a 4.676+ 0.13a

Control+ 3.6650+0.19a 4.125+0.13a 4.276+0.23 a 4.575 +0.33ab 4.556+ 0.15ab

I 3.6650+0.19a 3.600 +0.15 c 3.748+0.23 c 3.940 +0.33 d 4.075 +0.18ac

II 3.6650+0.19a 3.760+ 0.17bc 3.965+ 0.23bc 4.201+0.33c 4.370+ 0.15ab

III 3.6650+0.19a 4.021+ 0.19ab 4.136+0.33 b 4.228+ 0.33c 4.365+0.18ab

IV 3.6650+0.19a 3.965+0.15ab 4.190+ 0.23ab 4.266 + 0.33 c 4.431+0.14ab








Serum globulin

The data in table (9) indicated gradual increases in serum globulin of all the

experimental rat groups during the feeding period. Rats group II and III which were

fed on supplemented diet containing whey protein and whey protein hydrolysate

showed significantly the highest serum globulin concentration compared to all other

groups at each feeding period. Group I which was fed on supplemented diet

containing casein came in the second order in serum globulin value, being

statistically equal to those of other rat groups fed on other supplemented diets over

all the feeding period.

From the previous data it could be concluded that the elevation in serum total

protein was due to the increase in albumin more than that in globulin. Similar results

were reported previously by Pimento et al. (2006) who stated that diet containing the

WPH promoted the conservation of higher levels of total protein and albumin.


175

Table (9). Serum Globulin (mg/dl) of Hyperglycemia rats fed on supplemented diets containing









CONCLUSION

Based on the above mentioned results, it could be concluded that the whey protein

hydrolysate and soy protein are high-quality proteins, and can be used as a source of

protein to reduce the blood glucose level, and the health benefits of WPH are due to

their high concentration of amino acids that affect the level of blood glucose,

and reduce the levels of serum TG, TC, and LDL-C. Therefore, WPH and SP can be

used to support some private antidiabetic foods.

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181

Anti-diabetic effect of olive leaves extract in alloxan-diabetic rats

Mousa1* , H. M. ., Farahna2 M., Ismail1, M. S., Al-Hassan1 A. A. , Ammar3, A. S. and

Abdel-Salam1 A. M

1Food Science and Human Nutrition Department, College of Agriculture and Veterinary Medicine,

Qassim University, , P.O. Box 6622, 51452-Buraidah, Saudi Arabia 2 Basic Health sciences Departments, College of Applied Medical Sciences, Qassim University, P.O. Box

6622, 51452-Buraidah, Saudi Arabia

3Food Science and Technology Department, Faculty of Agriculture, Cairo University, Giza, Egypt. *Corresponding Author: , Email: [email protected]

Abstract. Diabetes is often accompanied by metabolic alterations , which contribute significantly to

morbidity and mortality in diabetic subjects .Olive leaves extract (OLE) are used in traditional medicine as hypoglycemic agent in many Arab countries. To examine the beneficial effect of OLE, it was

administered in drinking water of alloxan induced diabetic rats at 3% and 6% concentrations.Blood

glucose level decreased significantly (P<0.05) from 135.6±12.2 mg/dl in the control group to 48.3±2.73 and 64.8±14.62 mg/dl in 3% and 6% treated groups respectively .The concentrations of cholesterol,

triglycerides, total proteins and albumin were improved by the administration of OLE.The activities of

liver enzymes , Alanine aminotransaminase(ALT,), Aspartate aminotransaminase,AST) were elevated in diabetic rats, however , administration of OLE reduced the activities of these enzymes.The activity of

AST was significantly reduced from 204.7±13.2 IU/L in the positive control to 109.3±11.0 and

130.2±16.0 IU/L in 3% and 6% OLE respectively and the activity of ALT was also reduced significantly from 48.0±9.2 IU/L in the positive control to 24.7±10.4 ,34.8±6.2 IU/L in 3% and 6% OLE

respectively.In the brain,Cellular changes were noticed in untreated diabetic rats including generalized

and localized edema in the white matter of the cerebrum and cerebellum. Diabetes induced neuronal degeneration; neuronal necrosis, neuronal chromatolysis and axonal demylenation. All these changes

were improved by OLE administration.It can be concluded that OLE is having hypoglycemic effect,

Neuroprotective activity and improves changes associated with diabetes probably due to the many potentially bioactive compounds it contain.

Keywords : Olive leaves extract, Diabetes, hypoglycemic effect

Mousa, H. M. et al 182

INTRODUCTION

Olive leaves derived from olive tree (Oleaeuropaea ) are obtained as by- product

during oil extraction. It is traditionally used in Mediterranean countries as an

alternative source of nutrients for animals during feed scarcity (MartıńGarcıá et al.,

2003).The chemical analysis of leaves indicated that it is poor in N, rich in crude fat

and Acid detergent fiber and low in tannins.( Delgado Pertıń˜ ez 1994).Further

analysis indicated that olive leaf has many other constituents, including uropein and

several flavonoids (rutin,apigenin, luteolin ( Briante et al., 2002,Amiot et al.,1986).

One of these potentially bioactive compounds is the secoiridoiduropein, which can

constitute up to 6–9% of the dry matter in the leaves. Other bioactive components

found in olive leaves include related secoiridoids, flavonoids, and triterpenes.

