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In the Name of ALLAH, Most Gracious, Most Merciful

Volume (7) – NO.(2)

July 2014 – Ramadan 1435H

Scientific Publications & translation

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

Deposit No.: 1429/2022

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)

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

101 102 Khaled B. Alharbi

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.

104 Khaled B. Alharbi

SRAY

BREEDING EWES

SRAY

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.

A Control Program for Abscess Disease of Sheep 105

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.

106 Khaled B. Alharbi

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 A Control Program for Abscess Disease of Sheep 107 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 108 Khaled B. Alharbi

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.

Journal of Agricultural and Veterinary Sciences Qassim University, Vol. 7, No. 2, pp. 109-124 (July 2014/Ramadan 1435H)

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.

101 110 Aida E. Bayad et al.

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.

MATERIALS AND METHODS 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 112 Aida E. Bayad et al. 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 2nd week 4th week 8th week 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 Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = ciprofloxacin. Reproductive, Hematological and Hepato-renal Effects… 113

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 2nd week 4th week 8th week 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 Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = 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 % 2nd week 4th week 8th week 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) 2nd week 4th week 8th week 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.

114 Aida E. Bayad et al.

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 ± standard error (n=5). Treatment Sperm abnormalities % 2nd week 4th week 8th week 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 Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = 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) 2nd week 4th week 8th week 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) 2nd week 4th week 8th week Control 11.70±0.25 A 9.77±0.85 A 11.42±0.60 A Cipro.(30 mg/kg B. wt) 8.48±0.61 B 8.79±0.96 A 11.22±0.33 A Cipro.(60 mg/kg B. wt) 6.90±0.40 C 7.42±1.62 A 10.16±0.46 A Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = ciprofloxacin.

Reproductive, Hematological and Hepato-renal Effects… 115

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) 2nd week 4th week 8th week Control 8.48 ±0.47 AB 7.24±0.29 A 7.94±0.32 A Cipro.(30 mg/kg B. wt) 7.39±0.48 B 8.05±0.26 A 8.99±0.47 A Cipro.(60 mg/kg B. wt) 9.48±0.50 A 8.64±0.66 A 8.18±0.26 A Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = 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 % 2nd week 4th week 8th week 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 Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = 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 administration (Tables 11-17). 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 administration (Tables 11-17).

116 Aida E. Bayad et al.

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 ± standard error (n=5). Treatment Creatinine (mg%) 2nd week 4th week 8th week 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 Cipro.(60 mg/kg B. wt) 0.68±0.03 A 0.66±0.04 A 0.66±0.04 A Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = ciprofloxacin.

Table (12): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on serum urea level at different periods in adult male rats. Values are expressed as mean ± standard error (n=5). Treatment Urea (mg%) 2nd week 4th week 8th week 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 Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = ciprofloxacin.

Table (13): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on serum albumin level at different periods in adult male rats. Values are expressed as mean ± standard error (n=5). Treatment Albumin (g/dl) 2nd week 4th week 8th week 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.

Table (14): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on serum globulin level at different periods in adult male rats. Values are expressed as mean ± standard error (n=5). Treatment Globulin (g/dl) 2nd week 4th week 8th week Control 1.53±0.05 AB 1.20±0.35 A 1.46±0.31A Cipro.(30 mg/kg B. wt) 1.35±0.29 B 2.08±0.34 A 1.62±0.13 A Cipro. (60 mg/kg B. wt) 2.07±0.24 A 1.54±0.25 A 1.26±0.40 A Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = ciprofloxacin.

Reproductive, Hematological and Hepato-renal Effects… 117

Table (15): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on serum total protein level at different periods in adult male rats. Values are expressed as mean ± standard error (n=5). Treatment Total protein (g/dl) 2nd week 4th week 8th week 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 Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = ciprofloxacin.

Table (16): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on serum ALT level at different periods in adult male rats. Values are expressed as mean ± standard error (n=5). Treatment ALT (IU/L) 2nd week 4th week 8th week Control 23.40±1.31 B 24.00±0.70 B 20.76±1.30 B Cipro.(30 mg/kg B. wt) 51.15±3.48 A 38.95±4.48 A 30.32±2.04 A Cipro.(60 mg/kg B. wt) 49.89±6.66 A 43.43±2.43 A 33.56±1.46 A Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = ciprofloxacin.

Table (17): Effect of oral administration of ciprofloxacin (30 mg and 60 mg/kg B. wt) on serum AST level at different periods in adult male rats. Values are expressed as mean ± standard error (n=5). Treatment AST (IU/L) 2nd week 4th week 8th week 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 Cipro.(60 mg/kg B. wt) 138.40±8.04 A 126.40±2.01 A 87.40±6.46 A Values on the same column carrying the same letters are not significantly different (p< 0.05). Cipro. = 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 118 Aida E. Bayad et al. 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.

Reproductive, Hematological and Hepato-renal Effects… 119

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.

