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Page 1: ! Broj 2 Hrana i ishrana FINAL 3 ISPRAVKA 28-12-2018hrana-ishrana.org/.../2019/01/Hrana-i-ishrana-59-br-2.pdfHRANA I ISHRANA (BEOGRAD), VOL. 59, . 2/2018. II Društvo za ishranu Srbije

2BEOGRAD 2018

59

59

UDK: 613.2

59

Page 2: ! Broj 2 Hrana i ishrana FINAL 3 ISPRAVKA 28-12-2018hrana-ishrana.org/.../2019/01/Hrana-i-ishrana-59-br-2.pdfHRANA I ISHRANA (BEOGRAD), VOL. 59, . 2/2018. II Društvo za ishranu Srbije

2BEOGRAD 2018

59

59

UDK: 613.2

59

Page 3: ! Broj 2 Hrana i ishrana FINAL 3 ISPRAVKA 28-12-2018hrana-ishrana.org/.../2019/01/Hrana-i-ishrana-59-br-2.pdfHRANA I ISHRANA (BEOGRAD), VOL. 59, . 2/2018. II Društvo za ishranu Srbije

Vlasnik i izdavač / Owner and Publisher:

Društvo za ishranu Srbije / Serbian Nutrition Society11000 Beograd, Savska 9/III

Izdavački savet / Editorial Committee:

Ljiljana Trajković Pavlović, Dragojlo Obradović, Bato Korać, Ida Leskošek-Čukalović, Petrica Ružić , Anita Klaus, Ivan Stanković, Nađa Vasiljević, Nedeljko Radlović, Slavica Šiler-Marinković, Suzana Dimitrijević, Vera Katić, Jasna Mastilović, Vesna Vučić

Glavni i odgovorni urednik / Editor in Chief:

Prof. dr Bato Korać

Zamenik glavnog i odgovornog urednika:

Prof. dr Petrica Ružić

Uređivački odbor / Editorial Board:

Evangelos Polychronopoulos (Gračka), Heiner Boeing (Nemačka), Philip Calder (Velika Britanija), Stefaan de Henauw (Belgija), Marie Kunesova (Češka), Budimka Novaković (Srbija), Ana Stančić (Srbija), Aleksandra Korać (Srbija), Zorica Nikolić (Srbija), Sladjana Šobajić (Srbija), Bojana Vidović (Srbija), Miomir Nikšić (Srbija), Dušica Stojanović (Srbija), Milka Popović (Srbija), Sladjana Žilić (Srbija)

Lektor za engleski jezik: Danica Pavlović

Grafi čka obrada i lektura: Dušan Ćasić

Saradnik za UDK: Narodna biblioteka Srbije

Uredništvo i administracija:

11000 Beograd, Savska 9/II; p. fah: 333Tel.: ++381(0)11 420-2998

ČASOPIS IZLAZI DVA PUTA GODIŠNJE

U troškovima štampanja časopisa učestvuje Ministarstvo prosvete, nauke i tehnološkog razvoja Republike Srbije

Štampa:

Ton Plus, Danila Lekića Španca 3111077 Beograd, Tel.: 011/3016624

Tiraž: 100 primeraka

SADRŽAJ / CONTENTS

Reč urednika / Word from the editor ......................................................................... II

PREGLEDNI RADOVI / REVIEW PAPERS

Vesna Otašević, Aleksandra Korać, Ana Stančić, Aleksandra Janković,

Bato Korać

Impact of nutrition on human fertility / Uticaj ishrane na fertilitet kod ljudi ...................................................................................................... 53

Sanja Mijatović, Danijela Maksimović-Ivanić

Aloe emodin: from anti- to pro-tumor action / Aloe emodin u tretmanu tumora: saveznik ili protivnik ............................................................ 59

Marijana Simić, Slađana Žilić

Proteini pšenice sa tehnološkog, nutritivnog i zdravstvenog aspekta / The technological, nutritional and medical aspects of wheat proteins ........................................................................................................... 68

ORIGINALNI RADOVI / ORIGINAL PAPERS

Vanja Todorović, Anđelka Dančetović, Nevena Dabetić, Slađana

Šobajić, Bojana Vidović

Antioksidativna aktivnost odabranih začina sa tržišta Srbije / Antioxidant activity of selected spices from Serbian market .................... 74

Emilija Oreščanin, Ivana Perić, Mirjana Pešić, Slađana Stanojević

Koliko smo upoznati sa osobinama i prisustvom mikotoksina u hrani? / How much we know about properties and the presence of mycotoxins in the food? ......................................................................................... 80

STRUČNE VESTI / PROFESSIONAL NEWS

Iz prirode u susret Nobelovoj nagradi ............................................................... 85

PRIKAZ: LETNJA ŠKOLA, BEOGRAD, 2018. GODINE

Translating scientifi c fi ndings into nutritional recommendations .......... 86 Good practice for human dietary intervention studies .............................. 87 Assessment of dietary intake at the population level – methods, signifi cance, application ...................................................................... 88

Clinical study and evidence .................................................................................. 89 Approaches to monitor population dietary sodium/salt intake ............. 90

U SPOMEN / IN MEMORIAM ................................................................................. 91

UPUTSTVO AUTORIMA / INSTRUCTION TO AUTHORS .......................... 92

VOL. 59 BEOGRAD, 2018. BROJ 2

ISSN 0018-68727UDK: 613.2

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HRANA I ISHRANA (BEOGRAD), VOL. 59, №. 2/ 2018.

II

Društvo za ishranu Srbije je jedna od najstarijih zajed-nica naučnih radnika i stručnjaka u Srbiji koja ima za cilj da bude nacionalni servis stručne i šire javnosti. Od svog osnivanja 1956. godine objedinjuje istraživače iz oblasti agronomije, biohemije, hemije, farmacije, hu-mane i veterinarske medicine, dijetetike i drugih srod-nih naučnih disciplina koji brinu o proizvodnji i bez-bednosti hrane i zdravlju nacije, posebno dece.HRANA I ISHRANA je pregledni časopis Društva za ishranu Srbije koji od 1960. godine prati osnovne po-stulate Društva u cilju poboljšanja i unapređenja jav-nog zdravlja. Časopis objavljuje originalne istraživačke i pregledne čla nke, teme za diskusiju, vesti i informaci-je, kao i ekspertske izveštaje o svim aspektima hrane i ishrane.Časopis je mesto koje obezbeđuje da se glas naučne zajednice čuje i blagovremeno prenose informacije o hrani i ishrani stanovništvu, mesto za sve napredne ide-je i kritičko razmišljanje.Osnovni cilj časopisa HRANA I ISHRANA je integraci-ja znanja fundamentalnih istraživanja nauke o hrani i ishrani u zdravlju i bolesti. Paralelno sa istraživanjem molekularnih mehanizama delovanja hrane, časopis je usmeren na praćenje zakonodavstva i etičkih stan-darda, što je danas veliki izazov u svetu kada se radi o hrani.Urednički tim očekuje interaktivnu saradnju i pomoć svih naučnika kojima je hrana i ishrana više od znače-nja tih reči, etička kategorija koja ima za cilj poboljšanje zdravlja nacije.

Prof. dr Bato KoraćGlavni i odgovorni urednik

Serbian Nutrition Society is one of the oldest commu-nities of scientists in Serbia, which aims to be a national service and a service to all people. Since its establish-ment in1956, it unites researchers, doctors, educators and all followers who care about the health of the na-tion, especially children.The Food and Nutrition is a peer reviewed journal of the Serbian Nutrition Society which since 1960 follows the basic postulates of the Society, with the aim to improve and promote public health. The Journal pub-lishes original research and review articles, topics for discussion, news and information, and expert reports on all aspects of food and nutrition.The Journal is a place that ensures that the voice of the scientifi c community is heard and timely transmits information related to food and nutrition to the popu-lation, the place for all the advanced ideas and critical thinking.The primary aim of the Food and Nutrition journal is to integrate knowledge of fundamental research of food science and nutrition in health and diseases. In parallel with the research of molecular mechanisms of action of food, the scope of the Journal is to monitor legislatures and ethical standards, which today is a great challenge in the world when food is concerned.The editorial board expects interactive cooperation and assistance from all scientists to whom food and nutrition are more than the meaning of those words, an ethical category aimed at improving the health of the nation.

Prof. dr Bato KoraćEditor in Chief

DRUŠTVO ZA ISHRANU SRBIJE, ČASOPIS „HRANA I ISHRANA”

SERBIAN NUTRITION SOCIETY, JOURNAL “FOOD AND NUTRITION”

Beograd, Savska 9/II, tel: 011/420 2998; p. fah: 333Žiro račun: 355-1032408-17

Reč urednika / Word from the editor

Ukoliko želite da naručite određeni broj časopisa, možete se obratiti na sledeći mail: [email protected].

Ukliko želite da se učlanite u Društo za ishranu Srbije, neophodno je da popunite zahtev koji se nalazi na internet stranici Društva za ishranu Srbije www.hrana-ishrana.org i uplatite godišnju

članarinu u iznosu od 1000 dinara na sledeći žiro račun 355-1032408-17.

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Otašević et al.: Impact of nutrition on human fertility

INTRODUCTION

Infertility is one of the most serious medical issues that has dramatically increased in our country and world-wide, especially in a recent years [1]. Sterility is almost identically represented in both men and women (from 30% to 40%) and is caused by a various number of fac-tors [2,3]. Certain factors that cause infertility can only be successfully overcome by using assisted reproduc-tion methods and most often in vitro fertilization (IVF) is the only possible path to parenting. The development of medicine in the fi eld of infertility treatment and in artifi cial reproductive technologies (ART) allows 80% of couples to achieve their goal and become parents. However, despite the progress in reproductive medi-cine, infertility progresses too. Thus, а crucial direction in biomedicine today is the development of new ap-proaches for infertility treatment. Extensive research dedicated to this problem clearly shows that sterility is a disease of modern society and that lifestyle has a substantial impact on the development of sterility. In particular, it has been shown that bad nutrition, i.e. specifi c nutritional factors signifi cantly infl uence hu-man (in)fertility [4]. What is encouraging when we talk about nutrition is the fact that nutritional factors are easily interchangeable and that simple modifi cation of diet pattern can have a huge impact on (in)fertility.

Healthy dietary pattern among both men and women of reproductive age includes foods which are dense in specifi c nutrients needed for hormonal func-

tion, production and balance, fetal development, egg health, sperm health, blood health and much more. Although the meaning of unhealthy dietary pattern to some extent varies across the studies, it have remark-able overlaps in diets rich in calories [5], trans-fatty acids (TFAs), saturated fats [6] or cholesterol [7] that have been associated with testicular disruption, im-pairments in spermatogenesis, menstrual dysfunction, anovulation, infertility, miscarriage, and pregnancy complications [8,9]. On the other hand, diet rich in fi -ber, whole grains, fruits, vegetables, fi sh and olive oil, folate and lycopene; the use of vegetable rather than animal proteins, full-fat dairy products, food with re-duced glycemic index and unsaturated fats have been shown to improve fertility in women and quality of se-men in men, ART outcomes and chance of pregnancy [10–12] (Scheme 1).

How diet is important to fertility best exemplifi es the Harvard study [13] in which 18555 married women without a history of infertility were followed up as they attempted pregnancy or became pregnant during an 8 year period. Results showed that consuming 5% of total energy intake as vegetable protein rather than as animal protein was associated with a more than 50% lower risk of ovulatory infertility. In addition, women with higher intake of mono-unsaturated over trans-fat ratio, vegetable over animal protein, high-fat over low-fat diary, a decreased glycemic load, and an increased intake of multivitamins had lower rates of ovulation

Impact of nutrition on human fertility

Abstract

Infertility is one of the most serious medical issues that is dramatically ri-sing worldwide. Extensive research dedicated to this problem clearly shows that infertility is a disease of modern society and that a nutrition has a great infl uence on the development of sterility. Thus, the impact of specifi c nutri-tional factors, i.e. diet pattern on both male and female fertility is included in this review. It is encouraging that modifi cation of nutritional habits can help couples to conceive spontaneously, or increase their chances of con-ception with in vitro fertilization (IVF) treatment.Key words: Infertility, nutrition, obesity, fat, carbohydrates, proteins, micronutrients

UDK: 613.392:612.663

Vesna Otašević1, Aleksandra Korać2, Ana Stančić1, Aleksandra Janković1, Bato Korać1,2

1 University of Belgrade, Institute for Biological Research “Siniša Stanković”, Bulevar despota Stefana 142, Belgrade, Serbia;

2 University of Belgrade, Faculty of Biology, Belgrade, Serbia.

Address of correspondence: Professor Bato Korać, PhDUniversity of Belgrade, Institute for Biological Research „Siniša Stanković”, Department of Physiology, Bulevar despota Stefana 142, 11060 Belgrade, SerbiaTel.: (381-11)-2078-307Fax: (381-11)-2761-433e-mail: [email protected]

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disorders [13]. Diet consisting of the aforementioned food groups is Mediterranean diet which is clearly shown to be benefi cial on achieving clinical pregnancy and live birth among non-obese women, but specifi -cally those below the age of 35 [14]. The same holds true for men. Namely, it has been shown that healthy dietary pattern, such as Mediterranean type of diet has been strongly associated with better semen quality pa-rameters worldwide [15–17]. In addition, the healthy dietary pattern has been shown to be associated with decrease in sperm DNA fragmentation index [15].