Recent research in Australia showed that Olive Leaf Extract has up to 40 times more

antioxidants than the best Extra Virgin Olive Oils. http://www.olea.com.au/benefits

/antioxidant-power.

Leaves have been widely used in traditional remedies in European and

Mediterranean countries such as Greece, Spain, Italy, France, Turkey, Israel,

Morocco, and Tunisia. Interest in the olive leave beneficial effects has recently

being increasing. It was reported that Ancient Egyptians used olive leaf for

mummification and as a remedy against various diseases.The British used them to

treat Malaria in the 1800s(Leila Abaza et al.,2007). They have been used in the

human diet as an extract, herbal tea, and a powder, and they contain many

potentially bioactive compounds that may have anti inflammatory effects (Tuck and

IIayball ,2002, Bitler et al., 2005, Miles,2005) antithrombic actions (Carluccio et al

2003), prevention of LDL oxidation (Wiseman et al1996., Visioli and Galli 1994)

hypoglycemic effect (Sato et al., 2007, HedyaJemai et al.,2009) anti-ischemic and

hypolipidemic effects (Andreadou et al., 2006), antioxidant, antihypertensive. Sedef,

SibelKarakaya ,2009).

Furthermore, it has been reported that uropein and/or olive oil have Anti-

cancer effect by inhibiting tumor growth .It was found that A plethora of minor

constituents in olive oil have been identified as effective agents in mitigating the

initiation, promotion and progression of multistage carcinogenesis. Menendez et al.

(2007) showed that uropeinaglycone is the most potent phenolic compound in

decreasing breast cancer cell viability. It was reported that uropein is having

neuroprotective activity(German and Walzem,2000) mainly by preventing oxidative

injury to mitochondria and preventing cellular dysfunction

The primary objective of this study is to investigate the biological effects of

extracts of olive leaves against induced diabetes and its complications in rats .

http://pubs.acs.org/action/doSearch?action=search&author=Jemai%2C+H&qsSearchArea=author

Anti-diabetic effect of olive leaves extract in alloxan-diabetic rats 183


Preparation of olive leaves extract:

Green olive leaves were collected, dried and stored until use. A hot water extract of

olive leaves was prepared according the methods described by Abdel-Salam et al.

(2009 and 2010). Olive leaves were cut into small pieces and boiled for 10 minutes in

distilled water. The mixture was filtered twice: first through cheese cloth (50%

cotton/50% polyester), and then through filter paper (Whatman No.2). The solutions

obtained were preserved in sterile dark bottles in a cool environment (4° C) until use.

Animals

Male adult Wistar rats of eight weeks of age and 120-150 g body weight were

obtained from Faculty of Pharmacy, KingSaudUniversity. The animals were housed

in clean plastic cages and allowed to acclimatize to the laboratory environment for

two weeks under the same laboratory conditions of photoperiod (12-h light:12-h

dark cycle), a minimum relative humidity of 40%and room temperature 23 ± 2 ◦C.

During the experimental period animals had ad libitum access to food and

water.Animals were housed in cages and assigned to be given a pelleting basal diet

(Protein (12%), sugars (5%), fat (10%), vitamin mixtures (1%), salt mixtures (4%),

fiber (4%) and starch (64 %).

Experimental design

Diabetes was induced in rats by I/P injection of alloxan at a dose of

150mg/Kg body weight. Olive leaves powder was prepared in two

concentrations(3% and 6%) in the drinking water.

Rats were divided into four different groups (10 rats in each group n=10).

Group 1: Control group receiving basal diet and tap water ( negative control)

Group 2. Control group, Diabetic rats , Basal diet and tap water (Positive

controls).

Group 3: Diabetic rats receiving basal diet + 3% olive leaves solution as

drinking water

Group 4: Diabetic rats receiving basal diet+ 6% olive leaves solution as

drinking water.

The experiment continued for six weeks and at the end the rats , after an

overnight fast, were anesthetized with A.C.E. mixture (1:2:3, Ethanol : Chloroform:

Diethyl Ether, respectively) (Wawersik, 1991) and then killed by decapitation.Blood

was collected in dry hepranized tubes and centrifuged at 3000rpm for 10 min. Part

of the blood was used for haematological analysis. After sacrifying the animals,

samples of brain were taken.


Biochemical analysis:

Briefly, collected blood was centrifuged at 3000 X g for 10 min within 30

min after collection into heparinized tubes. Plasma samples were separated and

transferred in Eppendorf tubes for analysis.

The concentration of total proteins, albumin, glucose, cholesterol , triglycerides, ,

and Alanine aminotransaminase (ALT, GPT) and aspartate aminotransaminase (AST,

GOT) and alkaline phosphatase(ALP) activities were determined by commercial kits

(Bio-Merieux Laboratory Reagents and Products, France) and kits obtained from

NubencoInterprises INC, Paramus, New Jersey, USA.

Hemoglobin (Hb) concentration, packed cell volume (PCV), red blood cells

(RBC), white blood cells (WBC)count, mean corpuscular volume (MCV), mean

corpuscular hemoglobin concentrations(MCHC) were determined by the Abbott Cell

Dyn® 3500 (Abbott Diagnostic Division, California (USA).