120 Aida E. Bayad et al.

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, Reproductive, Hematological and Hepato-renal Effects… 121 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 122 Aida E. Bayad et al. 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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Culling, G.F.A. Handbook of Histological and Histochemical Techniques. 3rd Ed. Butterworth, London, Boston: (1974), 214. Demir, A.; Turker, P.; Onol, F.f.; Sirvanc, S; Findik, A. and Tarcan, T. Effect of experimentally induced Escherichia coli epididymo-orchitis and ciprofloxacin treatment on rat spermatogenesis. Intr. J. Urol.; 14: (2007), 268-272. Dollery, C. Therapeutic drugs, second edition. Churchill livingstone, Edinburgh London, NewYork. Philadelphia, San Francisco, Sydney, Toronto, P. C. (1999), 230-233. Drlica, K., and Zhao, X. DNA gyrase, topoisomerase IV and the 4-quinolones. Microbiol. Mol. Biol. Res.; 61: (1997), 377-392. Gilbersto,S. and Jones,D.R. Effect of nalidixic acid on blood. Brit. J. Urol; 44: (1972), 503. Grabe, M.; Forsgren, A. and Bjork, T. Concentration of ciprofloxacin in serum and prostate tissue in patients undergoing transurethral resection. Eur. J. Clin. Microbiol; 5: (1986), 211-212. Hooper, D.C. Quinolones. In, Mandell, Douglas, and Bennett's Principles and practice of Infectious diseases, 5th Edition (Mandell, G.L., Bennett, J.E., and Dolin, R., Eds) Churchill Livingstone, Inc., Philadelphia; (2000), PP.404-423. Khaki, A.; Heidari, M.; Novin, M.G. and Khaki, A.A. Adverse effect of ciprofloxacin on testes apoptosis and sperm parameters in rats. Iran.J.Repr. Med.; 6(2): (2008), 71-76. Lambert, T. and Jaupitre, A. Norfloxacin concentrations in human prostatic tissue. In proceeding of the 24th interscience conference on antimicrob. Agents Chemother. American Society for Microbiology, Washington. DC. (1984). Matousek, J. Effect on spermatogenesis in guinea pigs, rabbits and sheep after immunization with sexual fluid of bulls. J. Report. Fert., 19: (1969), 63-72. Matysiak, W.; Jodlowska-Jedrych, B.; Kifer-Wysocka, E.; Romanowska- Sarlej, J. and Czerny, K. Long term administration of ciprofloxacin as the cause of ultrastructural changes in the liver and disturbance of its functional balance; Microsc Microanal 14 (suppl.2): (2008), 1616-1617. Ng, E.Y.; Trucksis, M. and Hooper, D.C. Quinolone resistance mutations in topoisomerases IV: Relationship to the flg A locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase is the secondary target of fluoroquinolones in Staphylococcus aureus. Antimicrob. Agents Chemother.; 40: (1996), 1881-1888. Orgebin-Crist, M.C.; Danzo, B.J. and Gooper, T.G. Re-examination of dependence of the epididymal environment. J. Reprol. Fertil. Suppl. 24, (1976), 115-128. 124 Aida E. Bayad et al.

Paget, G. E and Barnes, J.M. (1964): Evaluation of Drug Activities, Toxicity Tests. Pharmacometrics. Academic Press, London and New York. Vol. I, (1964), P.135. Pfister, K.; Mazur, D.; Vormann, J. and Stahlmann, R. Diminished ciprofloxacin – induced chondrotoxicity by supplementation with magnesium and vitamin E in immature rats. Antimicrob. Agents and Chemother.; 51 (3): (2007), 1022-1027. Plumb, D.C. Plumb's Veterinary Drug Handbook, 5th Edition. Blackwell Publishing : (2005), P. 172. SAS Statistical Analysis System. User's Guide. SAS Institute Inc., Cary, NC, U.S.A. (1987). Schalm ,O.W. Veterinary Hematology, 4th edn. Lee and Febiger: Philadelphia. (1986). Walker, R.D.; Giguere, S.; Prescott, J.F.; Baggot, J.D. and Dowling, P.M. Antimicrobial Therapy in Veterinary Medicine. Fourth Edition, Blackwell Publishing, (2007), P. 263-284. Weyers, A.I.; Ugnia, L.I.; Garcia Ovando, H. and Gorla, N.B. Ciprofloxacin increase hepatic and renal lipid hydroperoxides levels in mice. Biocell; 26: (2002), 225-228. Wolfarm,C. Specific toxicologic aspects of quinolones. Rev. Infect. Dis. 10 (Suppl.1): (1988), 141-146.

Journal of Agricultural and Veterinary Sciences Qassim University, Vol. 7, No. 2, pp. 125-133 (July 2014/Ramadan 1435H)

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]

(Received 26/2/2014; accepted 11/3/1014)

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.

125 126 Saleh Altuwaijri

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 128 Saleh Altuwaijri 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).

A) B)

WBC Lymphocytes

2

2

l

l

µ

/

µ

/ cells

cells 1 *

cells 3

3 1

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0 0 TRAMP Wt TRAMP Wt Wt Wt 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.

Androgen ablation mitigates defect … 129

A) Spleen

B) C)

Wild Type TRAMP Wild Type

80.89 54.96

17.85 45.31

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 130 Saleh Altuwaijri 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.

A) B)

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TRAMP 4 0.8 Wt Androgen level TRAMP 0.6 Castration

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Surviving Surviving 0.4

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0.0 0 10 20 30 40 50 TRAMP TRAMP Age ( weeks) Wt Castration

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.

A) B)

TRAMP Wild Type TRAMP Castration 53.96 64.16

4.53 17.60

45.31 18.92

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

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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.

REFERENCE Altuwaijri S, Lin HK, Chuang KH, Lin WJ, Yet S, Hanchett LA, Rahman MM, Kang HY, Tsai MY, Zhang Y, Yang L, Chang C. Interruption of nuclear factor kappa B signaling by the androgen receptor. Cancer Research., 63: 7106-7112, 2003. Best CJ, Gillespie JW, Yi Y, Chandramouli GV, Perlmutter MA, GathrightY, Erickson HS, Georgevich L, Tangrea MA, Duray PH, Gonzalez S, Velasco A, Linehan WM, Matusik RJ, Price DK, Figg WD, Emmert-Buck MR, Chuaqui RF. Molecular alterations in primary prostate cancerafter androgen ablation therapy. Clin Cancer Res., 11: 6823-6834, 2005. 132 Saleh Altuwaijri