Generally, infertility caused by nutritional factors is associated with changes in the synthesis and activity of hormones, or quality of gametes. Estrogen, the main sexual hormone in women is produced in two places in the body, ovaries and adipose tissue. High or low lev-els of body fat aff ect the synthesis of estrogen. These changes in the production of estrogen may cause per-manent or temporary infertility, since the disorder of menstrual cycle or ovulation. The general rule is that people who have a body mass index (BMI) below 20 or over 30 are out of weight range for achievement of op-timal fertility. Underweight, namely, weighting 10–15% less than an ideal body weight can result in a disturbed menstrual cycle [12]. On the other hand, a slight in-crease in body weight in women with extremely low percentage of body fat can improve fertility. In obese women insulin resistance adversely aff ects fertility [18]. Women with a BMI over 30 have bad endometrial re-ceptivity, and thus a signifi cantly higher percentage of spontaneous abortions (13.6% compared to 10.7% in women with normal BMI) and lower rates of pregnancy (38.3% vs 45.5%) [19,20]. Obesity is common in wom-en of reproductive age, with 38.3% of women over 20 years of age classifi ed as obese [8]. In addition, assisted reproduction is less eff ective in overweight and obese women, with lower pregnancy and live birth rates, and increased incidence of pregnancy loss [8].

Obesity aff ects the fertility of men too. An increase in BMI of only 3 units signifi cantly reduces sperm pa-rameters [21], and increases the number of mutations

in the DNA of spermatozoa [22]. Also, the lack of body weight or excessive accumulation of fat in the abdomi-nal area is associated with reduced synthesis of testos-terone and consequently poor sperm quality [23]. Many factors may explain the relationship between over-weight and poor sperm parameters, which includes al-terations in hormones, such as decreased testosterone, increased estradiol, and elevated endorphins that can impact sperm production; hyperinsulinemia, which may mediate a decrease in sex hormone-binding glob-ulin in obese men; a rise in scrotal temperature caused by fat tissue accumulation; and an increased accumu-lation of toxic substances in fatty tissue [24].

Luckily, negative eff ects of obesity on the fertility of both men and women may be reversible. Numerous studies have shown that normalization of body fat in-fl uences the levels of reproductive hormones and con-sequently improved fertility. The best way to maintain/achieve an ideal body weight and good reproductive performance is to eat a healthy, balanced diet. Still, there are specifi c nutrients and food categories that are particularly important for reproductive function bot in women and men. First, macronutrients are required – fats, carbohydrates and protein. Then there are essen-tial vitamins and minerals (micronutrients), cholesterol and fi ber. Importance of this nutrients on human fertil-ity are summarized below.

MACRONUTRIENTS

Carbohydrates

Dietary carbohydrates impact fertility by infl uencing glucose homeostasis and insulin sensitivity, which in turn aff ects ovarian androgen production and ovarian function [25]. Data from the large prospective cohort of healthy women in the Nurse’s Healthy Study II (NHS-II), revealed that both total carbohydrate consumption and glycemic load were associated with higher risks of ovulatory infertility [26]. Accordingly, it has been shown that reduction in dietary carbohydrates among women with polycystic ovary syndrome improved in-sulin sensitivity and enhance ovulatory function [27]. In the line with this are results showing that constit-uents of whole grains, which have a benefi cial eff ects on glucose metabolism, increase fertilizing potential by increasing insulin sensitivity [28]. Also, women who had higher preconception intake of whole grains is as-sociated with higher rate of live birth [29]. High dietary load of carbohydrates negatively impact men fertility too. A recent study associated high intakes of refi ned carbohydrates and the ensuing high blood glucose levels with a decreased sperm count in young men [30]. According to systematic review that covers two decades, as carbohydrate intake and dietary glycemic load increase sperm concentration and semen quality decreased, unrelated to the male years [31].

Scheme 1. Impact of nutritional factors on human fertility

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Dietary fats

Fatty acids (saturated, SFA; monosaturated, MUFA and polyunsaturated, PUFA) are very important for numer-ous reproductive functions in humans. In men, compo-sition of sperm cell membrane is of utmost importance for proper sperm function, i.e. crucial sperm fertilizing processes – capacitation, acrosome reaction, sperm-oo-cyte fusion [1]. As sperm matures, the amount of PUFA (especially docosahexaenoic acid, DHA) increases. Con-sumption of food rich in these fatty acids modifi es com-position of sperm membrane and consequently, semen quality [32]. Higher intake of omega-3 PUFAs has been related to increased population of morphologically normal sperm [32], and increased sperm count [30]. Walnuts contain large concentration of omega-3 fatty acids, and supplementation of young men consuming Western diet with 75 g/d for 12 weeks signifi cantly im-proved all functional parameters of sperm, compared to control subjects [32]. In female, fatty acids are used as energy substrates during oocyte maturation and early embryo development [33]. As a precursor for prostaglandins and steroid hormones, they are also vi-tal for implantation and pregnancy maintenance [33]. Accordingly, intake of DHA has been shown to be asso-ciated with increase in total level of estradiol and lower risk of anovulation [34]. On the other hand trans fatty acids have deleterious eff ects on reproductive func-tions both in men and women. These fatty acids are pri-marily found in commercially baked and fried food, and after consumption they accumulate in the testis, lead-ing to defective spermatogenesis, poor semen quality and subfertility [32,34]. Trans fat intake was associated with hormonal disables [35], increased ovulatory infer-tility [12] and reduced fecundability [33].

Proteins

Proteins are micro nutrients that are vital both for conception and pregnancy. They are responsible for building and repairing cell, producing hormones and a healthy reproductive functions. However, proteins from diff erent foods are diff erent in their eff ects on reproductive functions in both sexes. The main sourc-es of protein in human nutrition are milk (dairy) and meat. Generally, literature on the relationship between dairy product intake and human fertility is inconclu-sive. While one study reported few associations be-tween preconception dairy intake and fertility in two populations of reproductive-aged women [33], NHS II reported no associations between total intake of dairy products and risk of infertility [36]. In terms of eff ects of fat percent in dairy products, it has been report-ed association of full-fat dairy products intake with a lower risk of ovulatory infertility and association of low-fat dairy products and a high ovulatory infertility risk [36]. Similar inconsistence was observed in male population. Here some studies suggested dairy as a possible risk for poorer semen parameters while other did not support this standpoint [32]. While few stud-

ies reported that intake of full-fat dairy products was adversely related to normal sperm morphology and motility, cross-sectional study among fertility patients in the Netherlands reported no correlation between dairy intake and semen quality [15]. Studies about re-lation between meat intake and semen quality gener-ally suggest that red meat have an adverse eff ects. Two cross-sectional studies reveled lower sperm concentra-tion and motility and morphology in men who con-sumed higher amounts of processed meat [30,32]. In women, results from an infertility cohort study showed that blastocyst formation during embryo development was aff ected negatively by consumption of red meat and positively by consumption of fi sh [33]. Also, ovula-tion was negatively aff ected by increased meat intake among NHS-II participants [12].

MICRONUTRIENTS

Folic acid

In addition to its benefi cial role in preventing neural tube defects in infants, folic acid is important for nor-mal reproductive functions both in women and men [32,33]. Namely, it has been shown that subfertile wom-en who took a multivitamin containing 400 μg of folic acid for three months had a 26% of pregnancy compar-ing to women in placebo group, who had 10% of preg-nancy [37]. Explanation for this relays in a fact that mul-tivitamin users had approximately one third lower risk for developing ovulatory infertility compared to non-users. Similarly, Biocycle study reveal that folate intake decreased frequency of anovulation of young healthy women, while Danish study reported shorter time to pregnancy in pregnancy planners [33]. In addition, favorable eff ect of folic acid supplementation on ART outcomes has been shown among subfertile women [37]. In men, folic acid intake improved sperm count, as well as proportion of motile spermatozoa [32,38]. Also, seminal plasma folate emerged as very important for sperm DNA integrity and sperm aneuploidy [39]. Nev-ertheless, dose-response benefi t of folate seems to be beyond the recommended for the prevention of neural tube defects and testing oriented to dose determina-tion still running. However, current evidence based on such benefi cial data, strongly recommend folic acid supplementation before and during pregnancy.

Vitamins A, C, D, E

Data concerning eff ects of vitamins on fertility have some limitations particularly in studies on females, where multivitamins rather than monosubstances, were tested. Thus, the eff ect cannot always be abso-lutely attributed to one substance. Anyway, positive eff ects of these substances on human fertility deserves further attention. Vitamin C seems to be very import-ant for sperm functioning. Namely, it was shown that administration of 200 and 1000 mg of vitamin C daily

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signifi cantly improved sperm quality [39], as well as administration of combination of vitamin C and E [39]. Also, a signifi cantly higher seminal vitamin C level in fertile vs. infertile individuals, as well as a correlation be-tween seminal vitamin C and the percentage of sperm with normal morphology was shown by Colagar and Marzony [40]. In women with luteal phase defects, dai-ly substitution of 750 mg of vitamin C for a six months signifi cantly increased level of progesterone and estro-gen, as well as pregnancy rate [39]. A several studies re-vealed an improvement of various sperm parameters, as well as a higher pregnancy rate after substitution with 300–600 mg per day of vitamin E [39]. Also, serum concentrations of retinol and α-tocopherol in men with normal sperm parameters were signifi cantly higher than in those with oligozoospermia and asthenozo-ospermia [41]. Prospective studies of vitamin A and hu-man fertility tested mixed substances only. Data from these studies revealed an increased sperm count after therapy with a mix of antioxidants containing 0.06 IU/kg vitamin A [39] and improved conception rate, pro-gesterone level and rate of mid luteal days in women supplemented with multivitamin containing 3600 IU of vitamin A [39]. Despite the shown positive correlation between vitamin D and sperm motility and morphol-ogy [32], as well as on various reproductive processes in women, these data are based on association and in vitro studies and should therefore be interpreted with caution.

Selenium, Zinc, Iodine, Coenzyme Q10

Both selenium and iodine are important for synthesis of thyroid hormones. Considering that thyroid dysfunc-tion lead to impaired spermatogenesis it is clear that both iodine and selenium defi ciency infl uence male infertility. Accordingly, it has been shown that combi-nation preparations containing 100–200 μg of these elements achieved benefi cial eff ects on several sperm parameters [42,43]. In women, patients with unex-plained infertility had signifi cantly decreased follicular selenium concentrations compared with those with a male-related cause of infertility [44]. Apart of its role in protein synthesis, zinc is very important for human re-production. Sperm motility, as well as sperm count and morphology signifi cantly increased after substitution with 400 mg of zinc sulfate alone [45] or along with 5 mg of folic acid [46]. Cross-sectional studies showed a direct link between the coenzyme Q10 concentrations in the seminal fl uid and sperm count and motility [47], while a prospective study showed an improvement in sperm motility after 6 months of therapy with 200 mg of coenzyme Q10 [48]. Studies performed on females have a limitation in the sense that multivitamins con-taining Zn have been used instead of monosubstance and due to that, benefi cial eff ects of these treatments on reproductive outcome of female, cannot be attribut-ed to one substance (Zn in this case).

CONCLUSION

Finally, taking all this into account it can be concluded that modifi cation of diet pattern may have huge impact on (in)fertility. Precisely, balanced healthy diet of each individual before and during conception attempts can be vital for improving fertility and achieving signifi cant positive impact on reproductive health of couples. Also, apart from nutritional modifi cations, supplements can support the quality of gametes and help to treat infertility. Thus, given the benefi cial eff ects of a healthy diet on reproductive outcome, there is an urgent need for integration of nutritional counseling into infertility treatments.

We do hope that this approach, which involves change in bad nutritional habits, especially in cou-ples who are undergoing IVF, could enhance their re-productive performance and effi ciency of treatment, perhaps even reduce the need for invasive and costly high-technological fertilization procedures.

Acknowledgment

This work was supported by the Ministry of Education, Science and Technological Development of the Repub-lic of Serbia, Grant no. 173054.

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UTICAJ ISHRANE NA FERTILITET KOD LJUDI

Kratak sažetak

Neplodnost je jedan od najtežih medicinskih problema koji je dramatično u porastu širom sveta. Obimna istraživanja posvećena ovom problemu jasno su pokazala da je neplodnost bolest modernog društva i da ishrana ima veliki uticaj na razvoj steriliteta. Iz tog razloga, uticaj specifi čnih nutri-tivnih faktora, tj. specifi čnih tipova ishrane je uvršćen u ovaj pregled. Ono što ohrabruje jeste činjenica da modifi kacija načina ishrane može pomoći parovima da spontano ostvare trudnoću, ili da povećaju šanse za trudnoću in vitro fertilizacijom (IVF).Ključne reči: Sterilitet, ishrana, gojaznost, masti, ugljeni hidrati, proteini, mikronutrijenti

Vesna Otašević1, Aleksandra Korać2, Ana Stančić1, Aleksandra Janković1, Bato Korać1,2

1 Univerzitet u Beogradu, Institut za biološka istraživanja “Siniša Stanković”, Bulevar despota Stefana 142, Beograd, Srbija;

2 Univerzitet u Beogradu, Biološki fakultet, Beograd, Srbija.

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INTRODUCTION

Cancer is a disease developed from permanent loss of balance between death and life at the intercellular level. Abnormal proliferation, ruined tissue, and organ archi-tecture lead to local and soon after, systemic dysfunc-tion. Because of rowdy tissue organization, the main impression is that tumor grows randomly and with-out any rules. Today is well recognized that strategies employed by tumor at cellular as well as intercellular level are fascinating, and altogether demonstrates the superiority toward all therapeutic approaches which ignored their “higher intelligence” and treat them as randomly generated proliferative mass of the cells. Their plasticity and an enormous capacity to change phenotype and avoid host immune defense became more and more powerful through the process of de-diff erentiation [1]. According to this parameter, tumors are classifi ed into grades. So, high-grade tumors are in-vasive/metastatic and, in general, almost incurable. The diff erence in sensitivity to treatment between well dif-ferentiated and low or non-diff erentiated forms of can-cer can be explained not only by the cell death resistant

phenotype of poorly diff erentiated cell subpopulation but even more by specifi c intercellular communication inside of the heterogeneous tumor mass. Tumor re-population frequently observed upon the chemo- or radiotherapies of metastatic tumors arose as a conse-quence of this specifi c and complex interplay between members of tumor commune [2,3]. Proliferation in response to death induction basically compromised healing protocols in high-grade tumor treatments [4]. As a consequence of this, traditional medicine and al-ternative approaches, which are not accepted by con-ventional medicine as legitimate, scientifi cally are very frequently explored during the last decades. Numerous data generated from investigation of antitumor poten-tial of diff erent herbs, fractions or isolated compounds give to biologist hard but important task – to select the most potent between them, to explain how they work at the molecular level, defi ne their targets and then as-sess possibility to improve them and use as a drugs in treatment of cancer. For all, scientist and people out of the science, the main task remains to understand – how people, starting from 4000 years ago were able to rec-ognize and treat diseases using certain herbs.