Histopathologicalexamination:,Brain samples were taken and quickly fixed in

10% formalin for 24 hours. Paraffin sections, 6 μm thick, were prepared and stained

with hematoxylin and eosin (H & E) for the histopathological examination.

(Humason, 1979)

RESULTS

Effects of adding OLE in drinking water on hematological parameters is shown in

table 1.As presented in the table there were no significant differences between

different groups in relation to RBCs, hemoglobin, hematocrit, and MCH. As for

MCV. However, results showed that the mean values for rats that received 6% olive

leaves extract were significantly (P<0.05) higher than negative control, positive

control, and rats fed 3% OLE.The mean values were 60.3±2.16 vs. 55.5±2.08,

58.3±2.50, and 58.3±2.52 fl respectively.Regarding MCHC, the mean values of rats

fed 3% and 6% olive leaves extract were significantly (P<0.05) lower than negative

and positive control groups by the mean of (32.2±0.74 and 32.2±0.66 vs. 34.2±0.82

and 34.5±0.60 g/dl respectively). Regarding platelet count, the results showed that

the mean values of groups fed 3% and 6% of OLE were significantly (P<0.05)

higher than negative and positive control groups, by the mean of 939.5±124.3 and

847.3±42.0 103 /ml vs. 657.3±9.5 and 794.7±83.6 103 /ml respectively). Finally, the

mean MPV values of rats fed different concentrations of OLE were significantly

higher than negative and positive control groups.


Table (1). The effect of different concentrations from OLE on hematological parameters

Negative control (n=6)

Positive control (n=6)

Olive 3 %

(n=6)

Olive 6% (n=6)

ANOVA

Mean±SD Mean±SD Mean±SD Mean±SD F

RBC (106 microliter) 8.0±0.46 a 8.0±0.51 a 7.9±0.15 a 8.0±0.36 a 0.06

Hemoglobin (g/dl) 15.1±1.10 a 16.0±0.66 a 15.2±1.17 a 15.9±1.10 a 0.69

Hematocrit (%) 44.3±3.77 a 46.4±1.62 a 45.9±2.32 a 48.0±3.75 a 1.30

MCV (fl) 55.5±2.08a 58.3±2.50ab 58.3±2.52ab 60.3±2.16 b 2.14

MCH (pg) 18.9±0.64 a 20.1±1.03 a 19.3±1.37 a 20.0±0.52 a 1.27

MCHC (g/dl) 34.2±0.82 ac 34.5±0.60 a 33.2±0.98bc 33.2±0.66 b 5.05**

RDW (%) 11.9±0.62 a 12.1±0.90ab 12.3±0.47ab 12.9±0.64 b 5.48**

Platelet count (103

ml) 657.3±9.5 a 794.7±83.6 b 939.3±42.0 c 847.8±68.1bc 6.59**

MPV (fL) 5.7±0.38 a 6.0±0.12 a 6.6±0.21 b 6.5±0.37 b 8.91***

n: Number of rats, SD: Standard Deviation, and ANOVA: Analysis of Variance

Values subscribed in the same row with different letters showed significant differences (P<0.05) between

these values as calculated by ANOVA and LSD

** P<0.01, and *** P<0.001

, RBC: Red blood cells, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC:

Mean corpuscular hemoglobin concentration

Results of the glucose, cholesterol, triglycerides, total proteins and albumin

are presented in table 2.

As shown in table (2) Blood glucose levels were significantly (P<0.05) lower

in rats that received 3% and 6% OLE when compared with the positive

control ,however, the 3% extract produced better lowering effect than the 6%

extract .The mean values are 48.3±2.73 and 64.8±14.62 mg/dl respectively.As for

cholesterol, the results were inconsistent , feeding 3% of OLE caused significant

(P<0.05) decrease when compared with negative control and 6% feeding. The levels of

triglycerides were significantly (P<0.05)reduced with 3% treatment .The level was

29.8±6.42 mg/dl in the negative control group and was reduced to16.6±3.12mg/dl with

3% OLE .however , drinking water containing 6% OLE increased insignificantly the

level of triglycerides. Regarding total proteins, there was no significant differences

between treated groups and positive control group, however, 6% treatment increased

significantly the level of total proteins in this group. Regarding albumin, the 6% OLE

increased significantly(P<0.05) the level of albumin (2.8±0.27 g/dl)and there was no

significant differences between the other groups.


Table (2). The effect of different concentrations from OLE on blood glucose, cholesterol,

triglycerides, total proteins, and albumin.

Negative

control (n=10)

Positive

control (n=10)

Olive 3 %

(n=10)

Olive 6%

(n=10) ANOVA

Mean±SD Mean±SD Mean±SD Mean±SD F

Glucose (mg/dl) 66.0±5.79 a 135.6±12.2±6

b 48.3±2.73 d 64.8±14.62 a 54.14***

Cholesterol (mg/dl) 48.0±6.40 a 37.8±6.65 b 39.3±2.73 b 54.8±8.08 ad 10.77***

Triglycerides

(mg/dl) 29.8±6.42 a 16.6±3.12 b 22.7±3.61 c 32.5±5.68 a 12.37***

Total Protein (g/l) 11.8±0.4 a 13.1±1.9ab 12.2±0.7 a 13.9±1.7 b 3.17*

Albumin (g/l) 2.4±0.03 a 2.4±0.18 a 2.6±0.22 a 2.8±0.27 b 3.55*

n: Number of rats, SD: Standard Deviation, and ANOVA: Analysis of Variance



*** P<0.001

Table (3). The effect of different concentrations of OLE on the activities of Alanine

aminotransaminase, aspartate aminotransaminase and alkaline phosphatase enzymes.