Cutolo M, Balleari E, Accardo S. Preliminary results of serum androgen level testing in men with rheumatoid artheritis.Arhritis Rheum., 27: 958–959.1984. Cutolo M, Balleari E, Giusti M.Androgen replacement therapy in male patients with rheumatoid arthritis. Arthritis Rheum., 34: 1–5, 1991. Cutolo M, Capellino S, Montagna P, Ghiorzo P, Sulli A, Villaggio B. Sex hormone modulation of cell growth and apoptosis of the humanmonocytic/macrophage cell line. Arthritis Research & Therapy 75:R1124-R1132, 2005. Dalgleish AG, O'Byrne K. Inflammation and cancer: the role of the immune response and angiogenesis.Cancer. Treat Res., 130:1-38, 2006. Gilardoni MB, Rabinovich GA, Oviedo M, Depiante-Depaoli M. Prostatecancerinduction in autoimmune rats and modulation of T cell apoptosis.J ExpClin Cancer Res., 18:493-504, 1999. Gray A, Garcia-Hernandez M, West MV, Kanodia S, Hubby B, Kast W.Blockade of TGF-β Signaling Greatly Enhances the Efficacy of TCR Gene Therapy ofCancer. J Immunol 1301270; published ahead of printAugust 12, 2013. Greenberg NM, DeMayo F, Finegold MJ, Medina D, Tilley WD, Aspinall JO,.Prostate cancer in a transgenic mouse.ProcNatlAcadSci U S A. 1995 Apr 11;92(8):3439–43. Greenberg NM, deMayo F, Finegold MJ, Medina D, Tilley WD, Aspinall JO,Cunha GR, Donjacour AA, Matusik RJ, Rosen JM.Prostate cancer in atransgenic mouse. Proc. Natl. Acad. Sci., 92:3439-3443, 1995. Hiroshi Miyamoto, Edward M Messing, Chawnshang Chang. Does androgen deprivation improve treatment outcomes in patients with low-risk andintermediate-risk prostate cancer? Nature Clinical Practice Oncol.2: 236- 237, 2005. Li L, Hsu P, Patel K, Saffari Y, Ashley I, Brody J. Polyclonal plasma cell proliferation with marked hypergammaglobulinemia and multiple auto antibodies. Ann Clin Lab Sci., 36:479-84, 2006. Maloney DG. Immunotherapy for non-Hodgkin's lymphoma: monoclonalantibodies and vaccines. J ClinOncol., 10:6421-6428, 2005. Medina, K. L. & P. W. Kincade: Pregnancy-related steroids are potential negativeregulators of B lymphopoiesis. ProcNatlAcadSci U S A., 91, 5382- 5386, 1994. Neeson, P. Paterson Y. Effects of the tumor microenvironment on the efficacy of tumor immunotherapy.Immunol Invest., 35:359-394, 2006. Androgen ablation mitigates defect … 133

Olsen NJ, Kovacs WJ.Effects of androgens on T and B lymphocyte development.Immunol Res., 23: 281-288, 2001. Olsen NJ, Gu X, Kovacs WJ.Bone marrow stromal cells mediate androgenic suppressionof B lymphocyte development.J Clin Invest., 108:1697-704,2001. Pope JE, Joneja M, Hong P.Anti-androgen treatment of prostatic carcinoma maybe a risk factor for development of rheumatoid arthritis. J Rheumatol., 29:2459- 2462, 2002. Smithson G, Couse JF, Lubahn DB, Korach KS, Kincade PW.The role of estrogen receptors and androgen receptors in sex steroid regulation of B lymphopoiesis.J Immunol. 161:27-34, 1998. Viselli, S. M., S. Stanziale, K. Shults, W. J. Kovacs & N. J. Olsen: Castration alters peripheral immune function in normal male mice. Immunology. 84:337-342, 1995. Viselli, S. M., K. R. Reese, J. Fan, W. J. Kovacs & N. J. Olsen: Androgens alter B cell development in normal male mice. Cell Immunol., 182: 99-104 1997. Wang RF. Functional control of regulatory T cells and cancer immunotherapy.Semin Cancer Biol., 2:106-114, 2006. Wilson, C. A., S. A. Mrose& D. W. Thomas: Enhanced production of B lymphocytes after castration. Blood., 85:1535-1539, 1995. Yi T, Wei YQ, Tian L, Zhao X, Li J, Deng HX, Wen YJ, Zou CH, Tan GH,Kan B, Su JM, Jiang Y, Mao YQ, Chen P, Wang YS. Humoral and cellular immunity induced by tumor cell vaccine based on the chickenxenogeneic homologous matrix metalloproteinase-2. Cancer Gene Ther.,14:158-164, 2007. Yin D, He Y, Hong SS, Marhefka CA, Stourman N, Kirkovsky L, Miller DD, and Dalton JT. Key Structural Features of Nonsteroidal Ligands for Bindingand Activation of the Androgen Receptor. Molecular Pharmacology, 63:211-223, 2003. Zhang Q, Jang TL, Yang X, Park I, Meyer RE, Kundu S, Pins M, JavonovicB, Kuzel T, Kim SJ, Van Parijs L, Smith N, Wong L, Greenberg NM, Guo Y, Lee C. Infiltration of tumor-reactive transforming growth factor-beta insensitive CD8+ T cells into the tumor parenchyma is associated withapoptosis and rejection of tumor cells. Prostate, 66:235-247, 2006

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Food science and human nutrition 136 Saleh Altuwaijri

Food science and human nutrition

Journal of Agricultural and Veterinary Sciences Qassim University, Vol. 7, No. 2, pp. 137-146 (July 2014/Ramadan 1435H)

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.

(Received 13/2/2014; accepted 2/4/2014)

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

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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 140 Amr M. Awad et al 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. Probable Atrophied Islet Cells Revival … 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. Body weight (gm) Groups Treatment mg/kg body Pretreatment 0 day Post treatment day 15 (Mean + SD) of mice wt. oral 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 .

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Table (2). Effect of O. eurpaea and L. sativum extracts on the blood glucose levels in normal and Alloxan treated diabetic mice Glucose levels mg/dl Groups Treatment mg/kg body Pretreatment 0 day Post treatment day 15 (Mean + SD) of rat wt. oral 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).