Aloe emodin: from anti- to pro-tumor action

Abstract

Poor response of highly invasive forms of cancer to the treatment can be explained not only by the cell death resistant phenotype of low/undiff e-rentiated cell subpopulation but even more, by tumor repopulation as a reaction to damage triggered by the chemo- or radiotherapy. Regarding the serious limits of regular healing protocols, one of the pivotal challenges for biologists is to prove the relevance and discover the mechanisms be-hind traditional medicine-based tumor healing. One of the oldest and most powerful plants with 4000 years long tradition in folk medicine, Aloe vera is intensively studied during last century due to the treasure of active compo-unds with proven biological potential attractive not only for physicians and patients but also for scientists. Anthraquinones, emodin and aloe emodin (AE), derived from Aloe vera and diff erent plants from Polygonaceae family are defi nitely the most researched constituents. Aloe emodin owns multiple anti-tumor properties realized through induction of cell cycle arrest, cell de-ath, diff erentiation and suppression of the malignant cell motility. However, its interaction with tumor cells is not unidirectional and due to the complex network of signals in the tumor microenvironment can be easily transfor-med from tumor destructive to tumor-stimulating. Therefore, this review will summarize direct tumoricidal eff ects as well as the interaction of AE with cells and mediators of the immune system. In addition, the potential of AE as chemo- or photosensitizer will be elaborated. Finally, new designs including chemical interventions on this molecule and nanotechnology will be discussed.Key words: Aloe emodin, anticancer properties, interplay with microenvi-ronment, cytostatic drugs, photodynamic therapy

Sanja Mijatović, Danijela Maksimović-IvanićDepartment of Immunology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Serbia.

Corresponding Author: dr Sanja MijatovicPostal address: Institute for Biological Research “Siniša Stanković”, Bulevar despota Stefana 142, 11000 Beograd, Serbia; E-mail address: [email protected]

Phone: +381 11 2078 452; Fax: +381 11 2761 433;

UDK: 615.322:582.573.41

616-006-085

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ALOE VERA- FROM ANCIENT TIME TO DATE

One of the oldest and most powerful plants with extra-ordinary potential to heal is, for sure, Aloe vera. Its po-tential to regenerate the wounds, inhibit infl ammation, suppress infection, stimulate the clearness of the gut and in general bring the refreshment for the whole body, and be used for beauty care, as well as preserva-tion, is described by old civilization such as Sumerian and Egyptians, starting from 4000 years BCE [5]. There are data indicating that the main motive of Alexander the Great for capturing Island of Socotra in the Indian Ocean was to come close to the famous Aloe plants growing there, required for healing the wounds of sol-diers [5]. Today, at the market, the most popular pro-duct made from this herb is aloe gel, which from the middle of the last century, became popular nutritional drink with a long list of benefi cial eff ects to the health maintenance but also in diff erent pathologies. This po-tent infl uence on human physiology is pivotally ascri-bed to diff erent carbohydrates but also numerous pro-teins, lipids, amino acids, vitamins, enzymes, inorganic compounds, and small organic molecules present in Aloe vera leaf pulp. Polysaccharide fraction is found to be very important in the stimulation of innate immu-nity and regarding this, Aloe vera gel was indicated as an immune booster [6]. Synergistic actions of numero-us other ingredients of Aloe vera leaf extract protect he-althy tissue from destruction and intoxication triggered by the chemotherapy. However, according to double edge sword principle, consumption of the gel conco-mitantly with chemotherapy can be questionable since compounds from Aloe vera gel can be cytoprotective even for malignant cells, decreasing the eff ects of che-motherapy.

In addition to numerous constituents listed above, many secondary metabolites anticipated as a product of III polyketides (PK) as well as lately discovered nov-el octaketide synthase, PKS4, and PKS5, were found to possess strong biological activities as an anti-infl am-matory, lipid-lowering, antioxidant, microbicidal and laxative [7]. Recently, this kind of molecules became the most famous concerning their potent direct anti-neoplastic eff ects [8–12]. The main merit for the antitu-mor potential of Aloe vera belongs to one of them-Aloe emodin (AE). Even the name suggested that its source is exclusively Aloe species, this anthraquinone is found in many plants frequently used in folk medicine from Asia to Balkan. Among them are A. barbadensis miller, rhubarb (Rheum palmatum), buckthorn (Rhamnus fran-gula), Senna etc. In addition to the direct eff ect on the viability of malignant cells, long list of AE biological ef-fects on mammalian cells, in general, should be taken into account when its anticancer features are assessed [8]. Additionally, potent infl uence of AE on diff erent signaling pathways involved in essential cellular pro-cesses, multiply refl ected on immune cell-mediated an-titumor activities basically through changed gene and protein expression, as well as the activity of diff erent

mediators implicated in the transfer of information at the intracellular level. According to this, the eff ective-ness of the drugs applied concomitantly with AE can be aff ected. How extreme oscillation in eff ects on tu-mor cells AE exerts, depending on circumstances, such as intercellular contact, the presence of proinfl amma-tory cytokines and the chemotherapeutic drug will be reviewed in this paper.

DIRECT ANTITUMOR EFFECTS OF AE- FROM CELL

DEATH TO DIFFERENTIATION

A major criterion for characterization of any com-pound as antitumor is the ability to induce cell death, preferentially apoptosis in transformed cells. Following this, AE for sure belongs to this list. It is intensively studied on a wide range of tumor cell lines and in ad-dition to numerous original papers its feature to pro-mote cell death in diff erent forms is also reviewed. Cell cycle arrest and apoptosis as a fi nal outcome are noted in various in vitro studies on glioma, melanoma, blad-der, breast, gastric, oral squamous cell carcinoma, co-lon, cervical, prostate, leukemia cell lines and even those that are resistant to conventional chemothera-peutic [8,12]. Cell cycle arrest is found to be connected with the AE interference with cyclin and cyclin-depen-dent kinases expression [11,13]. Apoptosis as a result of AE treatment, depending on the type of the tumor, has been realized through the receptor-mediated or mito-chondrial pathway, and regarding high correlation with oxidative stress, can be the consequence of reac-tive oxygen species (ROS) production. AE triggered the activation of caspase-3, -6, -7, -8 and -9, regulates the expression of numerous transcriptional factors like nu-clear factor kappa B (NF-κB), p53, pro/antiapoptotic members of B-cell lymphoma-2 (Bcl-2) family etc., af-fecting almost all intracellular pathways involved in the realization of death program [10–19]. Intensifi ed pro-duction of ROS upon AE exposure in colon cancer cell lines induces endoplasmic reticulum stress character-ized by glucose-related protein 78, phosphorylated protein kinase R-like ER kinase, phosphorylated eu-karyotic initiation factor-2α expression, and increased cytosolic calcium level [17]. Additionally, AE elevated the permeability of lysosomal membranes in cervical adenocarcinoma HeLa cells, followed by the release of cathepsins, showing that the drug initiates lysosomal pathway-dependent apoptosis [20]. AE targeted vari-ous signaling pathways included in tumor cell prolifer-ation, diff erentiation and death like the extracellu-lar-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, mammalian target of rapamycin/protein kinase B (mTOR/AKT) etc., revealing its strong antitumor competence but also, its potential to aff ect diff erent aspects of cell functioning [10,11,15,19]. During the last decades, more attention is devoted to AE ability to trigger autophagy. Its potential to pro-mote this process strongly refl ected on its antitumor

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but, importantly, also protumor activities generated in interplay with other agents or molecules. It is docu-mented that fundamentally autophagy has a bivalent role in homeostasis maintains and ballast between death and self-renewal. An addition to apoptosis, its involvement in drug cytotoxicity realization is recog-nized as highly important since can vary from “safe” mode to being the type of cell death. According to this, the defi nition of its contribution to any kind of cancer treatment became a very important task. For the fi rst time, it was observed that the treatment of glioma cells with AE, apart from apoptosis, is tracked with the ap-pearance of acidic vesicles in the cytoplasm, indicative for the autophagy. Regarding this, we speculated that both type of programmed cell death – type I (apopto-sis) and II (autophagic cell death) were triggered by AE [10]. However, AE triggered autophagy as opposed to the apoptotic process in concomitant treatment with other molecule or agent with apoptosis-inducing po-tential [21]. So far, only one study showed that AE is able to lead to mitotic catastrophe. Treatment of HeLa cells with AE resulted in the appearance of multinucle-ate cells, giant and micronuclear cells. Mitotic index was diminished and the prevalence of cells in the metaphase was noted [22]. Serious facts about lack of success and even contraindications of killing based protocols in the treatment of highly invasive tumors, underline the advantage of diff erentiation based ther-apies, even with all diffi culties in research settings and time frames needed for the evidence-based assess-ment of this kind of approach [23]. The most intriguing feature of AE, in this context, is its potential to act as a diff erentiation-inducing agent. Conversion of un/low diff erentiated malignant phenotype into a more ma-ture stage might be of great benefi t from multiple points of view. First, this phenotype conversion is ac-companied with the decreased proliferation rate and the reversion of the higher grade to lower, less aggres-sive form. In parallel, the process of change into a more mature stage remarkably enhances their sensitivity to chemotherapy. This eff ect became profoundly import-ant in vivo and in the context of high grade, heteroge-neous tumor mass consisted of tumor cells with diff er-ent phenotype and level of diff erentiation. Presence of “stem” cells is a critical point for undesirable tumor re-population in reaction to aggressive treatment since these cells start to proliferate in response to damage in the surrounding [23]. Regarding this, it could be spec-ulated that AE has the potential to promote diff erenti-ation and subsequently, minimize tumor tissue prolif-eration in response to apoptosis induced by itself or other agents. Tabalocci et al. found that AE endorsing macrophage diff erentiation from a human U937 mono-blastic leukemia cell line, followed with amplifi ed trans-glutaminase activity. Therefore, AE can serve as a diff er-entiation-inducing agent in the treatment of leukemia [24]. Beside hematological malignancies, abundant evidence in vitro showed its potential to diff erent cell lines representing solid cancers. We found that treat-

ment of B16 cells, derived from solid melanoma tumor, with AE, resulted in inhibited cellular proliferation, ac-quisition of fl attened enlarged morphology and at the biochemical level, intensifi ed melanin production and tyrosinase activity. Such transformed the melanoma cells lost their potential to induce tumors in syngeneic C57/BL6 animals confi rming once again that AE might be useful in diff erentiation based therapy of melanoma [11]. Alteration of B16 metastatic clone isolated from lung metastasis – B16-F10, by AE was followed with an elevation of the activity of the transamidating form of TG2 while the invasiveness and production of matrix metalloproteinase-9 were inhibited [25]. Similarly, in-tensifi cation of transamidating activity of transglutam-inase was found upon the treatment of human mela-noma SK-MEL-28 and A375 cells. AE signifi cantly diminished the proliferation, stemness, and invasive potential of melanospheres indicating its activity against cancer stem cells [26]. Treatment of rat astrocy-toma C6 with AE beside apoptotic and autophagic cell death resulted in phenotype change of survived cells that display elongated morphology accompanied with elevated glial fi brillary acidic protein (GFAP) expression. While GFAP is well-known marker of astrocyte lineage, upregulated expression of this protein clearly indicated the process of glioma cell maturation upon AE. Having in mind that inhibitor of ERK1/2, PD98059, imitated the diff erentiation eff ects of AE on glioma cell without trig-gering tumor cell death, we concluded that diff erenti-ation of astrocytoma cells was connected with the in-hibition of this signaling pathway [10]. There are also other types of tumors that underwent diff erentiation in the presence of this herbal anthraquinone. It was found that cervical carcinoma HeLa and oral KB tumor cells displayed a panel of molecules that can be connected with diff erentiation. For example, alkaline phosphatase activity was increased by AE treatment, while prolifer-ating cell nuclear antigen (PCNA) expression, cyclin A and cyclin-dependent kinase 2 (CDK2) were diminished [27,28]. Interestingly but not so surprisingly, AE ability to induce diff erentiation and inhibit proliferation of gastric cancer cells was synchronized with repression of alkaline phosphatase, oppositely to oral cancer cells [29]. This cell-specifi c feature of AE to regulate the same protein in the opposite manner is also observed in the comparative analysis of its infl uence on ERK1/2 in two diff erent melanoma cell lines. The same dose and time frame exposure of less invasive B16 clone and the inducible nitric oxide synthases+ (iNOS+) highly in-vasive amelanotic melanoma cell line-A375 resulted in phosphorylation of p44/42 in mirror image mode- strong time-dependent inhibition in B16 cells opposite to remarkable, also time-dependent, potentiation in A375 cell line [11].