(Mean±SD)

Negative

control (n=6)

Positive control

(n=6)

Olive 3 %

(n=6)

Olive 6%

(n=6) ANOVA

Mean±SD Mean±SD MeanSD Mean±SD F

AST (U/l) 142.8±16.7 ad 204.7±13.2 b 109.3±11.0 c 130.2±16.0 a 33.64**

*

ALT (U/l) 35.4±8.3 ac 48.0±9.2 b 24.7±10.4 c 34.8±6.2 ac 5.45**

ALP (U/l) 604.6±97.4ab 660.8±183.4bc 660.3±37.6bc 471.2±87.8 a 4.96**

ALP: Alkaline Phosphatase, n: Number of rats , SD: Standard Deviation, and ANOVA: Analysis of Variance



* P<0.05,** P<0.01 and *** P<0.001

As shown in table 3 there was a significant (P<0.05) decrease in the activities

of AST in rats that received both 3% and 6% of OLE when compared with the

positive control. The same trend was noticed in ALT, where the mean value for rats

fed 3% OLE (24.7±10.4 U/l) was the lowest among all groups but the difference was

only significant (P<0.05) between this value and values of positive control group

(48.0±9.2 U/l) .Olive leaves extract at 6% level resulted in a significant(P<0.05)


reduction in the activities of ALP enzyme(471.2±87.8 U/l) when compared with the

positive control 660.8±183.4 U/l .

The histopathological examinations of the brain was presented in table 4 and

it revealed cellular and tissue changes in the positive control, including generalized

and localized edema in the white matter of cerebrum and cerebellum, neuronal

necrosis and axonal demylenation. Administration of OLE improved the histological

picture of diabetic rats.

Table (4). Effect of administration of Olive extract (3& 6 %) in the brain of Diabetic rats compared

with of positive control.

Perivascul

ar Edema

Congeste

d blood vessels

Neuron

al necrosis

Neuronal

chromatolysis

Axonal

demylenation

Haemorrhag

e

Control

negative + + + + +

-

Control positive

++++ +++ +++ +++ +++ -

Olive 3 % - + - - - -

Olive 6% + + - - - -

++++ Severe +++ Moderate – Normal

DISCUSSION

Diabetes mellitus(DM) is widely spread in Saudi Arabia(KSA) and previous surveys

from KSA suggested that diabetes is present in epidemic proportions. The overall

prevalence of Diabetes mellitus in adults inKSA is 23.7%. (Al-Nozha, et al.,2004).

There are so many complications of diabetes including loss of sight, coronary artery

diseases, leg amputation and kidney failure.

One reason for this high incidence of DM in KSA is the change of the lifestyle

of people towards modern diets that increase the rate of obesity, overweight ,low

physical activity. Healthier eating habits may partly contribute to reducing the

incidence and/or reduce the complications associated with DM. The so-called

Mediterranean diet which is rich in olive products has become associated with health

benefits particularly against cardiovascular diseases ,colon, breast and skin cancer and

diabetes (Andreadou et al., 2006, HedyaJemai et al.,2009, Briante et al.,2002,

Menendez et al.,2007) and protection of the nervous system (Zhao et al.,2013)

In the present investigation, OLE at the two concentrations, in drinking

water, were tested against changes in some blood metabolites, enzyme activities and

brain histology in induced DM in rats.

Administration of OLE reduced significantly the blood glucose levels

particularly with the 3% OLE dose .This reduction of blood glucose level by OLE

http://pubs.acs.org/action/doSearch?action=search&author=Jemai%2C+H&qsSearchArea=author


may be achieved by two mechanisms: OLE may cause more glucose to be utilized

by the body and it may also stimulate the release of insulin.(Eidi,et al., 2009)

Furthermore OLE was found to inhibit the activities of α-amylases from human

saliva and pancreas . In Animal models studies the hypoglycemic effect of OLE may

be facilitated through the reduction of starch digestion and absorption (Wainstein et

al.,2012).In the present investigation there was improvement in the level of

cholesterol, triglycerides,total proteins and albumin.Similar results were reported by

Eidi,et al., (2009), they reported that the oral administration of the olive leaves

extract (0.1, 0.25 and 0.5 g/kg body wt) for 14 days significantly decreased the

serum glucose, total cholesterol, triglycerides, urea, uric acid, creatinine, aspartate

amino transferase (AST) and alanine amino transferase (ALT) while it increased the

serum insulin in diabetic rats but not in normal rats.

Consumption of OLE resulted in non significant changes in RBCs,

hemoglobin, hematocrit, and MCH. values. It was reported that many hematologic

abnormalities have been defined in diabetic subjects however, there is lack of classic

hematologic pathologic findings in this condition(Jones and Peterson (1981).