A

B C

Fig (1). Microscopic examination of normal Fig (2). Normal Mice Pancreas showing exocrine control showing normal pancreatic glands A, interlobar septa B and Islets of tissues formed of normal exocrine Langerhans C (H & E x 40). glands and interlobar septa .(H&E×40) Probable Atrophied Islet Cells Revival … 143

B

A

Fig (3). Mice Pancreas treated with Alloxan after 7 Fig (4). Pancreatic islet showing moderate days showing atrophied islets A and reduction in size and cellularity severe vascular congestion B (H & E x40) (arrow), mild degenerative changes (H&E x40).

A

A B

Fig (5). Microscopic examination of pancreatic Fig (6). Mice pancreas given Olea eurpaea tissues by treated Olea e-urpaea leaves leaves showing moderately showing mildly regenerating islets A and regenerating islets A and mild vascular decrease interlobar septa (H & E x 40) congestion B and There is still mild recovery of the islets as well as mild inflammation (H &E x 40).

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B A A

Fig(7). Microscopic examination of pancreatic Fig (8). Mice pancreas given L. sativum showing tissues by treated Lepidium Sativum moderate vascular congestion A and showing mildly regenerating islets A and mild infiltrates of lymphocytes B (H & decrease interlobar septa (H & E x 40) 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 Probable Atrophied Islet Cells Revival … 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. 146 Amr M. Awad et al

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.

Journal of Agricultural and Veterinary Sciences Qassim University, Vol. 7, No. 2, pp. 147-161 (July 2014/Ramadan 1435H)

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] )

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

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

147 148 Gadallah, Mohamed G.E et al

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). 150 Gadallah, Mohamed G.E et al

2.6. Swelling power and solubility: Swelling power and solubility were determined at 50, 70 and 90 oC 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 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) 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 oC 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 Evaluation of different modification methods … 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).

Crude Nitrogen Starch samples Moisture protein Lipids Ash free extract (N x 6.25) (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. 152 Gadallah, Mohamed G.E et al

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/cm3. 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. Bulk density Starch samples WBC (g/g) OBC (g/g) pH (g/cm3)

Native potato starch 0.866d 1.42b 1.98a 6.48d

Modified potato starch 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

Values followed by the same letters in the same column are not significantly different (p ≤ 0.05). 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 Evaluation of different modification methods … 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).

Native potato starch Pregelatinized potato starch

Acid-thinned potato starch Dextrinized potato starch

30.00

25.00

20.00

15.00

10.00 Swelling power (g/g) power Swelling 5.00

0.00 50 70 90 Temperature oC

Fig. (1). The swelling power as a function of heating treatment of native and modified potato starches.

154 Gadallah, Mohamed G.E et al

Native potato starch Pregelatinized potato starch Acid-thinned potato starch Dextrinized potato starch 50.00 45.00 40.00

35.00 30.00 25.00 20.00

Solubility (%) Solubility 15.00 10.00 5.00 0.00 50 70 90 o Temperature C 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 64.30oC 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 95oC, 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 Evaluation of different modification methods … 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.

Maximum Viscosity Viscosity Viscosity Break- Viscosity Viscosity Setback Gelatiniza- after 15 after 15 down Starch samples at 95 oC at 50 oC (B.U.) tion temp. oC min at 95 min at 50 (B.U.) (B.U.) o (B.U.) o oC B.U* C (B.U.) C (B.U.) (1) (2) (1) (2)

Native 63.25 95.00 1030 1030 675 660 700 0 355 -370 -15

Modified potato starch

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 156 Gadallah, Mohamed G.E et al 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.

Crude Nitrogen Caloric Samples Moisture protein Lipids Ash free extract values (N x 6.25) (NFE)* Control (without 47.40ab 1.16a 75.99a 2.06b 20.79b 771.69 starch)

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 Values followed by the same letters in the same column are not significantly different (p ≤ 0.05). 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.

Evaluation of different modification methods … 157

Table (5). Quality attributes of salad dressing incorporated with native and modified potato starches. Emulsion stability Samples pH (Oil recovered %)

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

Values followed by the same letters in the same column are not significantly different (p ≤ 0.05). 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). 158 Gadallah, Mohamed G.E et al

Control (without starch) Native potato starch Pregelatinized potato starch

Acid-thinned potato starch Dextrinized potato starch 10 9 8

7 6 5 4

Sensory score Sensory 3 2 1 0 Appearance Color Flavor Consistency Acceptability Sensory characteristics

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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Journal of Agricultural and Veterinary Sciences Qassim University, Vol. 7, No. 2, pp. 163-185 (July 2014/Ramadan 1435H)

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

(Received 1/2/2014; accepted 10/4/2014)

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

163 164 El- Hofi, M.A.; et al

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.

MATERIALS AND METHODS 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 166 El- Hofi, M.A.; et al 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.

Impact of Different Dietary Proteins … 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).

Initial body weight Final body weight Body weight gain Groups (g) (g) (%)

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%

a d II 107± 5.19 121.31± 7.15 13.29% a a III 107± 5.19 154.30± 4.88 44.11% IV 107± 5.19 a 150.20± 5.14b 39.43%

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 168 El- Hofi, M.A.; et al 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). Feeding period (days) Groups Initial 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). 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 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.

Impact of Different Dietary Proteins … 169

Table (3). Serum Triglyceride (mg/dl) of Hyperglycemic rats fed on supplemented diets containing different protein sources for 28 days. (n = 7 rats). Feeding period (days) Groups Initial 7 14 21 28

a e e d c Control- 86.48±2.1 84.24±2.3 85.63±2.8 85.23±2.8 85.66±2.8 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). 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

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 170 El- Hofi, M.A.; et al 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 different protein sources for 28 days. (n = 7 rats). Feeding period (days) Groups Initial 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 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

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. Impact of Different Dietary Proteins … 171

Table (5). Serum HDL (mg/dl) of Hyperglycemic rats fed on supplemented diets containing different protein sources for 28 days. (n = 7 rats(. Feeding period (days) Groups Initial 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 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

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.