Finally, AE interfered with migration, invasion, and adhesion of tumor cells infl uencing their dissemina-tion. AE repressed cancer metastasis through the inhi-bition of epithelial-mesenchymal transitions transition. Exposure of human epidermal growth factor recep-

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tor-2 (HER-2) overexpressing breast cancer cells to AE blocked their motility in vitro, suppress YB-1 expression through the down-regulation of the intracellular inte-grin-linked kinase (ILK)/protein kinase B (Akt)/mTOR signaling pathway, diminishing further downstream HER-2 expression. Moreover, its activity was confi rmed in vivo, in xenograft model induced in nude mice [30]. In a colon cancer cell, WiDr cells, AE inhibited migra-tion and invasion induced by the phorbol-12-myris-tyl-13-acetate. AE targeted a lot of proteins responsible for the mentioned eff ects like matrix metalloprotein-ase (MMP)-2/9, Ras-homologous (Rho) B and vascular endothelial growth factor (VEGF) expression. The de-scribed eff ect might be due to suppressing the nuclear translocation and DNA binding of NF-κB [31]. AE also inhibits invasion of nasopharyngeal carcinoma cells (NPC) by suppressing the expression of MMP-2 via the p38, mitogen-activated protein kinase (MAPK)-NF-κB signaling pathway [32].

INTERPLAY WITH MICROENVIRONMENTAL

FACTORS: FROM ANTI- TO PROTUMOR ACTION

Today is well recognized that micro-environmental fac-tors are able to transform signals triggered by the drug into unexpected and in an unpredictable direction. There are more and more data about the sensitive and very fragile relationship between signals leading to death and those leading to proliferation [2]. Only one additional molecule possesses a power to completely convert apoptotic into dividing stimulus, and subse-quently, the outcome of the drug application turns into opposite than anticipated. Numerous molecules created with the purpose to induce apoptotic death of cancer cells, under certain conditions promoted their growth [23]. Plenty of data obtained from clinical trials showed that eff ectiveness of tested compounds was dramatically lower or even opposite than expected at least partly due to this fascinating biological phenom-enon that extremes- death and life, pro- and anti- al-ways go together [3]. Data obtained about AE antican-cer activities greatly illustrated how context defi nes the outcome. Many studies describe its potential to reduce the number of tumor cells in culture and few of them confi rmed this in vivo. Mechanistically, AE works as an intercalating agent classifi ed as topoisomerase II inhibitor [33]. According to this feature and many other data about its infl uence on main signaling pathways involved in cell proliferation, death, and diff erentiation, AE is characterized as an agent with strong antitumor potential. However, it was clearly showed that even the simplest variation in cultivation conditions can radical-ly change the outcome of the treatment with this com-pound. Cell density in cultures at the beginning of the treatment, for example, can be of crucial importance for AE eff ectiveness. While in low-density cultures AE was effi cient in the range from 20 to 40 μM, applied on the subconfl uent/confl uent state of the same cell type,

it became completely ineffi cient [9]. It means that in-tracellular features previously declared as essential for the sensitivity to the treatment with AE easily become irrelevant when cells reached the confl uence. Close intercellular contact dramatically aff ected AE infl u-ence on the tumor cells in vitro, making questionable compound eff ectiveness in vivo. Since AE potential to reduce tumor volume in vivo was already documented [16,34], it is clear that the spectrum of its infl uence on tumor progression is more complex than it is possible to be simulated in vitro. Loss of AE potential to direct-ly aff ect tumor cell viability in confl uent cell cultures was discovered by the case in experiments designed in order to explore its eff ectiveness in the presence of proinfl ammatory cytokines in rat astrocytoma C6 and mouse fi brosarcoma L929 cell cultures [9]. Namely, the presence of proinfl ammatory cytokines in mentioned cell cultures triggered production of endogenous ni-tric oxide (NO) through enhanced expression of iNOS. To quantify NO and to determine the AE infl uence on its production, cells were exposed to the compound when they were in the high-density state. In addition to the fact that AE remarkably inhibited iNOS expres-sion and NO production in the presence of cytokine stimulation, it becomes clear that even unstimulated, but AE treated cells were insensitive to the treatment when the drug is applied on subconfl uent/confl uent culture, oppositely to their low-density counterpart. Furthermore, generated NO negatively regulates vi-ability of its own producers – C6/L929 cell, through induction of apoptosis. This mechanism is known as suicidal and is established by our immune cells and their products- proinfl ammatory cytokines, as a part of a defensive mechanism against the tumor. Under these circumstances, the iNOS inhibiting the potential of AE consequently abolished interleukin 1(IL1)/inter-feron-γ (IFNγ) antiglioma and antifi brosarcoma eff ects (Scheme 1A). Not too far, our group discovered that AE neutralizes tumoricidal potential of tumor necro-

Scheme 1. Interplay of AE with cytokines in tumor microenvi-ronment. A) AE inhibited on IL1/IFNγ triggered NO producti-on in tumor cells. B) AE diminished TNFα mediated tumorici-dal action.

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sis factor α (TNFα) through induction of autophagy, which opposes to TNFα mediated apoptosis (Scheme 1B) [21]. In addition, since ERK1/2 is very important for the propagation of TNFα triggered signal, diminished activation of this protein upon AE became, at least partly, responsible for the observed tumor protective activities of the agent. Here we arrived to one more paradoxical level of antitumor/protumor potential of AE, facing with the fact that same signal, like the in-hibition of ERK1/2, mediates both- tumor destructive activities such as blockage of proliferation, induction of diff erentiation and even chemo-sensitization, and con-comitantly, seriously opposes to some of the import-ant cytokine-mediated aspects of antitumor immune response. A long list of contradictions is further ex-tended by AE infl uence on macrophages (Mf) (Scheme 2). In concordance with its ability to down-regulate iNOS expression and NO production in C6 and L929 cells, as well as their healthy counterpart- astrocytes and fi broblasts, cultivation of Mf in the presence of the drug resulted in suppression of NO release [35]. This result indicated the diminished antitumor capac-ity of Mf when they are exposed to AE. Instead of this, co-cultivation of macrophages with glioma cells in the presence of AE revealed even enhanced the cytotoxic potential of Mf and, concordantly, enhanced nitrite ac-cumulation in culture supernatants, as a consequence of the increased release of NO. The observed contradic-tion between AE infl uence on Mf NO production when cells were cultivated alone and in co-cultures with tu-mor cells can be connected with some additional sig-nal generated from the contact between tumor cells and Mf, which in combination with AE resulted in hy-per- instead of hypo-production of NO. However, the same eff ect was observed in high-density cultures of peritoneal Mf alone, indicated ones more, the pivotal importance of cell to cell contact and its infl uence on

signals triggered with AE. Importantly and diff erently to the eff ect of AE on NO production determined in C6/L929 cells as well as primary astrocytes and fi broblasts, hyper-production of NO observed in the high-den-sity culture of mouse Mf was iNOS independent and resistant to treatment with inhibitors of transcription or translation, actinomycin, and cycloheximide, respec-tively [35, unpublished data]. It is clear that AE is able to interfere with NO production at the level of iNOS gene as well as protein expression. In addition to NO, it was discovered that AE down-regulated production of interleukin-6 and interleukin-1β in macrophage cell line RAW264.7 cells upon stimulation with bacterial li-popolysaccharide and realized the suppressive eff ect on leukocytes isolated from Sprague-Dawley rats, de-creasing the phagocytic potential of Mf and NK cells activity. Contradictory, measurement of cytokine pro-duction showed that AE augmented the interleukin-1 β and TNFα [36,37]. Altogether, it is clear that outcome of the treatment with AE presents the net eff ect of the complex network of hardly predictable interactions generated through collection of signals triggered at the level of DNA and genes, proteins and cell mem-brane. More than that, further integration of the stimuli modifi ed by AE happens in communication between diff erent cells in heterogeneous tumor mass- tumor cells in the diff erent stage of diff erentiation and their non-transformed counterparts, stromal cells, and var-ious immune cells.

INTERPLAY WITH CYTOSTATIC DRUGS: FROM

SYNERGISM TO ANTAGONISM

Usage of aloe derived anthraquinones as chemosen-sitizing agents are well described in the literature. Mostly literature data evaluate the eff ect of emodin as an amplifi er of chemotherapy, especially cisplatin. The interaction with the drug basically refers to ROS production and their sensitizing features, interference with multi-drug resistant transporters and induction of autophagy [38,39]. Even though the antitumor action of AE is well studied there are only a few pa-pers describing its interaction with chemotherapeutic drugs. The co-treatment of Merkel cell carcinoma with cis-platinol (abiplastin), doxorubicin (adriablastin), and 5-fl uorouracil (5-Fu), and AE resulted in potentiation of their cytotoxicity [40]. Similarly, AE amplifi ed the cyto-toxicity of tamoxifen against MCF-7 breast cancer cells through reduction of epidermal growth factor recep-tor (EGFR), rat sarcoma (Ras), ERK, c-Myc, and mTOR protein expression. In addition, the activity of Ras/ERK and phosphatidylinositol 4,5-bisphosphate 3-ki-nase (PI3K)/mTOR pathways was suppressed. Produc-tion of ROS and enhanced cell death in co-treatment was potentiated indicating that AE can act as a strong chemosensitizer [41] The potentiation of 5-fl uorouracil was also achieved in epidermoid carcinoma A431 and head and neck squamous cell carcinoma SCC25A431 through regulation of caspase-8, -9, and -3 expression

Scheme 2. Infl uence of AE on macrophage NO production depending on cell-cell contact. AE inhibited NO production in low-density macrophage cultures while in high-density or in co-cultivation with tumor cells hypo production of NO is converted to hyper production.

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[42]. Oppositely to the benefi cial chemosensitizing ef-fect of AE, there are few data indicating opposite ef-fect. The possibility that the concomitant application of AE and certain cytostatic can lead to neutralization of tumoricidal eff ects of chemotherapy, underlines that special caution is needed in the eventual design of combined treatment with conventional drugs. We already described that AE as a single agent possesses strong antitumor potential on two diff erent melanoma cell lines, human A375 and mouse B16. However, par-allel treatment with doxorubicin or paclitaxel resulted in antagonistic action in vitro [11]. In line with this, AE neutralized cisplatin-induced apoptosis and necrosis on murine L929 fi brosarcoma and C6 glioma cell lines. The counteracted action of cisplatin was due to op-posite regulation of ERK in tumor cells, but not c-Jun N-terminal kinase [43].

ALOE EMODIN AS A SENSITIZER IN

PHOTODYNAMIC THERAPY

Photodynamic therapy (PDT) is a clinically approved, noninvasive therapeutic method that includes the ap-plication of a photosensitizing agent concomitantly with a particular type of light [44]. This procedure can be valuable particularly for early-stage tumors but also can extend survival and quality of life of patients with inoperable cancers. Its advantage is marginal toxicity for normal tissues, minor systemic eff ects, diminished morbidity and development of resistance mechanisms. Photosensitizing substances are activated by a specifi c wavelength that usually triggers the production of ROS that is harmful to cancer cells. Besides the direct killing of cancer cells, such treatment aff ects blood vessels in the tumor and triggered an immune response against the tumor. Also, repetition of PDT and concomitant treatment with surgery, radiation, and chemotherapy is possible. So far, PDT is approved by the Food and Drug Administration for the treatment of esophageal cancer and non-small cell lung cancer. Recent fi ndings indicate that AE can be a useful drug as photosensi-tizer during PDT. Therefore, it was shown that AE, as the part of this therapeutic approach, inhibited prolif-eration of oral mucosa carcinoma KB cells in G1 phase and triggered apoptotic cell death due to the massive ROS production. Apoptotic cell death was followed with an up-regulated expression of caspase-3 and Bax together with strong suppression of Bcl-2 expression. Moreover, AE-PDT had marked inhibitory eff ect in vivo and extended the survival time of animals without no-table side eff ects [45]. Similar fi ndings were observed in human gastric cancer SGC-7901 cells where the AE-induced PDT resulted in the activation of mitochon-drial-dependent apoptosis [46]. Apoptosis is also the main outcome of AE-PDT of osteosarcoma MG63 cells. Fast intracellular ROS production led to the disruption of mitochondrial membrane potential, the release of cytochrome c, enhancement of caspase-3, -9, and -12, CCAAT-enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) [47].