Results obtained in the present investigation revealed that the nervous system

is affected by diabetic toxicity.Cellular changes were noticed in untreated diabetic

rats including generalized and localized edema in the white matter of the cerebrum

and cerebellum, Diabetes induced neuronal degeneration; neuronal necrosis,

neuronal chromatolysis and axonal demylenation. Similar findings were reported by

Lee et al (2013), Zhao et al.,( 2013) and Reno et al.(2013) .These changes could be

due to oxidative stress secondary to diabetic neurotoxicity

Treatment of diabetic rats with different concentrations of OLE improved the

histological picture; this could be due to anti oxidative stress or ant-excitotoxic

effect of the olive leave extracts.

The potential beneficial promoting health effect of Olive leaf extract appear

to be linked to its antioxidant activity which was found to be helpful in the

prevention of diabetic complications associated with oxidative stress.

Few results are available from human studies, however. More research into

the possible blood sugar-lowering effects of olive leaf extract is needed before it can

be recommended for this use.

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Plant production



193

Screening of thirteen garlic (Allium sativum L.) Genotypes for characteristics of

Yield and Quality under Sohag Conditions

Hazem A. Obiadalla Ali1,2

1Horticulture Department, Faculty of Agriculture, Sohag University, Sohag 82786, Egypt.

2Plant Production and Protection Department, College of Agriculture and Veterinary Medicine, Qassim

University, P. O. Box 6622, Buraidah 51452, Saudi Arabia.

Corresponding Author: Hazem A. Obiadalla-Ali, Department of Plant Production and Protection,

Faculty of Agriculture and Veterinary Medicine, Qassim University, Buraidah, Saudi Arabia.

E-mail: [email protected]


ABSTRACT. Thirteen Egyptian accession of garlic (Allium sativum L.) were assessed for yield, earliness and some quality characteristics at the Experimental Farm, Faculty of Agriculture, Sohag University,

Sohag, Egypt, during two successive seasons, 2010/2011 and 2011/2012. There were significant

differences among genotypes for all studied characters. Bani Sweif, El-Minia and Sids 40 landraces were the earliest in maturity while, Assiut accession was the latest. Plant of Elgharbia accession was the

longest while, Sids 40 accession was the shortest. Aswan and El-faiyum accessions were the largest for

bulb diameter while, El-Minia landrace was the smallest. Elgharbia accession had the greatest total soluble solids while, Gehena landrace had the lowest. Sids 40 accession exceeded all other genotypes in

plant fresh weight, weight of cloves/bulb and total yield. However, Sids 40 accession had the least

number of cloves/bulb. The results of this study could be useful for improving garlic production under south valley condition.

Keywords: accessions, yield, bulb ratio, fresh weight, correlation coefficient

Hazem A. Obiadalla Ali 194

INTRODUCTION

Garlic (Allium sativum L.) is one of the important crops in Egypt for local market

and export. Evidence of garlic cultivation can be found as far back as 3000 B.C. in

Egypt ((Figliuolo et al., 2001). It has been widely used throughout history as a food

additive for both its flavor and medicinal effects. Recent research indicates that fresh

and processed garlic may have some health benefits on human health such as anti-

carcinogenic, anti-fungal, and anti-bacterial properties (Clemente et al., 2011). It is

currently used for its unique flavor as a food ingredient as well as a dietary

supplement (Khanum et al., 2004). Furthermore, a liquid garlic spray has been used

as an insect repellent for other crops.

Many investigators studied the growth and yield variations among garlic

genotypes (Waterer and Schmitz, 1994; Dickerson and Wall, 1997; Kevresan et al.,

1997; Gvozdanovic-Varga et al., 2002; Islam et al., 2004; Baghalian et al., 2005;

Panthee et al., 2006; Stavelikova, 2008; Volk and Stern 2009; Aly, 2010; Clemente

et al., 2011; Dawood et al., 2011; Gouda Anwar; 2012; Ankur and Tiwari, 2013)

Because garlic does not produce seed, breeders cannot breed and develop

cultivars specific to growing regions. So, garlic can be improved by selection and

planted the accession (ecotypes) most productive, qualitative and earliest maturity

under Upper Egypt condition. The objective of this work was to evaluate the

performance of 13 local garlic genotypes for yield and some quality characteristics.


Plant Materials

Thirteen local garlic genotypes (Sids 40, Gehena, Tahrir, Sohag, Qena, Elbehera,

Elgharbia Bani Swief, Elminia, Aswan, Assiut, Elwady El-Gaded and El-Faiyum)

were used in the present study. All genotypes were collected from various provinces

in Egypt where they have been commonly grown for several decades. Landraces of

garlic was a kind gift of Mr. Sayed Gebril Mahmoud (Assistant Lecture, of

vegetable crops, Department of Horticulture, Faculty of Agriculture, Sohag

University, Sohag, Egypt)

Field Trial Layout

Two years field trial (2010/2011 and 2011/ 2012) was executed at the

Experimental Farm of Faculty of Agriculture, Sohag University, Sohag, Egypt

where the soil is newly reclaimed (Sandy Clay Loam as show in Table 1) to evaluate

the 13 garlic entries for earliness, yield and quality characteristics.

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Screening of thirteen garlic… 195

Table (1). Soil characterization of the experimental site.