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Table (6). Serum LDL (mg/dl) of Hyperglycemic rats fed on supplemented diets containing different protein sources for 28 days. (n = 7 rats). Feeding period (days) Groups Initial 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 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

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.

Impact of Different Dietary Proteins … 173

Table (7). Serum Total protein (mg/dl) of Hyperglycemic rats fed on supplemented diets containing different protein sources for 28 days. (n = 7 rats). Feeding period (days) Groups Initial 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 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

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.

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Table (8). Serum Albumin (mg/dl) of Hyperglycemic rats fed on supplemented diets containing different protein sources for 28 days. (n = 7 rats). Feeding period (days) Groups Initial 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 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

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.

Impact of Different Dietary Proteins … 175

Table (9). Serum Globulin (mg/dl) of Hyperglycemia rats fed on supplemented diets containing different protein sources for 28 days. (n = 7 rats). Feeding period (days) Groups Initial 7 14 21 28 Control- 2.505 ±0.03 a 2.725 ±0.31ab 2.510 ±0.27bc 2.380 ±0.21 b 2.468±0.21 c Control+ 2.505 ±0.03 a 2.373 ±0.31bc 2.393 ±0.28c 2.398 ±0.21 b 2.401±0.24 c I 3.005± 0.03 a 2.255 ±0.33c 2.591±0.30bc 2.600±0.24 b 2.773± 0.25 b II 2.505± 0.03 a 2.948±0.34 a 2.960 ±0.29a 2.906 ±0.26 a 2.970± 0.31a III 2.505± 0.03 a 2.621±0.21b 2.751 ±0.27ab 2.881 ±0.21 a 3.108±0.31a IV 2.505 ±0.03 a 2.190±0.34 c 2.095±0.30d 2.368 ±0.25 b 2.463±0.25c 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

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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Buko, V.; ukivskaya O.L.; Nikitin V.; Tarasov Y.; Zarodnik L.; Borodassky A.; Gorenshetin B.; Janz B. and Gundermann K.J. (1996). Hepatic and pancreatic effects of polyenoylphtidyl choline in rats with alloxan induced diabetes. Cell Biochem. Funct; 14(2):131-137. Carrol, K.K. and Kurowska E.M. (1995). Soy consumption and cholesterol reduction: review of animal and human studies. J. Nutr; 125:594S–597S. Chaan, A.Y.M.; Cheng M.L.L.; Keil L.C. and Myers B.D. (1988). Functional response of healthy and diseased glomeruli to a large, protein-rimeal. J. Clin. Invest, 81:245-254. Chapman, M.J. (1980). Animal lipoproteins: chemistry, structure and comparative aspects. J. lipid Res, 21:789-792. Claessens, M.; Calame W.; Siemensma A.D.; VanBaak M.A. and Saris W.H. (2007). The effect of different protein hydrolysate/carbohydrate mixturesposprandial glucagon and insulin response in healthy subjects. Eurjclin Nutr., 63:48-56. Cynthia, M and Kristin (1991). The effect of pravastatin on serum cholesterol Levels in hypercholesterolemic diabetic rabbits. Biochemical elbiophystea acid 238-244 Elsevier Science Publishers B.V.0925-4439/91 Doumas, B.T. (1971). Standard methods of clinical chemistry. Academic Press, New York, 4:85. Elizabeth, A.P.; Colleen P.G.; Sarah E.C.; Gerada A.D.; Johanna W.L. and Alison M.D (2012). Soy protein reduces serum LDL cholesterol and the LDLcholesterol: HDL cholesterol and apolipo protein B: apolipoprotein – Ratios in adults with type 2 diabetes. The journal of nutrition, 3:1700-1706. Fossati, P. and Prencipe L. (1982). The determination of triglyceride using enzymatic methods. Clin. Chem. 28:2077-2080 Friedman, M. (1996) Nutritional value of proteins from different food sources: a review. J Agric Food Chem., 44:6 –29 Fruhbeck, G. (1998). Slow and fast dietary proteins. Nature; 39:843–845. Gannon, M.C.; Nuttall F.Q.; Neil B.J. and Westphal S.A. (1988). The insulin and glucose responses to meals of glucose plus various proteins in type II diabetic subjects. Metabolism, 37:1081– 1088. Gannon, M.C.; Nuttall F.Q.; Krezowski P.A.; Billington C.J. and Parker S. (1986).The serum insulin and plasma glucose responses to milk and fruit products in type 2 (non-insulin-dependent) diabetic patients. Diabetologia, 29: 784–791. Henry, R. (1964). Determination of total protein. Clin Chem. Principles and Technics. Harper Row, N.Y., p. 182 Impact of Different Dietary Proteins … 177