Apart from apoptosis, AE-PDT induced the autopha-gy of human osteosarcoma cell line MG-63 through the activation of the ROS-JNK signaling pathway [48]. In addition, AE as a sensitizer for PDT interferes with the metastatic spreading of MCF-7 breast cancer cells. Oxidative stress targeted the expression of MMP2, MMP9, VEGF, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) as well as cytoskeleton organization after treatment with AE-PDT [49]. In lung cancer, H460 cells photo-activated AE provoked a disturbance of cyto-skeleton and triggered anoikis, as the programmed cell death that occurs in adherent cells detached from the extracellular matrix. Anoikis is related to α-actinin and MAPK expression and led to the mitochondrial perme-ability transition pore opening and elevated expres-sion of apoptosis-related proteins [50]. Aloe emodin and irradiation stimulated the expression of protein kinase Cδ (PKCδ) in H460 cells and its translocation of to the nucleus. Additionally, AE-PDT triggered numer-ous proteins important for cytoskeleton organization like RAS, ras homolog gene family member A (RHO), p38, heat shock protein 27 (HSP27), focal adhesion ki-nase (FAK), α-actinin and tubulin [51]. Finally, AE-PDT interfered with angiogenesis process that is crucial for both growth and metastatic spreading of cancers. Aloe emodin photodynamic therapy suppressed formation branching points, tubule number, and length. Also, the number of capillary structures was signifi cantly re-duced by AE-PDT treatment. Migration and invasion of human umbilical vein endothelial cells were also aff ect-ed by the treatment. Mention processes are connected with the activation of p38, ERK, but not JNK, while the expression of vascular endothelial growth factor was diminished upon the treatment. In addition, AE-PDT altered the organization of F actin cytoskeleton [52].

NEW CHEMICAL DESIGNS FOR IMPROVEMENT OF

AE ANTICANCER POTENTIAL

The antitumor activity of AE is highly compromised by its rapid degradation and low bioavailability. A lot of eff ort was made to make a diff erent formulation of AE to improve its features. One of the approaches includes the application of nanotechnology. Wu et al. made a poly (lactic-co-glycolic acid) based AE nanoparticles. The application of AE in this form was more effi cient than an original compound in inhibition of human lung squamous cell carcinoma proliferation, induction of cell cycle arrest and further apoptotic cell death. Apoptosis was followed with activation of Caspase-3, poly (ADP-ribose) polymerase (PARP), Caspase-8 and Caspase-9. In parallel, ROS production was elevated. Nano-AE stimulated MAPK activation and suppressed PI3K/AKT signaling pathway. More importantly, na-no-AE suppressed the tumor growth in vivo with in-signifi cant toxicity [53]. The other group prepared surface-functionalized polyethylene glycol liquid crys-talline nanoparticles (PEG-LCNPs) of AE to enhance its water solubility and enable its anticancer use. Particle size was 190 nm and their stability in serum was ele-

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vated. Further studies showed a good safety profi le of PEG-LCNPs of AE [54]. One more strategy involves solid lipid nanoparticles with a stable particle size at approx-imately 90 nm with good drug entrapment effi ciency and stability. AE loaded in solid lipid nanoparticles displayed amplifi ed cytotoxicity against human breast cancer MCF-7 cells and human hepatoma HepG2 cells in comparison to the AE. The toxicity toward human mammary epithelial MCF-10A cells was diminished. AE loaded in solid lipid nanoparticles induced apop-tosis in MCF-7 cells probably due to increased cellular uptake of AE [55]. Similar improvement was achieved by integrating AE into the liposomal formulation. That formulation augmented cell death of A431 and SCC25 cells and improved transdermal delivery of AE [42]. Other approaches aimed to make water-soluble for-mulation consider the chemical modifi cation through coupling with various amino acid esters and substitut-ed aromatic amines. Derivate signifi cantly reduced the growth of human liver cancer cells HepG2, and lung cancer cells NCI-H460, human epithelial carcinoma cells HeLa and prostate cancer cells PC3 more potently than AE alone [56]. The attachment of an amino-sugar unit to AE formed a new class of AE glycosides (AEGs) with improved cytotoxic potential even in doxorubi-cin-resistant cell lines probably through interference with P-glycoprotein effl ux pumps [57]. Finally, with an attempt to improve tumoricidal potential hybrid mole-cule from rhein and AE was synthesized. This chimeric molecule was more effi cient against human hepato-ma HepG2, human nasopharyngeal carcinoma CNE, human lung cancer NCI-H460, human ovarian cancer SK-OV-3, and human cervical cancer HeLa cells than separate compounds [58].

CONCLUSION

Summarizing all the mentioned eff ects of AE, it is cle-ar that its direct tumoricidal potential and synergistic action with some of the conventional drugs is undo-ubting (Scheme 3). However, it is not possible to over-view it separately and independent from the other ac-tors in tumor microenvironment or applied therapy. It is more than clear that serious observation is necessary because the line between the cure and harm is so tiny and hard to predict. “Magic bullet” for cancer therapy still not exists but the defi nition of conditions and pro-tocols when the phytotherapeutics can be benefi cial is highly valuable. Thousands of years old ethnic- medi-cine can defi nitely mark the right way, but gathering an experimental knowledge and clinical experience is also necessary. The study of Lissoni et. on 240 patients with metastatic lung, colorectal and pancreatic tumors who have received chemotherapy in parallel with Aloe arborescens treatment showed signifi cant tumor re-gressions, disease control and increased survival rate, claiming that this class of drugs might have a clinical potential [59]. We can have multiple benefi ts from phytotherapy if we understand and utilize it correctly.

Acknowledgment. Financial support by the Ministry of Education, Science and Technological Development of the Republic of Serbia (project No. 173013) is grate-fully acknowledged.

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Scheme 3. Infl uence of AE on tumor is a net eff ect of com-plex network involving diff erent members of tumor commu-ne. In addition to direct eff ect on tumor cell AE infl uence tumor growth through modulation of immune cells, normal cells, heterogeneous population of tumor cells as well as che-motherapy.

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47. Li KT, Chen Q, Wang DW, Duan QQ, Tian S, He JW et al. Mitochondrial pathway and endoplasmic reticulum stre-ss participate in the photosensitizing eff ectiveness of AE-PDT in MG63 cells. Cancer Med 2016; 5(11):3186–93.

48. Tu P, Huang Q, Ou Y, Du X, Li K, Tao Y et al. Aloe-emodin-mediated photodynamic therapy induces autophagy and apoptosis in human osteosarcoma cell line MG-63 through the ROS/JNK signaling pathway. Oncol Rep 2016; 35(6):3209–15.

49. Chen Q, Tian S, Zhu J, Li KT, Yu TH, Yu LH et al. Exploring a Novel Target Treatment on Breast Cancer: Aloe-emodin Mediated Photodynamic Therapy Induced Cell Apopto-sis and Inhibited Cell Metastasis. Anticancer Agents Med Chem 2016; 16(6):763–70.

50. Lee HZ, Yang WH, Hour MJ, Wu CY, Peng WH, Bao BY et al. Photodynamic activity of aloe-emodin induces resen-sitization of lung cancer cells to anoikis. Eur J Pharmacol 2010; 648(1–3):50–8.

51. Chang WT, You BJ, Yang WH, Wu CY, Bau DT, Lee HZ. Pro-tein kinase C delta-mediated cytoskeleton remodeling is involved in aloe-emodin-induced photokilling of human lung cancer cells. Anticancer Res 2012; 32(9):3707–13.

52. Chen Q, Li KT, Tian S, Yu TH, Yu LH, Lin HD et al. Pho-todynamic Therapy Mediated by Aloe-Emodin Inhibited

Angiogenesis and Cell Metastasis Through Activating MAPK Signaling Pathway on HUVECs. Technol Cancer Res Treat 2018; 17:1533033818785512.

53. Wu YY, Zhang JH, Gao JH, Li YS. Aloe-emodin (AE) nano-particles suppresses proliferation and induces apoptosis in human lung squamous carcinoma via ROS generation in vitro and in vivo. Biochem Biophys Res Commun 2017; 490(3):601–7.

54. Freag MS, Elnaggar YS, Abdelmonsif DA, Abdallah OY. Stealth, biocompatible monoolein-based lyotropic liquid crystalline nanoparticles for enhanced aloe-emodin deli-very to breast cancer cells: in vitro and in vivo studies. Int J Nanomedicine 2016; 11:4799–818.

55. Chen R, Wang S, Zhang J, Chen M, Wang Y. Aloe-emodin loaded solid lipid nanoparticles: formulation design and in vitro anti-cancer study. Drug Deliv 2015; 22(5):666–74.

56. Thimmegowda NR, Park C, Shwetha B, Sakchaisri K, Liu K, Hwang J et al. Synthesis and antitumor activity of natural compound aloe emodin derivatives. Chem Biol Drug Des 2015; 85(5):638–44.

57. Breiner-Goldstein E, Evron Z, Frenkel M, Cohen K, Meiron KN, Peer D et al. Targeting anthracycline-resistant tumor cells with synthetic aloe-emodin glycosides. ACS Med Chem Lett 2011; 2(7):528–31.

58. Yuan YF, Hu XY, He Y, Deng JG. Synthesis and anti-tumor activity evaluation of rhein-aloe emodin hybrid molecu-le. Nat Prod Commun 2012; 7(2):207–10.

59. Lissoni P, Rovelli F, Brivio F, Zago R, Colciago M, Messi-na G et al. A randomized study of chemotherapy versus biochemotherapy with chemotherapy plus Aloe arbo-rescens in patients with metastatic cancer. In Vivo 2009; 23(1):171–5.

ALOE EMODIN U TRETMANU TUMORA: SAVEZNIK ILI PROTIVNIK

Kratak sažetak

Loš odgovor visoko invazivnih formi kancera na tretman se ne može obja-sniti samo fenotipom ćelija rezistentnih na indukciju smrti već i repopulaci-jom tumora u odgovoru na oštećenja nastala usled primene hemio- ili ra-dioterapije. Ozbiljna ograničenja regularnih terapeutskih pristupa jedan su od glavnih izazova za biologe da utvrde relevantnost i istraže mehanizme u osnovi lečenja malignih bolesti zasnovanih na tradicionalnoj medicini. Jedna od najstarijih i najmoćnijih biljaka sa 4000 godina dugom tradicijom u narodnoj medicini, Aloe vera, je intenzivno izučavana u poslednjem veku zbog čitave riznice aktivnih komponenti privlačnih kako lekarima i pacijen-tima, tako i naučnicima. Antrahinoni, emodin i aloe emodin (AE), izolovani iz Aloe vera i drugih biljaka iz porodice Polygonaceae su defi nitivno najvi-še izučavani konstituenti. Aloe emodin poseduje vešestruka antitumorska svojstva realizovana kroz indukciju zastoja u ćelijskom ciklusu, ćelijske smr-ti, diferencijacije i supresije motiliteta malignih ćelija. Međutim, njegova in-terakcija sa tumorskom ćelijom nije jednosmerna i zbog kompleksne mreže signala u mikrosredini tumora može lako biti konvertovana sa destruktivne u promovišuću za tumor. Ovaj pregledni članak će sumirati direktne tumo-ricidne efekte ali će razmatrati i interakcije AE sa imunskim ćelijama i njiho-vim medijatorima. Takođe, biće elaboriran i potencijal AE kao hemio- i fo-tosenzitizatora. Konačno, diskutovaće se i novi pristupi u modifi kaciji ovog molekula koji uključuju hemijske intervencije i primenu nanotehnologije. Ključne reči: Aloe emodin, antikancerska svojstva, interakcija sa mikrosre-dinom, citostatici, fotodinamička terapija

Sanja Mijatović, Danijela Maksimović-IvanićOdeljenje za imunologiju, Institut za biološka istraživanja “Siniša Stanković”, Univerzitet u Beogradu, Srbija.

Autor za korespondenciju:dr Sanja MijatovićAdresa: Institut za biološka istraživanja „Siniša Stanković”, Bulevar despota Stefana 142, 11000 Beograd, Srbija; E-mail: [email protected].: +381 11 2078 452;Faks: +381 11 2761 433.

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UVOD

Opšte je prihvaćeno da je pšenica prva žitarica koja se gajila kao usev za hranu još od 10000–8000 god-ine p.n.e. [1]. Značaj pšenice uglavnom se pripisuje njenoj sposobnosti da može biti samlevena u brašno i griz, koji čine osnovne sastojke hleba, drugih pekarskih proizvoda i testenina. Mlevenjem pšenice razdvajaju se anatomski delovi zrna i ono se usitnjava. Postepenom redukcijom zrna pšenice, uz višefazno usitnjavanje i mlevenje dobijaju se čestice različitog promera koje se prosejavaju, a spajanjem različitih pasaža formiraju se tipovi brašna sa različitim udelom endosperma, klice i mekinja. S obzirom na to da sadržaj proteina, uglje-nih hidrata, mineralnih materija, vitamina i antioksida-tivnih fi tonutritienata varira između različitih frakcija zrna pšenice [2], nutritivna, tehnološka i funkcionalna svojstva raznih tipova brašna su različite, kao i njihova podobnost za različite prehrambene proizvode.

Pšenično brašno se pretežno sastoji od skroba (70–75%), vode (12–14%), proteina (8–16%) i drugih komponenata kao što su prehrambena vlakna (2–3%), lipidi (2%) i pepeo (1%). Kvalitet pšeničnog brašna za-visi od sadržaja i karakteristika ovih komponenata koje se razlikuju u zavisnosti od sorte pšenice [3]. S obzirom na to da belo pšenično brašno ima nizak sadržaj vita-mina, jedinjenja sa antioksidativnim karakteristikama i dijetalnih vlakana koja se gube u procesu mlevenja, poslednjih godina se vrši njegovo obogaćenje različi-tim prirodnim izvorima ovih jedinjenja čime se može poboljšati nutritivna, ali i funkcionalna vrednost pekar-skih proizvoda [4].

PROTEINI PŠENICE

Sadržaj proteina u pšeničnom zrnu se kreće od 8 do 11% u hlebnoj pšenici i od 10 do 15% u durum pšenici [5] i uslovljen je kako genetičkim tako i faktorima spolj-ne sredine [6].