Sampling

depth

E.C

. (1

:5)

dS

m-1

pH

(H

2O

)

(1:2

.5)

O.M

%

CaC

O3

%

Cla

y

%

Sil

t

%

San

d

%

Soil

Textu

re

Tota

l N

(%)

NaH

CO

3-P

pp

m

Avail

ab

le K

pp

m

0 - 25 0.21 7.35 2.51 11.27 29.70 23.12 47.18 SCL 0.199 8.3 374

25 - 45 0.15 7.73 0.09 52.15 3.19 6.00 90.81 S 0.053 19.5 178

45 - 65 0.19 7.90 0.40 55.49 2.90 7.18 89.92 S 0.004 19.9 144

65 - 80 0.20 7.85 0.31 22.50 2.60 7.22 90.18 S 0.004 6.5 102

SCL= Sandy Clay Loam, S= Sand, NaHCO3-P= NaHCO3-P extractable-P.

The two years trial was conducted using cloves from the same cloves lots.

The experimental design was a randomized complete block with 3 replications. Each

experimental unit (plot) consisted of five ridges giving about 10.5 m2 areas. Cloves

were planted on the both sides of ridge at about 7 cm apart on October 1st in both

seasons. Fertilization, irrigation and other cultural practices were carried out as

recommended for garlic commercial production (Hassan, 1991).

In mid March in both seasons, ten garlic plants were randomly taken from the

outer two ridges of each plot to record the following characters: 1) Plant height

(cm), 2) Number of leaves per plant, 3) Bulbing ratio (neck diameter/bulb diameter);

calculated as described by Mann (1952).

Each plot was observed to record time of harvest maturity (garlic is ready to

harvest when leaves begin to yellow or brown and fall over, but there are still about

3-4 or 30-50% green leaves on the plant). As plant reached harvest maturity stage,

all the plants were uprooted from plot to record the Plant fresh weight (g) character.

After harvesting, plants were left to cure before cutting off dry leaves and roots and

total yield (ton/feddan) character was recorded. Ten garlic head bulb cured were

randomly taken from each plot to record the following characters: 1) Cured bulb

diameter (cm), 2) Cured bulb height (cm), 3) Number of cloves per bulb, 4) Weight

of cloves per bulb (g), 5) Total soluble solids (TSS).

Statistical analysis

All recorded data were statistically analyzed (Gomez and Gomez, 1984) and

treatment means were compared using the Duncan’s multiple range test (DMRT,

Duncan, 1955) at 0.05 probability level. Also, phenotypic correlation coefficients

among traits were calculated in the second season.

RESULTS AND DISCUSSION

As shown in Table 2 significant differences were recorded among garlic entries in

both seasons for all studied traits except in the second season for number of leaves

per plant. Plant of landrace Elgharbia was the longest, while, Sids 40 accession was


the shortest in both seasons. Sohag landrace had the highest mean values for number

of leaves per plant, while, Bani Swief accession had the lowest in both seasons. El-

Faiyum and Bani-Sweaf landraces had the highest mean value for neck bulb

diameter, while, Assuit accession had the lowest mean value for this character. El-

Faiyum and Aswan landraces gave the highest mean value for cured bulb diameter,

while, El-Minia accession gave the lowest mean value for this character.

Table (2). Plant height, Number of leaves/plant, Neck bulb diameter and Curd head bulb diameter

for the 13 garlic genotypes sown during 2010/2011 and 2011/2012 seasons.

Genotypes

Plant height

(cm)

Number of

leaves/plant

Neck bulb diameter

(cm)

Cured head bulb

diameter (cm)

2010/2011 2011/2012 2010/2011 2011/2012 2010/2011 2011/2012 2010/2011 2011/2012

Sids 40 54.80 l 54.40 h 10.70

abcd 11.10 a 2.900 ab 2.767 a 5.100 b

4.950

cde

Gehena 80.60 i 80.30 f 10.63

bcd 10.20 a 2.467 de 2.333 d 5.000 b 4.850 e

Tahrir 90.20 b 90.10 ab 11.20

abc 10.90 a 2.633 cd 2.500 bc 5.050 b 4.900 de

Sohag 85.50 e 85.30 d 11.40 a 11.40 a 2.733 bc 2.600 b 5.133 b 5.000

bcde

Qena 89.40 c 89.20 b 11.33 ab 11.00 a 2.700 c 2.633 b 5.100 b 5.050

bcd

Elbehera 87.70 d 87.40 c 11.17

abc 11.30 a 2.500 de 2.400 cd 5.200 b 5.100 bc

Elgharbia 91.60 a 91.50 a 10.93

abc 11.20 a 2.633 cd 2.567 b 5.200 b 5.133 b

Bani

Swief 81.20 h 81.10 f 10.20 d 10.10 a 2.967 a 2.900 a 5.150 b

5.050

bcd

El-Minia 83.40 g 83.20 e 10.60 cd 10.40 a 2.733 bc 2.600 b 4.700 c 4.600 f

Aswan 58.50 k 58.20 g 10.70

abcd 10.50 a 2.733 bc 2.633 b 5.600 a 5.500 a

Assiut 84.00 f 83.70 de 11.20

abc 10.60 a 2.000 f 1.900 e 5.000 b 4.900 de

Elwady 81.30 h 81.10 f 10.90

abc 11.10 a 2.400 e 2.333 d 5.050 b

5.000

bcde

El-faiyum 59.20 j 59.10 g 11.03

abc 10.80 a 3.000 a 2.900 a 5.500 a 5.400 a

Means followed by the same letters are not significantly different from each other at 0.5% level