Jacobucci, H.B.; Sagarbieri V.C.; Dias N.F.; Borges P. and Tanikawal C. (2001). Impact of different dietary protein on rat growth blood serum lipids, protein and liver cholesterol Nutrition Research, 21:905-915. John, F.M. (1978).Resistance of alloxan-Diabetic rats to the behavioral Activation induced by d-Amphetamine partial Restoration with a High fat protein Diet. Physiology and Behavior, 20:319-322. Kawase, M.; Hashimoto H. and Hosoda M. (2000). Effect of administration of fermented milk containing whey protein concentrate to rats and healthy men on serum lipids and blood pressure. J. Dairy sci; 83:255-263. Krebs, M.; Brehm A. and Krssak M. (2003). Direct and indirect effects amino acids on hepatic glucose metabolism in humans. Diabetologia, 46:917-925. Layman, D.K. (2003): The role of leucine in weight loss diets and glucose homeostasis. J Nutr, 133(1):261S-267S Leila, A. and Ahmad E. (2009). Soy protein consumption and kidney-Related biomarhers among type 2diabetes: A crossover randomized clinical trial .J. of Renal Nutrition, 19(6):479-486. Lopez-Virella, M.F. (1977). Determination of blood triglycérides using an oxidation peroxidase system. Clin. Chem. 21, 882. Lovali, M.R.; mest C.E. and Beynen C.A. (1990). Dietary animal's proteins and cholesterol metabolism in rabbits. Brit. Nut, 64:473-485. Manders, R.J.; Wagenmakers A.J.; Koopan R.; Zorenc A.H.; Menheere P.P. and Schaper N.C. (2005). Co-ingestion of a protein hydrolysate and amino acid mixture with carbohydrate improves plasma glucose disposal in patients with type 2 diabetes. A.M.J. clin. Nutr., 82:76-83. Nagaoka, S.; Kanamaru Y. and Kojima T. (1992). Comparative studies on the serum Cholesterol lowering action of Whey protein and soy bean protein in rats. Biotech Biochem., 56:1484-1450. Nelson, L.A.; Colner C.M.; Kalman D.S. and Swain M. (2002) A double bind comparative pilot trial evaluating the effect of whey protein isolate and soy protein isolate in healthy adults. Today’s Dietition 4:389-396. Nilsson, M.; Stenberg M.; Frid A.H.; Holst J.J. and Björck I.M.E. (2004). Glycemia and insulinemia in healthy subjects after lactose equivalent meals of milk and other food proteins: the role of plasma amino acids and in cretins. Am. J. Clin. Nutr., 80:1246-1253. Nogaoka, S. (1996). Studies on regulation of cholesterol metabolism induced by dietary food constituents or xenobiotis J.Jpn,Soc. Nutr. Food sci (in Japanese), 49: 303-313. 178 El- Hofi, M.A.; et al

Norton, S.A.; Beanes C.G.; Matwell C.V. and Morgan G.L. (1987). Effect of dietary whey upon the serum cholesterol of the pig .Nutr.Rep.Int., 36:273- 279. Park, M.S.C.; Kudchodkar B.J. and lipea G.U. (1987). Effects of dietary animal and plant proteins on the cholesterol metabolism in immature and mature rats.J.Nutr.117:130. Pimento, V.M.F.; Soria M.I.A.; Auler F.; and AFarfan J. (2006). Physical performance of exercising young rats fed hydrolysed whey protein at a sub- optimal level. Int. Dairy J., 16:984-999 Potter, S.M.; Bakhit R.M.; Essex-Sorlie D.L.; Wlingartner K.E.; Chapman K.M; Nelson, R.A.; Prabhudesai M.; Savage W.D.; Nelson A.I.; Winter L.W. and Erdman J.W. (1993). Depression of plasma cholesterol in men by consumptionof baked products containing soy protein. Am. J Clin. Nutr., 58:501– 506. Rocha, D.M.; Faloona G.R. and Unger R.H. (2004).Glucagon-Stimulating Active- ity of 20Ammino Acids in Dogs. J. Clin. Invest., 51:2346-2351 Saeed, A.; Jones S.A.; Nuttall F.Q. and Gannon M.C. (2002).Afasting-induced decrease in plasma glucose concentration does not affect the insulin response to ingested protein in people with type 2 diabetes. Metabolism, 51:1027–1033. Sandra, R.T.; Kelly A.T.; Leaann C.; Richard J.; Mukund P.; William, P and Maid John, W.E. (2004). Isolated soy protein consumption reduces urinary albumin excreation and improves the serum lipid profile in men type 2diabetes mellitus and nephropathy. J. Nutr., 143:1874-1880. SAS, (1999). Statistical Analysis System, SAS Users Guide: Statistics. SAS Institute Inc. Editor, Cary. NC. Sautier, C.; Dieng K.; Flament C.; Doucet C.; Suquet J.P. and Lemonnier D. (1993). Effect of whey protein, casein, soya-bean and sunflower protein on the serum, tissue and faecal in rats. Br. J. Nut., 49:313-319. Sendecor, G.W. and Cocharn, W. (1980). Statistical Methods 7th Ed, Aiwa Sate Univ., Press. Ames Aiwa, USA. P. 507. Shah, S.; Iqbal M.; Karam J.; Salifu M. and Mcfarlane S.I. (2007). Oxidative stress glucose metabolism and the prevention of diabetes: pathophysiological insights antioxid , Redox signaling, 9:911-929. Shermer, S. (1967). The blood morphology of laboratory animal's 3rd ed. p. 42 (Ed.F.A.Daviscompany), Philadelphia, US.A. Teixeira, S.R.; Potter S.M. and weigle R. (2000). Effects of feeding 4levels of soy protein for 3 and 6week on blood lipids and apolipoproteins in moderately hypercholesterolemic .Am. J. clin. Nutr., 71:1077-1084. Impact of Different Dietary Proteins … 179

Thomas, A.W.; Robert J.N.; Timothy K. and Brent F. (2007). Soy protein without isoflavones reduces aortic total and cholesterol ester concentrations greater than soy protein with is flavones compared with casein in hypercholesterolemic hamsters. Nutrition Research 27:498-504. Torres, N.; Torre-Villalavazo I. and Tovar A.R. (2006). Regulation of lipid metabolism by soy protein and its implication in diseases mediated by lipid disorders. J. Nutr. Biochem., 17:365-373. Trinder, P. (1969). Enzymatic colorimetric methods. Ann. Clin. Biochem, 6-24. Williams, A.J. and Walls J. (1987).Metabolic consequences of differing protein diets in experimental renal disease. Eur. J. Clin. Invest., 17:117-122.

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Journal of Agricultural and Veterinary Sciences Qassim University, Vol. 7, No. 2, pp. 181-196 (July 2014/Ramadan 1435H)

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

181 182 Mousa, H. M. et al

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 (Martı´nGarcı´a 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 Pertı´n˜ 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 .

Anti-diabetic effect of olive leaves extract in alloxan-diabetic rats 183

MATERIALS AND METHODS 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.

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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.