Prema Osbornovoj klasifi kaciji iz 1924. godine, pro-teini pšenice se na osnovu rastvorljivosti dele na četiri glavne grupe: albumini (rastvorljivi u vodi i razblaženim puferima), globulini (rastvorljivi u rastvorima soli), glija-dini (rastvorljivi u 70–90% etanolu) i glutenini (rastvor-ljivi u razblaženim kiselinama ili bazama). Molekulska masa pšeničnih proteina kreće se od 30.000 do više od 10 miliona Da [7] i mogu se podeliti na strukturno/me-taboličke (neglutenske) i rezervne (glutenske) proteine [8]. Strukturni/metabolički proteini sastoje se od albu-mina, globulina i amfi fi linih proteina. Ne-membranski amfi fi lni proteini imaju veliki uticaj na strukturno me-haničke karakteristike zrna i reološka svojstva testa [9]. Drugi sistem klasifi kacije deli glutenske proteine (prolamine) u tri grupe: sumporom bogate, sumporom siromašne i proteine visoke molekulske mase.

ALBUMINI I GLOBULINI

Albumini i globulini predstavljaju veoma raznovrsnu grupu proteina zbog svojih fi zičko-hemijskih svojstava u smislu aminokiselinskog sastava, izoelektrične tač-ke i molekulske mase. Albumisko-globulinska frakcija proteina zrna pšenice karakteriše se bogatom protein-skom šemom. Broj traka na gelu može da varira od 19 do 23, a molekulska masa izolovanih proteina kreće se

Proteini pšenice sa tehnološkog, nutritivnog i zdravstvenog

aspekta

Kratak sažetak

Pšenica je jedna od najvažnijih biljnih kultura koja se koristi u ishrani ljudi kao glavni izvor energije, proteina i dijetalnih vlakana. Uprkos relativno ni-skom sadržaju proteina hranljivu vrednost proteina pšenice ne bi trebalo potcenjivati. Kvalitet brašna, reološke i funkcionalne karakteristike testa i pekarskih proizvoda umnogome zavise od proteina pšenice. Proteini pše-ničnog zrna pokazuju visoku kompleksnost i različit međusobni stepen interakcije zbog čega je njihova karakterizacija teška. Uprkos njihovom ključnom uticaju na kvalitet testa i tehnološki kvalitet različitih proizvoda, glutenski proteini mogu uticati na zdravlje genetski podložnih osoba. Ključne reči: pšenica, proteini, gluten, tehnološka svojstva, testo, celijakija

Marijana Simić, Slađana Žilić Institut za kukuruz „Zemun Polje“, Laboratorija za prehrambenu tehnologiju i biohemiju, Slobodana Bajića 1, 11085 Beograd-Zemun, Srbija

Autor za korespodenciju: Marijana Simić

Institut za kukuruz „Zemun Polje“, Laboratorija za prehrambenu tehnologiju i biohemiju, Slobodana Bajića 1, 11085 Beograd – Zemun, SrbijaTel.: 065370 60 11E-mail: [email protected]

UDK: 633.11:612.392

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od 12,4 do 76,4 kDa [10]. Nutritivno, albumini i globu-lini imaju veoma dobar balans aminokiselina. Imaju re-lativno visok sadržaj triptofana i metionina [11] i sadrže oko 50% ukupne količine lizina koja se nalazi u zrnu [12]. Niskomolekulski albumini posebno su bogati vali-nom, glutaminskom kiselinom, a zatim cisteinom, ala-ninom, glicinom, prolinom, asparaginskom kiselinom i leucinom, dok su siromašni izoleucinom, fenilalaninom i histidinom [13]. Sadržaj albumina+globulina u pše-ničnom brašnu se kreće oko 25% od ukupnih proteina [14], ali njihov sadržaj se može kretati i do 39% [15]. Po-limerni globulini, takozvani triticin, koji čini svega oko 5% od ukupnih skladišnih proteina pšenice smatra se nutritivno važnim jer sadrži jedinstven lizinom-bogat dekapeptidni ponavljajući motiv. S druge strane, ovaj protein ima nizak sadržaj cisteina i metionina, dok pri-sustvo triptofana nije utvrđeno [13].

GLUTEN

Jedinstvene karakteristike pšenice se zasnivaju na svojstvu rezervnih proteina da formiraju gluten. Nji-hova unutrašnja viskoelastična svojstva su odgovorna za karakteristike različitih proizvoda od pšenice – hle-ba, testenina, nudli, keksa, kolača, peciva i drugih [16] i upotrebu proteina pšeničnog glutena u različitim prehrambenim proizvodima [17]. Pored uticaja na teh-nološka svojstva pšenice, gluten ima i antioksidativne karakteristike. Tako su istraživanja pokazala da kom-pleksna struktura proteina glutena uslovljava njihov visok antioksidativni kapacitet [18], dok su u drugim detektovani antioksidativni peptidi u hidrolizatima glutena [19].

Glutenski proteini čine oko 60–75% od ukupnog sadržaja proteina pšeničnog brašna i glavni su rezervni proteini pšenice [15]. Glutenski proteini predstavljaju kompleks sačinjen od prolina (10%), glicina (20%) i glu-tamina (35%) kao najzastupljenijih aminokiselina koje su odgovorne za karakteristike proteina glutena [20]. U aminokiselinskom sastavu glutena, cisteinski osta-ci čine mali udeo (~2%), ali veoma bitan za strukturu i funkcionalnost glutena. Niska rastvorljivost glutena u vodi se pripisuje niskim sadržajem ostataka lizina, arginina, glutaminske i asparaginske kiseline, koji za-jedno čine manje od 10% ukupnog broja aminokise-linskih ostataka. Glutenski proteini se na osnovu svoje rastvorljivosti mogu podeliti na glijadine i glutenine [7] a njihov odnos i udeo u pšeničnom zrnu je varijabilan i uslovljen je faktorima spoljne sredine i genetičkom predispozicijom [21].

GLIJADINI

Glijadini su monomerni proteini koji čine od 20% do 40% ukupnih proteina pšeničnog brašna [15]. Glijadini su proteini koji su najiscrpnije ispitivani elektrofore-zom. Molekulske mase glijadina se kreću od 31,4 do 73,6 kDa [22] i klasifi kovani su u četiri grupe α-, ß-, γ- i

ω-glijadine na osnovu svojih biohemijskih i genetičkih karakteristika i pokretljivosti na niskim pH vrednostima u poliakrilamid gel elektroforezi [23]. Kasnije studije na aminokiselinskim sekvencama su svrstale α- i β- glija-dine u jednu grupu (α/β-). Odnos α/β- i γ-glijadina sa ω-glijadinima utiče na sadržaj aminokiselina sa sum-porom, kvalitet proteina, strukturu i funkcionalnost glutena. α/β-glijadini su zastupljenija grupa u odno-su na γ-glijadine i njihov sadržaj se kreće od 47,57% do 59,12%, u brašnu hlebne pšenice [15]. Kako ova grupa glijadina ima velikog uticaja na povećanje vo-lumena hleba [24], genotipovi sa visokim sadržajem α/β-glijadina se mogu koristiti kao poboljšivači u se-lekcionarskom programu [5]. ω-glijadini (2,70– 6,50% u brašnu hlebne pšenice) imaju visok sadržaj glutamina i prolina, a siromašni su cisteinom, pa nemaju moguć-nost disulfi dnog umrežavanja već u formaciji polimera tokom procesa formiranja testa učestvuju preko neko-valentnih veza, dok α/β- i γ-glijadini mogu biti inkor-porirani u polimer glutena sa međumolekulskim S-S vezama [25]. Stoga se α/β-glijadini i γ-glijadini zovu prolamini bogati sumporom, a ω-glijadini se zovu pro-lamini siromašni sumporom. ω-glijadini u brašnu hleb-ne pšenice imaju jedan do dva polipeptida sa molekul-skim masama od 65,7–76,2 kDa u zavisnosti od sorte pšenice [5] (Slika 1).

GLUTENINI

Glutenini su poznati kao najveći polimeri u prirodi [26]. U pšenici se nalaze dve klase gluteninskih podjedinica, glutenini velikih molekulskih masa (HMW-GS) i glute-

Slika 1. SDS-PAGE analiza glijadinske frakcije proteina brašna hlebne pšenice [15]

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nini malih molekulskih masa (LMW-GS). LMW-GS ima-ju molekulsku masu od 30–55 kDa i njihovu strukturu čine cisteinski ostaci koji pomažu formiranje glute-ninskih polimera. Prosečna vrednost ukupne koncen-tracije LMW-GS je oko 5,6 puta veća od koncentracije HMW-GS [15]. HMW-GS čine 5–10% ukupnih proteina brašna [27] i imaju molekulsku masu od 80–160 kDa [28]. Gluteninske podjedinice HMW-GS mogu se kla-sifi kovati na dva tipa: x i y [29]. Njihov procentualni udeo u ukupnom sadržaju glutena se kreće u opsegu od 4–9%, dok je zastupljenost y-tipa podjedinica od 3–4% [7]. Glutenini se takođe dodatno klasifi kuju u četiri podgrupe (A, B, C i D) na osnovu elektroforetske mobilnosti na natrijum dodecil sulfat – poliakrilamid gel elektroforezi (SDS-PAGE). Podgrupa A je determinisana kao HMW-GS, a podgrupe B, C i D pripadaju LMW-GS [30]. B i C podgrupe sadrže oko 70% od ukupnih LMW-GS, imaju molekulsku masu 43,5–50,3 kDa odnosno 30,7–41,5 kDa i predstavljaju sumporom bogate pod-grupe LMW-GS. D podgrupa ima molekulsku masu od 52,6–74,8 kDa i spada u sumporom siromašnu podgru-pu LMW-GS [5] (Slika 2).

Slika 2. SDS-PAGE analiza gluteninske frakcije proteina braš-na hlebne pšenice [15]

TEHNOLOŠKA SVOJSTVA BRAŠNA I NJIHOV

ODNOS SA PROTEINIMA

Za pecivost brašna, pravi odnos i interakcija svih komponenata koje čine brašno su od suštinskog značaja [31]. Međutim, kvalitet brašna, reološke i funkcionalne karakteristike testa i pekarskih pro-izvoda najviše zavise od proteina pšenice. Meša-njem pšeničnog brašna i vode dobija se testo sa viskoelastičnim svojstvima koje je pogodno za izradu hleba i drugih pekarskih proizvoda. Testo spada u jedno od najsloženijih reoloških sistema i predstavlja viskoelastični sistem koji pri protica-

nju ispoljava pseudoplastično i tiksotropno ponašanje [32], a njegovo kompleksno reološko ponašanje je po-sledica njegove složene strukture.

Neglutenski proteini, albumini i globulini, imaju značajan uticaj na obradu i reološka svojstva pšenič-nog brašna [33] uglavnom kao funkcionalni proteini, pa se tako loš kvalitet pšeničnog brašna lako može poboljšati dodatkom različitih enzima, kao što su ami-laze i/ili ksilanaze. α-amilaze smanjuju viskozitet testa, poboljšavaju obradu testa, utiču na strukturu sredine hleba i dobijanje mekše i veće vekne hleba. Sa druge strane visok sadržaj α-amilaze u pšeničnom brašnu nije poželjan i takva brašna se ne mogu koristiti u procesu pripreme hleba. Enzimi kao što su izoenzimi lipoksi-genaze smanjuju vreme mešanja testa, mogu davati ukus „nalik orahu“ u nekim sistemima [34] i povećavaju toleranciju testa na mešanje i vreme odmaranja testa, što uslovljava poboljšanje volumena vekne hleba [35]. Aktivnost enzima pentozanaza poboljšava elastičnost glutena i fi nalni kvalitet hleba stvarajući promene u re-ološkim karakteristikama i/ili distribuciji vode [36].

Glijadini se mogu udruživati međusobno ili sa glu-teninima posredstvom hidrofobnih interakcija i vodo-ničnih veza [37], pa je vrlo teško tumačiti zasebne efek-te glijadina na kvalitet testa. Hidratisani glijadini imaju manju elastičnost i manje su kohezivni od glutenina. Dodavanjem glijadinskih frakcija pšeničnom brašnu bitno se smanjuje maksimalan otpor, a povećava ra-stegljivost testa [38], što je i očekivano imajući u vidu njihovu viskoznu prirodu. Iako glijadini generalno uti-ču na volumen hleba, uloga pojedinačnih glijadina još uvek nije dobro shvaćena [39]. Međutim, istraživanja su pokazala da γ-glijadini smanjuju vreme mešanja testa i maksimalni otpor pri rastezanju, a ω-glijadini najviše utiču na smanjenje volumena vekne [40] (Slika 3).

Glutenini visokih molekulskih masa čine testo ela-stičnim i omogućavaju mu da zadrži mehuriće gasa koje stvara kvasac i da raste. Ovo je veoma važno za kvalitet fi nalnog proizvoda [42], s obzirom na to da svojstvo zadržavanja gasa određuje zapreminu vekne i strukturu pora dobijenog hleba [43]. Utvrđeno je i da je uticaj x-tipa HMW-GS na pecivost znatno veći od y-tipa HMW-GS. LMW-GS koji formiraju velike agregate

Slika 3. Modifi kovana slika viskoelastičnih svojstva rehidratisanog glutena, glijadina i glutenina [41]

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utiču na jačinu testa, ali je jačina i otpor koji obezbe-đuju LMW-GS upola manja od one koju obezbeđuju HMW-GS [44]. Suprotno tome, u drugim istraživanjima je pronađena veza između LMW-GS i rastegljivosti te-sta i slaba korelacija između ovih proteina sa drugim svojstvima testa [45]. Dostupni podaci takođe ukazuju da je specifi čan način disulfi dnog umrežavanja između LMW-GS i HMW-GS u glutenu daleko važniji za pecivost od količine ovog proteina [13].