Table (3) present bulbing ratio, cured bulb height, plant fresh weight (gm)

and number of cloves per bulb. El-Minia, Bani-Sweaf and Sids 40 landraces had the

highest mean value for bulbing ratio, while, Assuit accession had the lowest mean

value for this character. Aswan landrace had the highest mean value for cured bulb

height, while, Tahrir accession had the lowest. Sids 40 landrace produced the

highest plant fresh weight, while, El-Minia accession gave the lowest plant fresh


weight. Bani Sweif ecotype had the highest number of cloves per bulb, while, Sids

40 ecotype produced the lowest number of cloves per bulb.

Table (3). Bulbing ratio, Cured bulb height, Plant fresh weight and Number of cloves/bulb for the

13 garlic genotypes sown during 2010/2011 and 2011/2012 seasons.

Genotypes Bulbing

ratio

Cured bulb height

(cm)

Plant fresh weight

(gm)

Number

of cloves/bulb

2010/2011 2011/2012 2010/2011 2011/2012 2010/2011 2011/2012 2010/2011 2011/2012

Sids 40 0.5700 a 0.5600 a 3.050 b 3.100 b 59.17 a 59.10 a 14.93 f 14.87 j

Gehena 0.4933 ef 0.4800 e 2.750 d 2.800 cd 41.07 h 41.00 h 30.50 d 30.47 g

Tahrir 0.5200 cd 0.5100 cd 2.750 d 2.700 d 49.83 d 49.80 d 35.63 b 35.60 c

Sohag 0.5300 bc 0.5200 c 2.900 bcd 2.850 cd 48.57 e 48.50 de 31.90 cd 31.83 f

Qena 0.5300 bc 0.5200 c 2.950 bc 2.900 c 45.67 f 45.60 f 32.90 c 32.83 de

Elbehera 0.4800 f 0.4700 e 2.800 cd 2.850 cd 48.77 de 48.70 de 30.17 d 30.10 g

Elgharbia 0.5067 de 0.5000 d 2.950 bc 2.850 cd 48.17 e 48.10 e 32.70 c 32.60 e

Bani

Swief

0.5767 a 0.5667 a 2.750 d 2.800 cd 45.03 f 45.00 f 38.70 a 38.63 a

El-Minia 0.5833 a 0.5733 a 2.850 cd 2.750 cd 36.17 i 36.10 i 33.23 c 33.20 d

Aswan 0.4900 ef 0.4800 e 3.300 a 3.400 a 51.50 c 51.40 c 15.90 f 15.83 i

Assiut 0.4000 g 0.3900 f 2.800 cd 2.900 c 42.57 g 42.47 g 36.40 b 36.33 b

Elwady 0.4767 f 0.4667 e 2.950 bc 2.850 cd 43.00 g 42.90 g 30.33 d 30.30 g

El-faiyum 0.5467 b 0.5400 b 2.950 bc 2.900 c 54.07 b 54.00 b 17.83 e 17.80 h

Means followed by the same letters are not significantly different from each other at 0.5% level

Table (4) present weight of cloves per bulb (gm) total soluble solids (%) and

total yield (ton/feddan) of the 13 garlic studied entries during two successive

seasons. Significant differences were found for all studied measurements in both

seasons. Sids 40 ecotype produced the highest weight of cloves per bulb, while,

Gehena ecotype produced the lowest weight of cloves per bulb. El-Gharbia

accession had the highest total soluble solid of curd head bulb, while, El-Gharbia

accession had the lowest total soluble solid. Sids 40 accessions had the highest total

yield, while the least total yield was recorded for El-Minia accession. This finding

could be explained in the light of the induced increment in weight of cloves/bulb as

previously reveled


Table (4). Weight of cloves/bulb, Total soluble solids and Total yield for the 13 garlic

genotypes sown during 2010/2011 and 2011/2012 seasons.

Genotypes Weight of cloves/bulb

(gm)

Total soluble solids

(%)

Total yield

(ton/feddan*)

2010/2011 2011/2012 2010/2011 2011/2012 2010/2011 2011/2012

Sids 40 54.70 a 54.60 a 40.40 c 40.30 c 8.300 a 8.233 a

Gehena 31.87 k 31.77 k 34.57 i 34.47 i 5.800 gh 5.733 g

Tahrir 34.97 h 34.87 h 38.07 e 38.03 e 7.033 c 6.967 c

Sohag 37.37 f 37.30 f 39.57 d 39.50 d 6.800 cd 6.733 d

Qena 35.97 g 35.87 g 35.90 g 35.83 g 6.400 e 6.333 e

Elbehera 39.07 e 39.00 e 35.07 h 35.00 h 6.867 cd 6.800 cd

Elgharbia 37.73 f 37.67 f 41.73 a 41.63 a 6.767 cd 6.700 d

Bani Swief 40.03 d 39.97 d 36.73 f 36.63 f 7.700 b 7.600 b

El-Minia 37.90 f 37.80 f 36.57 f 36.53 f 5.567 h 5.500 h

Aswan 48.07 b 47.97 b 40.90 b 40.83 b 6.900 cd 6.833 cd

Assiut 32.70 j 32.63 j 36.57 f 36.50 f 6.000 fg 5.933 f

Elwady 33.70 i 33.63 i 36.57 f 36.53 f 6.200 ef 6.100 f

El-faiyum 46.03 c 45.97 c 38.07 e 38.13 e 6.700 d 6.633 d

*Feddan is 4200 m2, approximately 0.42 ha.