Anti-diabetic effect of olive leaves extract in alloxan-diabetic rats 185

Table (1). The effect of different concentrations from OLE on hematological parameters

Negative Positive Olive 3 % Olive 6% ANOVA control (n=6) control (n=6) (n=6) (n=6) 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 (103 657.3±9.5 a 794.7±83.6 b 939.3±42.0 c 847.8±68.1bc 6.59** ml) 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.

186 Mousa, H. M. et al

Table (2). The effect of different concentrations from OLE on blood glucose, cholesterol, triglycerides, total proteins, and albumin.

Negative Positive Olive 3 % Olive 6% ANOVA control (n=10) control (n=10) (n=10) (n=10) Mean±SD Mean±SD Mean±SD Mean±SD F 135.6±12.2±6 Glucose (mg/dl) 66.0±5.79 a 48.3±2.73 d 64.8±14.62 a 54.14*** b 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 29.8±6.42 a 16.6±3.12 b 22.7±3.61 c 32.5±5.68 a 12.37*** (mg/dl) 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 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<0.001

Table (3). The effect of different concentrations of OLE on the activities of Alanine aminotransaminase, aspartate aminotransaminase and alkaline phosphatase enzymes. (Mean±SD)

Negative Positive control Olive 3 % Olive 6% ANOVA control (n=6) (n=6) (n=6) (n=6) Mean±SD Mean±SD MeanSD Mean±SD F 33.64** AST (U/l) 142.8±16.7 ad 204.7±13.2 b 109.3±11.0 c 130.2±16.0 a * 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 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<0.05,** 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) Anti-diabetic effect of olive leaves extract in alloxan-diabetic rats 187 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. Congeste Neuron Neuronal Axonal Haemorrhag Perivascul d blood al chromatolys demylenati e ar Edema vessels necrosis is on 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 188 Mousa, H. M. et al 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.

References Al-Nozha, M M., Mohammed A. Al-Maatouq, , Yaqoub Y. Al-Mazrou, Saad S. Al-Harthi, Facharzt, Mohammed R. Arafah, , Mohamed Z. Khalil,Nazeer B. Khan, Akram Al-Khadra, , Khalid Al-Marzouki, Facharzt, Mohammed S. Nouh, Moheeb Abdullah, Omer Attas, , Maie S. Al-Shahid, , Abdulellah Al-Mobeireek,(2004). Diabetes mellitus in Saudi Arabia Saudi Med J 2004; Vol. 25 (11): 1603-1610 Anti-diabetic effect of olive leaves extract in alloxan-diabetic rats 189

Andreadou I, Iliodromitis EK, Mikros E, Constantinou M, Agalias A, Magiatis P, Skaltsounis AL, Kamber E, Tsantili-Kakoulidou A.(2006). The olive constituent uropein exhibits anti-ischemic, antioxidative, and hypolipidemic effects in anesthetized rabbits. J Nutr.;136:2213–2219. Bitler CM, Viale TM, Damaj R, Crea R.( 2005). Hydrolyzed olive vegetation water in mice has anti-inflammatory activity. J Nutr.;135:1475–9. Briante R, Patumi M, Terenziani S, Bismuto E, Febbraio F, Nucci R. a europaea L. (2002). leaf extract and derivatives: antioxidant properties. Journal of Agriculture and Food Chemistry;50(17):4934-4940. Carluccio MA, Siculclla L, Ancora MA, Scoditti E, Storelli C, Visioli F, Distante A, DeCaterina R. (2003). Olive oil and red wine antioxidant polyphenols inhibit endothelial activation. ArteriosclerThrombVasc Biol.;23:622–9. Eidi A, Eidi M, Darzi R.(2009).Antidiabetic effect of Oleaeuropaea L. in normal and diabetic rats. Phytother Res. 23(3):347-50. German JB, Walzem RL.(2000). The Health Benefits of Wine. Annu Rev Nutr. ;20:561–593. HedyaJemai , Abdelfattah El Feki and Sami Sayadi (2009).Antidiabetic and Antioxidant Effects of Hydroxytyrosol and uropein from Olive Leaves in Alloxan-Diabetic RatsJ. Agric. Food Chem., 57 (19), 8798–8804 Humason, G. L. (1979). Animal Tissue Techniques. (4th ed) W. N. Freeman and Company, San Francisco. Jones RL and Peterson CM.(1981) Hematologic alterations in diabetes mellitus Am J Med.;70(2):339-52. Lee JH, Yoon S, Renshaw PF, Kim TS, Jung JJ, Choi Y, Kim BN, Jacobson AM, LyooIK. (2013). Morphometric Changes in Lateral Ventricles of Patients with Recent-Onset Type2 Diabetes Mellitus. PLoS One. 2013 Apr4;8(4):e60515. Print 2013. PubMed PMID:23593231. Martı´nGarcı´a, A. I., A. Moumen, D. R. Ya´n˜ ez Ruiz, and E. Molina Alcaide. (2003). Chemical composition and nutrients availability for goats and sheep of two-stage olive cake and olive leaves. Anim. Feed Sci. Technol. 107:61– 74. Menendez JA, Vazquez-Martin A, Colomer R, Brunet J, Carrasco-Pancorbo A, Garcia-Villalba R, Fernandez-Gutierrez A, Segura-Carretero A. (2007). Olive oil's bitter principlereversesacquired autoresistance to trastuzumab (Herceptin™) in HER2-overexpressing breast cancer cells. BMC Cancer. ;7:80.doi:10.1186/1471-2407-7-80. 190 Mousa, H. M. et al