ZDRAVSTVENI EFEKAT

Uprkos njihovom ključnom uticaju na kvalitet testa i tehnološki kvalitet različitih proizvoda, glutenski prote-ini mogu uticati na zdravlje genetski podložnih osoba. Visok sadržaj prolina čini gluten otpornim na degrada-ciju gastrointestinalnim enzimima i tako omogućava da veliki imunogeni peptidi glutena dopru do površine sluznice tankog creva i uzrokuju razvoj upalne reakcije [46]. Gluten može izazvati nekoliko različitih poremeća-ja: celijakiju, alergiju na brašno i osetljivost na gluten, a za nastanak ovih poremećaja odgovorni su različiti patomehanizmi [47]. U okviru različitih epitopa glutena koje su identifi kovane, α-glijadini imaju najveću imu-nogenost [48].

Celijakija ili gluten senzitivna enteropatija je hro-nična autoimuna bolest koju karakteriše doživotna nepodnošljivost glutena. Unosom hrane koja sadrži gluten dolazi do oštećenja sluznice tankog creva, koja gubi resičast izgled i postaje zaravnjena, dok broj tkiv-nih limfocita i epitelnih ćelija raste. Zadebljana sluzni-ca ima smanjenu moć apsorpcije što uzrokuje malap-sorpciju hranjivih materija, minerala i vitamina [49]. Poremećeni imunološki odgovor koji nastaje prilikom unosa glutena u organizam osoba s genetskom pre-dispozicijom trajan je i ne može se izlečiti privremenim izostavljanjem glutena iz ishrane. Iako se smatra da je genetska predispozicija najodgovornija za razvoj celi-jakije, poznato je da je celijakija snažno povezana sa specifi čnim humanim leukocitnim genom HLA DQ2 i HLA DQ8 [47].

Alergija na brašno pripada grupi alergija na hra-nu koje su rezultat pogrešnog imunološkog odgovo-ra na antigen unesen oralnim putem. Glavnu ulogu igraju antitela imunoglobulina E (IgE) koja učestvuju u patogenezi ove bolesti. Alergija na brašno može se manifestovati širokom lepezom simptoma kao što su urtikarija/angioedem, anafi laksija, atopijski dermatitis, respiratorni simptomi ili probavni poremećaji [50].

Osetljivost na gluten je poremećaj koga karakterišu simptomi nepodnošenja glutena, ali su testovi na aler-giju negativni pa nema atrofi je sluznice tankog creva i specifi čnih antitela u krvi za dijagnostikovanje celija-kije.

ZAKLJUČAK

Poznavanjem sastava i strukture proteina pšenice i us-postavljanjem veze sa tehnološkim svojstvima brašna omogućava se primena adekvatnog tehnološkog po-stupka proizvodnje pekarskih proizvoda i uticaj na nji-hove senzorne karakteristike, kao i izmena imunogene sekvence glutena kako bi se izbegla reakcija imunog sistema kod genetski podložnih osoba i omogućila pri-prema prihvatljive i zdravstveno bezbedne hrane.

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48. Camarca A, Anderson RP, Mamone G, Fierro O, Facchiano A, Costantini S i sar. Intestinal T cell responses to gluten peptides are largely heterogeneous: implications for a peptide-based therapy in celiac disease. J Immunol 2009; 182:4158–66.

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THE TECHNOLOGICAL, NUTRITIONAL AND MEDICAL ASPECTS OF WHEAT PROTEINS

Abstract

Wheat is one of the most important cereal crops worldwide and it is a major source of energy, protein, and dietary fi bre in human nutrition. Despite its relatively low protein content the nutritional importance of wheat proteins should not be underestimated. Wheat fl our quality, rheological and tech-nological properties of dough and bakery products are largely determined by the proteins. Wheat proteins show high complexity and diff erent interac-tions with each other, thus making them diffi cult to characterise. Despite from their key role in dough quality, gluten proteins can aff ect health in ge-netically susceptible individuals.Key words: wheat, proteins, gluten, technological properties, dough, coe-liac disease

Marijana Simić , Slađana ŽilićMaize Research Institute Zemun Polje, Laboratory of Food Technology and Biochemistry, Slobodana Bajića 1, Belgrade, Serbia

Tel.: + 381 (0)65 370 60 11E-mail: [email protected]

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UVOD

Začini su proizvodi biljnog porekla, svojstvenog mirisa i ukusa, koji se dodaju prehrambenim proizvodima radi postizanja odgovarajućeg mirisa i ukusa ili radi pobolj-šanja njihove svarljivosti. U promet se stavljaju kao ra-zličiti delovi aromatičnih biljaka (koren, list, kora, plod i dr.) a mogu biti u obliku većih ili manjih delova ili u obliku praha [1].

Poslednjih godina sve je veće interesovanje usme-reno ka proučavanju hemijskog sastava kao i aktivno-sti biološki aktivnih sastojaka začinskog bilja. Naime, brojna istraživanja su pokazala da sastojci začinskih biljaka poseduju karminativno, antimikrobno, antiin-fl amatorno, imunomodulatorno kao i antiproliferativno delovanje [2]. Pokazano je da jedinjenja prisutna u za-činima mogu imati pozitivan efekat na prevenciju i tok bolesti izazvanih narušenom redoks ravnotežom, kao što su ateroskleroza, dijabetes, katarakta i karcinom [3]. Posebna pažnja je usmerena na proučavanje sadržaja polifenolnih jedinjenja i njihovih antioksidativnih svoj-stava, što može biti značajno ne samo sa aspekta pro-

duženja roka upotrebe naročito upakovanih namirnica, već i u smislu upotrebe začinskog bilja kao funkcional-nih sastojaka hrane [4].

Naime, polifenoli su velika grupa strukturno različi-tih jedinjenja koja se mogu klasifi kovati na osnovu više kriterijuma: prema hemijskoj strukturi, rastvorljivosti, poreklu i lokaciji u biljkama, kao i biološkoj funkciji. Jedna od najjednostavnijih klasifi kacija jeste da se poli-fenolna jedinjenja dele na fenolne kiseline, fl avonoide, stilbene i lignane. Flavonoidi su najzastupljenija grupa fenolnih jedinjenja u biljkama i na osnovu hemijske strukture razlikuju se: antocijani, halkoni, fl avanoni, fl a-voni, fl avonoli, i izofl avonoidi [5].

U začinskim biljkama predominantno su prisutni fl avonoidi (fl avoni i fl avonoli) i fenolne kiseline. Neki od začina se odlikuju prisustvom karakterističnih polife-nolnih jedinjenja, pa su tako furanokumarini prisutni u peršunu, dok je kurkuma bogata kurkuminoidima. Iako se koriste u relativno malim količinama, začini mogu značajno doprineti ukupnom unosu dijetarnih anti-oksidanasa, naročito polifenolnih jedinjenja [6]. Ipak,

Antioksidativna aktivnost odabranih začina sa tržišta Srbije

Kratak sažetak

Začini su aromatični delovi začinskih biljaka, karakterističnog mirisa i uku-sa, koji se dodaju tokom pripreme hrane radi postizanja odgovarajućih organoleptičkih osobina, kao i zbog produženja roka trajanja namirnica. S obzirom na to da predstavljaju izvore prirodnih antioksidanasa, začini se smatraju i funkcionalnim sastojcima hrane. Osnovni cilj ovog rada jeste analiza sadržaja ukupnih polifenola i fl avonoida odabranih začina sa na-šeg tržišta, kao i komparativna procena njihove antioksidativne aktivnosti. Istraživanje je sprovedeno na komercijalno dostupnim uzorcima deset ra-zličitih vrsta začinskog bilja. Nakon ekstrakcije etanolom, sadržaj ukupnih polifenolnih jedinjenja (Total Polyphenol Content, TPC), kao i sadržaj fl a-vonoida (Total Flavonoid Content, TFC) određen je spektrofotometrijskim metodama. Procena antioksidativnog potencijala izvršena je korišćenjem tri različita testa (FRAP, DPPH, ABTS). Na osnovu dobijenih rezultata, račun-skim putem određene su vrednosti antioksidativnog kompozitnog indeksa (ACI). Najveći sadržaj TPC utvrđen je za cimet (61,3 ± 3,1 mg GAE/g), slede ruzmarin (30,2 ± 3,6 mg GAE/g) i origano (21,0 ± 0,8 mg GAE/g), dok je naj-manji sadržaj bio prisutan u uzorku vlašca (4,3 ± 0,0 mg GAE/g). Sadržaj TFC kretao se u rasponu od 3,2 ± 0,4 μmol CE/g (kim) do 133,7 ± 6,1 μmol CE/g (cimet). U pogledu antioksidativnog potencijala, sva tri testa su pokazala konzistentne rezultate. Utvrđena je statistički značajna povezanost između sadržaja TPC i ACI vrednosti (r= 0,976; p< 0,01). Na osnovu dobijenih rezul-tata može se zaključiti da cimet, ruzmatin i origano predstavljaju bogat izvor polifenolnih jedinjenja i poseduju izražen antioksidativni potencijal.Ključne reči: začini, polifenoli, fl avonoidi, antioksidativni kompozitni in-deks

Vanja Todorović*, Anđelka Dančetović, Nevena Dabetić, Slađana Šobajić, Bojana VidovićKatedra za bromatologiju, Farmaceutski fakultet, Univerzitet u Beogradu, Vojvode Stepe 450, 11221 Beograd, Srbija

* Autor za korespodenciju:Vanja TodorovićKatedra za bromatologiju, Farmaceutski fakultet, Univerzitet u Beogradu, Vojvode Stepe 450, 11221 Beograd, [email protected]

UDK: 615.322:633.81/.85

615.279

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prilikom ispitivanja njihove biološke aktivnosti, treba uzeti u obzir različite nivoe unosa, zatim uticaj pripre-me, uticaj metaboličkih procesa kojima začinske biljke podležu u organizmu, kao i uticaj drugih namirnica s obzirom da se retko konzumiraju samostalno.

Osnovni cilj ovog rada bio je analiza sadržaja uku-pnih polifenola i fl avonoida odabranih začina sa našeg tržišta kao i da se izvrši poređenje njihove antioksida-tivne aktivnosti.

MATERIJALI I METODE

Istraživanje je obuhvatilo deset različitih vrsta komer-cijalno dostupnih začina. Uzorkovanje je izvršeno u triplikatu, svi uzorci su bili osušeni i nisu dodatno usit-njavani. Pregled ispitivanih uzoraka kao i njihove karak-teristike prikazani su u Tabeli 1.

Tabela 1: Spisak analiziranih začina

Uzo-

rak

Naziv, opis, la-

tinski nazivProizvođač

Deo

biljkeFamilija

1Cimet, mleveni(lat. Cinnamo-mum zeylancium)

A.D. Prehram-bena industrija „Aleva“, Srbija

Kora Laura-ceae

2Biber, crni, mle-veni (lat. Piper nigrum)

A.D. Prehram-bena industrija „Aleva“, Srbija

Plod Pipera-ceae

3Ruzmarin, usit-njeni, (lat. Rosma-rinus offi cinalis).

Nestle Adriatic S d.o.o. Beograd, Srbija

List Lamia-ceae

4Origano, mleveni(lat. Origanum vulgare)

Nestle Adriatic S d.o.o. Beograd, Srbija

Nad-zemni deo

Lamia-ceae

5Mirođija, seckana(lat. Anethum graveolens)

Nestle Adriatic S d.o.o. Beograd, Srbija

List Apiaceae

6Kurkuma, mleve-na (lat. Curcuma longa)

AWT Internati-onal d.o.o. Beo-grad, Srbija

Rizom Zingibe-raceae

7Peršun, sušeni, sečeni (lat. Petro-selinum crispum)

A.D. Prehram-bena industrija „Aleva“, Srbija

List Apiaceae

8Bosiljak, mrvljeni, sušeni (lat. Oci-mum basilicum)

AWT Internati-onal d.o.o. Beo-grad, Srbija

Nad-zemni deo

Lamia-ceae

9 Kim, celo zrno(lat. Carum carvi)

Delhaize Serbia, d.o.o. Beograd, Srbija

Plod Apiaceae

10Vlašac, seckani(lat. Allium schoe-noprasum)

Delhaize Serbia, d.o.o. Beograd, Srbija

List Alliaceae

Priprema ekstrakata

Za potrebe ispitivanja pripremljeni su etanolni (60%) ekstrakti začina, prema postupku koji je opisan u lite-raturi [4]. Naime, odmereno je po 2,5 g svakog začina i preneseno u plastične tube. Dodato je po 50 ml 60% etanola i posle 24h ekstrakcije izvršeno je centrifugira-nje. Nakon fi ltriranja supernatanta, ekstrakti su dopu-njeni do zapremine od 50 ml etanolnim rastvorom.