Means for genotypes followed by the same letter(s) are not significantly different at the 5% level.

Our results in this investigation along with previous studies of local garlic

accessions/ landraces (Gowda et al., 2007; Hossny and mahmoud 2009; Moustafa et

al., 2009; Aly 2010; Gouda Anwar 2012) seems to be in well agreement on the

notion of needs for breeding efforts. As garlic does not produce seed, breeders

cannot breed and develop cultivars specific to growing regions. So, selection and

planted the domestic accessions/landraces, which are fully adapted to local

conditions and are important genetic resources and initial breeding material (should

be to improve local Egyptian garlic.

In our investigation we can observe variations among all ecotypes in number

of leaves/plant which effect in bulb weight. Also, we can see variations among these

ecotypes clearly in plant fresh weight, number of cloves, weight of cloves/bulb,

percentage of T.S.S in cloves and total yield characters. These differences could be

attributed to genetic architecture of the ecotype. These results are in agreement with

those reported by Kevresan et al., 1997; Gvozdanovic-Varga et al., 2002; Tiwari et

al., 2002; Patil et al., 2003; Pardo and Martin 2003; El-sayed 2004; Islam et al.,

2004; Mohamed 2004; Baghalian et al., 2005; Gowda et al., 2007; Hossny and

mahmoud 2009; Moustafa et al., 2009; Aly, 2010; Gouda Anwar 2012.


Table (5) present phenotypic correlation coefficients among 9 characters of

garlic genotypes sown during two successive seasons. The data showed that plant

height was positively significant correlated with number of cloves character but

negatively significant correlated with bulb diameter, bulb height, plant fresh weight,

weight of gloves per bulb, total soluble solids and total yield characters. Number of

leaves per plant was positively significant correlated with plant fresh weight and

total yield characters. Bulb diameter was positively significant correlated with bulb

height, plant fresh weight, weight of cloves per bulb, total soluble solids and total

yield characters but negatively significant correlated with number of cloves per bulb

character. Bulb height was positively significant correlated with plant fresh weight,

weight of cloves per bulb and total soluble solids (TSS) characters but negatively

significant correlated with number of cloves per bulb character.

Table (5). Phenotypic correlation coefficients among 9 characters of garlic genotypes sown during

2010/2011 and 2011/2012 seasons

Characters

Pla

nt

heig

ht

(cm

)

No

. o

f

lea

ves/

pla

nt

Bu

lb d

iam

ete

r

(cm

)

Bu

lb h

eig

ht

(cm

)

Pla

nt

fresh

weig

ht

(gm

)

Nu

mb

er o

f

clo

ves

/bu

lb

Weig

ht

of

clo

ves

/bu

lb

(gm

)

To

tal

solu

ble

soli

ds

(T.S

.S)

To

tal

yie

ld

(to

n/f

ed

da

n)

Plant height

(cm) - 0.141NS

-

0.493** -0.595**

-

0.609** 0.910**

-

0.848** -0.380*

-

0.414**

No. of

leaves/plant - 0.143NS 0.162NS 0.502**

-

0.0699NS 0.126NS 0.314NS 0.392*

Bulb diameter

(cm) - 0.582** 0.606** -0.561** 0.479** 0.406** 0.347*

Bulb height

(cm) - 0.446** -0.703** 0.604** 0.592** 0.203NS

Plant fresh

weight (gm) - -0.699** 0.777** 0.600** 0.793**

Number of

cloves/bulb -

-

0.837**

-

0.464** -0.354*

Weight of

cloves/bulb

(gm)

- 0.580** 0.697**

Total soluble

solids (T.S.S) - 0.491**

Total yield

(ton/feddan) -

* = Correlation is significant at the 0.05 level, ** = Correlation is significant at the 0.01 level


Data also showed a positive significant correlation for plant fresh weight with

weight of cloves per bulb, total soluble solids and total yield characters but

negatively significant with number of cloves/blub. Number of cloves per bulb was

negatively significant correlation with weight of cloves per bulb, total soluble solids

and total yield characters. Weight of cloves per bulb was positively significant

correlated with both total soluble solids and total yield characters. Finally, a total

soluble solid was positively significant correlated with total yield character. Many

investigators found one or more from these correlations between garlic traits, El-

Muraba et al. 1983; Metwally et al. 1990; El-Ghamriny 1991; El-Mansi et al. 1999;

Singh and Tiwari 2001; El-Mahdy and Mohamed 2003; Pardo et al. 2007 and

Hosseny and Mahmoud 2009.

Conclusion

Sids 40 landrace may directly be adopted in the production system in Sohag and

other regions of similar conditions as a high yielding and earliest genotype. Both

Sids 40 and Elgharbia are nominated germplasm for breeding programs to improve

productivity and quality of the studied Egypt local garlic accessions

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