Miles EA, Zoubouli P, Calder PC. (2005). Differential anti-inflammatory effects of phenolic compounds from extra virgin olive oil identified in human wh blood cultures. Nutrition.;21:389–94. Reno CM, Tanoli T, Bree A, Daphna-Iken D, Cui C, Maloney SE, Wozniak DF,Fisher SJ (2013).. Antecedent Glycemic Control Reduces Severe Hypoglycemia-Induced Neuronal Damage in Diabetic Rats. Am J PhysiolEndocrinolMetab., 15;304(12):E1331-1337. Sato H, Genet C, Strehle A, Thomas C, Kobstein A, Wahner A, Mioskowski C, Auwerx J, Saladin R. (2007). Anti-hyperglycemic activity of a TGR5 agonist isolated from a europaea. BiochemBiophys Res Commun.;362:793– 798. Sedef N El, SibelKarakaya(2009). Olive tree (a europaea) leaves: potential beneficial effects on human health Nutrition Reviews 67,( 11), 632–638. Tuck KL, IIayball PJ.( 2002). Major phenolic compounds in olive oil: metabolism and health effects. J Nutr Biochem.;13:636–644. Visioli F., Galli C. (1994). uropein protects low density lipoprotein from oxidation. Life Sci.;55:1965–71 Wainstein J, Ganz T, Boaz M, Bar Dayan Y, Dolev E, Kerem Z, Madar Z.(2012). Olive leaf extract as a hypoglycemic agent in both human diabetic subjects and in rats. J Med Food. 15(7):605-10. Wawersik, J. (1991). "History of Anesthesia in Germany". Journal of Clinical Anesthesia 3 (3): 235–244 Zhao B, Pan BS, Shen SW, Sun X, Hou ZZ, Yan R, Sun FY. (2013). Diabetes- inducedcentralneuritic dystrophy and cognitive deficits are associated with theformation of oligomeric reticulon-3 via oxidative stress. J Biol Chem., 31;288(22):15590-15599.

Anti-diabetic effect of olive leaves extract in alloxan-diabetic rats 191

Plant production

192 Mousa, H. M. et al

Journal of Agricultural and Veterinary Sciences Qassim University, Vol. 7, No. 2, pp. 193-206 (July 2014/Ramadan 1435H)

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]

(Received 27/2/2014; accepted 23/3/2014)

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

193 194 Hazem A. Obiadalla Ali

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.

MATERIALS AND METHODS 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.

Screening of thirteen garlic… 195

Table (1). Soil characterization of the experimental site.

P

-

1

- Sampling

% % % % %

Silt

Soil

(%)

ppm ppm

O.M

Clay

Sand

depth dSm

(1:2.5)

CaCO3

Total Total N

Texture

E.C. (1:5)

pH pH (H2O)

NaHCO3

Available K

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 196 Hazem A. Obiadalla Ali 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.

Plant height Number of Neck bulb diameter Cured head bulb Genotypes (cm) leaves/plant (cm) diameter (cm) 2010/2011 2011/2012 2010/2011 2011/2012 2010/2011 2011/2012 2010/2011 2011/2012 10.70 4.950 Sids 40 54.80 l 54.40 h 11.10 a 2.900 ab 2.767 a 5.100 b abcd cde 10.63 Gehena 80.60 i 80.30 f 10.20 a 2.467 de 2.333 d 5.000 b 4.850 e bcd 11.20 Tahrir 90.20 b 90.10 ab 10.90 a 2.633 cd 2.500 bc 5.050 b 4.900 de abc 5.000 Sohag 85.50 e 85.30 d 11.40 a 11.40 a 2.733 bc 2.600 b 5.133 b bcde 5.050 Qena 89.40 c 89.20 b 11.33 ab 11.00 a 2.700 c 2.633 b 5.100 b bcd 11.17 Elbehera 87.70 d 87.40 c 11.30 a 2.500 de 2.400 cd 5.200 b 5.100 bc abc 10.93 Elgharbia 91.60 a 91.50 a 11.20 a 2.633 cd 2.567 b 5.200 b 5.133 b abc Bani 5.050 81.20 h 81.10 f 10.20 d 10.10 a 2.967 a 2.900 a 5.150 b Swief 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 10.70 Aswan 58.50 k 58.20 g 10.50 a 2.733 bc 2.633 b 5.600 a 5.500 a abcd 11.20 Assiut 84.00 f 83.70 de 10.60 a 2.000 f 1.900 e 5.000 b 4.900 de abc 10.90 5.000 Elwady 81.30 h 81.10 f 11.10 a 2.400 e 2.333 d 5.050 b abc bcde 11.03 El-faiyum 59.20 j 59.10 g 10.80 a 3.000 a 2.900 a 5.500 a 5.400 a abc 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 Screening of thirteen garlic… 197 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 Cured bulb height Plant fresh weight Number ratio (cm) (gm) 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 0.5767 a 0.5667 a 2.750 d 2.800 cd 45.03 f 45.00 f 38.70 a 38.63 a Swief 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

198 Hazem A. Obiadalla Ali

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 Total soluble solids Total yield (gm) (%) (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. Screening of thirteen garlic… 199

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

(cm) (cm) (cm)

(gm)

No. of

Weight Weight of

Total Total yield

Number of

Plant Plant fresh

cloves/bulb

cloves/bulb

Bulb Bulb height

leaves/plant weight (gm)

Plant Plant height

(ton/feddan)

Total Total soluble

solids (T.S.S)

Bulb Bulb diameter

Plant height - - - - - 0.141NS -0.595** 0.910** -0.380* (cm) 0.493** 0.609** 0.848** 0.414** No. of - - 0.143NS 0.162NS 0.502** 0.126NS 0.314NS 0.392* leaves/plant 0.0699NS Bulb diameter - 0.582** 0.606** -0.561** 0.479** 0.406** 0.347* (cm) Bulb height - 0.446** -0.703** 0.604** 0.592** 0.203NS (cm) Plant fresh - -0.699** 0.777** 0.600** 0.793** weight (gm) Number of - - - -0.354* cloves/bulb 0.837** 0.464** Weight of cloves/bulb - 0.580** 0.697** (gm) Total soluble - 0.491** solids (T.S.S) Total yield - (ton/feddan) * = Correlation is significant at the 0.05 level, ** = Correlation is significant at the 0.01 level

200 Hazem A. Obiadalla Ali

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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