Određivanje sadržaja ukupnih fenolnih jedinjenja

(TPC)

Sadržaj ukupnih polifenolnih jedinjenja određen je korišćenjem Folin Ciocalteu reagensa [7]. U razblaže-ni etanolni ekstrakt (25 μl) dodat je komercijalni Folin Ciocalteu reagens (2,5 ml) koji je prethodno razblažen 10x i vodeni rastvor Na2CO3 (2 ml, 7,5%). Sadržaj je ostavljen da se inkubira 2h zaštićen od svetlosti. Na-kon mešanja, apsorbancija reakcione smeše merena je na talasnoj dužini od 760 nm (UV-vis J.P. SELECTA spek-trofotometar), u odnosu na slepu probu koja je pripre-mljena identično kao reakciona smeša, sa razlikom što je umesto 25 μl uzorka uzeto 25 μl 60% etanola. Sadr-žaj ukupnih polifenola određivan je metodom standar-dne krive koja je konstruisana upotrebom standarda – galne kiseline (0,1 g/l) u opsegu koncentracija 10–80 mg/l. TPC je izražen kao mg ekvivalenta galne kiseline po gramu uzorka (mg GAE/g).

Određivanje sadržaja ukupnih fl avonoida (TFC)

Sadržaj ukupnih fl avonoida u ekstraktima začinskog bilja meren je spektrofotometrijskom metodom uz upotrebu AlCl3, koja se bazira na stvaranju komplek-sa fl avonoid-aluminijum [8]. U razblaženi ekstrakt za-čina (500 μl) dodat je AlCl3 (150 μl, 10%), NaNO2 (150 μl, 5%), NaOH (1 ml, 1 M) i destilovana voda (3,2 ml). Reakciona smeša je promešana i njena apsorbanci-ja je odmah merena na talasnoj dužini od 510 nm, u odnosu na slepu probu za čiju pripremu je umesto uzorka korišćena ista zapremina 60% etanola. Sadržaj ukupnih fl avonoida određivan je metodom standardne krive koja je konstruisana uz upotrebu katehina (1 g/l) kao standarda u opsegu koncentracija 100 – 700 mg/l. TFC je izražen kao μmol katehin ekvivalenata po gramu uzorka (μmol CE/g).

Određivanje antioksidativne aktivnosti DPPH

testom

DPPH test se zasniva na redukciji ljubičastog DPPH radikala (1,1-difenil-2-pikrilhidrazil), koji primanjem protona vodonika ili redukcijom sa drugim radikalom prelazi u žuto obojeni difenilpikrilhidrazin [9]. Razbla-ženim uzorcima (200 μl) dodat je DPPH radni rastvor (2,8 ml) nakon čega je intenzitet boje meren spektro-fotometrijski na 517 nm. Izračunavanja su vršena meto-dom standardne krive koja je konstruisana na osnovu apsorbancija rastvora standarda (eng. Trolox) u etanolu

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u opsegu koncentracija 0,2 – 0,7 mmol/l. Procenat in-hibicije je matematički dobijen prema formuli: % Inh

= (Asp – Au) / Asp (Inh – inhibicija; Asp – apsorbanci-ja slepe probe; Au – apsorbancija uzorka). Rezultati su izraženi kao μmol Trolox ekvivalenta po gramu uzorka (μmol TE/g).

Određivanje antioksidativne aktivnosti ABTS+

(TEAC) testom

TEAC (eng. Trolox Equivalent Antioxidant Capacity) je test koji se zasniva na reakciji plavo obojenog ABTS+ [2,2’-azino-bis-(3-etilbenzotiazolin-6-sulfonska kiseli-na)] radikala sa antioksidativnim jedinjenjima pri čemu dolazi do njegovog obezbojavanja. Stepen obezboja-vanja se određuje spektrofotometrijski na 734 nm [10].

Za analizirane uzorke, napravljena su različita ra-zblaženja. Uzorcima (30 μl) je dodat ABTS radni rastvor (3 ml), i ostavljen da se inkubira 6 minuta u vodenom kupatilu na 37°C. Nakon mešanja merene su apsor-bancije. Korišćenjem izmerenih apsorbancija odgo-varajućih razblaženja, izračunati su procenti inhibicije po formuli: % Inh = (Asp – Au)/Asp. Zatim je za svaki uzorak konstruisana kalibraciona kriva koja predstavlja zavisnost procenta inhibicije od koncentracije. Upore-đivanjem jednačina krive za svaki pojedinačni uzorak i jednačine krive za standard (eng. Trolox), u opsegu koncentracija od 0,2 do 1,5 mmol/l, dobijeni su rezul-tati koji su izraženi kao μmol Trolox ekvivalenta po gra-mu uzorka (μmol TE/g). Preciznije, rezultati su dobijeni deljenjem koefi cijenata pravca jednačine kalibracione krive uzorka i jednačine kalibracione krive dobijene za standard.

Određivanje antioksidativne aktivnosti

FRAP testom

U toku FRAP analize (eng. Ferric Reducing Antioxidant Power) dolazi do doniranja elektrona od strane antioksidanasa i po-sledične redukcije kompleksa feri-tripiri-diltriazina [Fe3 +-TPTZ] do intenzivno pla-vog komleksa fero-tripiridiltriazina [Fe2

+-TPTZ ] pri niskim pH vrednostima [11].Analiza je izvedena na sledeći način: u

razblaženi uzorak (100 μl) dodat je FRAP reagens (3ml) nakon čega je sadržaj ostav-ljen da se inkubira u vodenom kupatilu 40 minuta na 37°C. Nakon završene inku-bacije i mešanja, merena je apsorbancija reakcione smeše na spektrofotometru na 593 nm u odnosu na slepu probu. Izraču-navanja su vršena metodom standardne krive koja je konstruisana korišćenjem ra-stvora Trolox-a kao standardne supstance u opsegu koncentracija 0,1 – 0,8 mmol/l. Antioksidativna aktivnost izražena je kao μmol Trolox ekvivalenta po gramu uzorka (μmol TE/g).

Antioksidativni kompozitni indeks (ACI)

Antioksidativni kompozitni indeks određivan je na osnovu rezultata dobijenih u sva tri primenjena testa (DPPH, ABTS, FRAP), pri čemu je vrednost indeksa od 100 dodeljivana najvišoj vrednosti antioksidativnog potencijala u svakom testu, a zatim je izračunavanje indeksa za sve ostale uzorke u okviru pojedinačnog testa vršeno po formuli: Indeks Antioksidativnog Potencijala (%) = [(rezultat

za uzorak/najbolji rezultat) x 100]

Srednja vrednost indeksa antioksidativnog potencijala ova tri testa predstavljala je ACI vrednost za određeni uzorak začina [12].

Statistička analiza

Sva merenja su vršena u triplikatu, a prikazani rezulta-ti predstavljaju njihovu srednju vrednost i standardnu devijaciju.

Pearson-ov koefi cijent korelacije primenjen je za procenu korelacije između vrednosti antioksidativnog kompozitnog indeksa i sadržaja ukupnih polifenola i fl a-vonoida. Statistička analiza urađena je korišćenjem kom-pjuterskog programa SPSS (Version 20, Chicago, IL, USA).

REZULTATI I DISKUSIJA

Spektrofotometrijskim metodama uz korišćenje stan-darda za konstruisanje kalibracione krive, kao što je opisano u poglavlju „Materijal i metode“, određen je sadržaj ukupnih polifenolnih jedinjenja (TPC) kao i sa-držaj ukupnih fl avonoida (TFC) u ispitivanim uzorcima. Dobijeni rezultati prikazani su u Tabeli 2.

Tabela 2: Sadržaj ukupnih polifenola, fl avonoida i antioksidativna aktivnost analiziranih začina

uzo-

rak

TPC

(mg

GAE/g)

TFC

(μmol

CE/g)

DPPH

(μmol

TE/g)

FRAP

(μmol

TE/g)

ABTS

(μmol TE/g)

1 61,3±3,1 133,7±6,1 208,2±1,1 332,4±4,8 1215,8±11,6

2 6,2±0,2 8,8±0,5 0,8±0,0 78,1±1,1 76,6±0,3

3 30,2±3,6 52,7±1,7 107,0±0,2 306,0±11,5 283,8±0,8

4 21,0±0,8 32,0±1,6 106,7±0,3 248,0±3,3 286,4±1,1

5 9,1±0,6 5,8±0,4 85,0±1,6 89,7±1,7 121,5±0,2

6 9,3±0,8 57,3±1,3 41,0±0,9 89,3±6,7 191,2±0,6

7 7,9±0,2 7,1±0,1 14,7±2,8 51,9±1,3 132,3±0,9

8 9,1±0,8 14,2±0,5 103,1±0,2 78,1±0,5 138,5±0,8

9 4,5±0,1 3,2±0,4 41,8±1,7 67,9±3,3 106,4±0,5

10 4,3±0,0 18,4±0,0 4,4±0,6 54,8±1,3 58,0±0,3

TPC – sadržaj ukupnih fenolnih jedinjenja; TFC – sadržaj ukupnih fl avonoida; DPPH – (1,1-difenil-2-pikrilhidrazil) test; FRAP – ferric reducing ability of plasma test; ABTS – 2,2’-azino-bis-(3-etilbenzotiazolin-6-sulfonska kiselina) test; GAE – ekvivalent galne kiseline; CE – katehin ekvivalent; TE – troloks ekvivalent.

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Sadržaj TPC kretao se u rasponu od 4,3 do 61,3 mg GAE/g, pri čemu je najviša vrednost određena za cimet, a zatim slede ruzmarin i origano, dok je najmanju vred-nost imao vlašac. Poređenja radi, dobijene vrednosti su višestruko veće od vrednosti koje se u literaturi mogu naći za sadržaj TPC odabranog zelenog lisnatog povrća, voća i orašastih plodova [13]. Takođe, dobijeni rezultati sugerišu da je cimet bogatiji izvor polifenolnih jedinje-nja u poređenju sa zelenim čajem i kakao prahom koji su poznati kao značajni izvori antioksidanasa [14]. Sa-držaj ukupnih polifenola u cimetu može se uporediti sa heljdom, žitaricom koja obiluje polifenolnim jedinje-njima [15]. Približno slične vrednosti TPC za ruzmarin i origano, a dva odnosno tri puta manje u odnosu na cimet (respektivno), mogu se objasniti istom familijom- Lamiaceae kojoj ove dve začinske biljke pripadaju. Slič-ni rezultati su dobijeni u prethodnom istraživanju Zen-ga i saradnika u kome se TPC u analiziranom začinskom bilju kretao u opsegu 3,53–58,25 mg GAE/g, i u kome je takođe utvrđeno da je cimet začinska biljka sa izrazito visokim sadržajem ukupnih polifenola [4]. Drugo istra-živanje [16] obuhvatilo je različite delove začinske bilj-ke vlašac pri čemu je zaključeno da je sadržaj ukupnih polifenolnih jedinjenja i stepen antioksidativne aktiv-nosti viši u korenu i listu u poređenju sa stabljikom. U pomenutoj studiji, uzorak je bio svež biljni materijal, dok je u ovom istraživanju korišćen osušen vlašac. Pre-ma Opara i Chohan, više vrednosti TPC primećene su u svim osušenim uzorcima začinskog bilja u poređenju sa svežim materijalom, što predstavlja moguće objašnje-nje dobijenih viših vrednosti za sadržaj TPC u vlašcu u ovoj studiji u odnosu na rezultate istraživanja Štajner i sar [16, 17]. Neophodno je naglasiti i da se sadržaj TPC razlikuje u zavisnosti od izbora rastvarača kao i uslova ekstrakcije, što je i pokazano u prethodnim studijama [18].

Na osnovu analize vrednosti dobijenih za sadržaj ukupnih fl avonoida koji se kretao u rasponu od 3,2 do 133,7 μmol CE/g, može se zaključiti da su dobijeni re-zultati u saglasnosti sa prethodno publikovanim istraži-vanjima [19]. Najviša vrednost TFC je utvrđena za cimet, dok je najniži sadržaj fl avonoida pokazao uzorak kima. Utvrđene različite vrednosti za TPC i TFC analiziranog začinskog bilja mogu se objasniti potencijalnim razlika-ma u sadržaju fenolnih kiselina koje, pored fl avonoida, predstavljaju drugu veliku grupu polifenolnih jedinje-nja u ovim biljkama [20].

Za in vitro ispitivanje antioksidativne aktivnosti ra-zličitih namirnica, najboljim pristupom smatra se pri-mena kombinacije nekoliko metoda koji se zasnivaju na različitim principima određivanja antioksidativnog potencijala analiziranog materijala kroz različite meha-nizme delovanja [21,22]. Upravo su DPPH, FRAP i ABTS najčešć e metode za određivanje in vitro antioksidativ-nog kapaciteta hrane i pića. DPPH esej zasnovan je na najjednostavnijem mehanizmu antioksidativne zaštite (DPPH• + RH (antioksidans) → DPPH-H + •R). FRAP me-toda se bazira na sposobnosti antioksidanasa, rastvor-nih u vodi, da redukuju gvožđe iz Fe3+ na Fe2+. Suprotno

od DPPH metode, koja je pogodna za određivanje anti-oksidansa rastvornih u organskim rastvaračima (etanol, metanol), FRAP metodom mogu se odrediti antioksida-tivna svojstva hidrosolubilnih jedinjenja [23]. Metoda ABTS u osnovi predstavlja merenje procenta inhibicije ABTS radikala u poređenju sa komercijalnim standar-dom Trolox-a. Što se same reakcije tiče, FRAP test se odvija preko SET (single electron transfer) mehanizma, dok DPPH i ABTS predstavljaju reakcije u kojima ova dva radikala mogu da budu neutralisana putem SET ili HAT (hydrogen atom transfer) mehanizma [24]. Što se anti-oksidativnog potencijala ispitivanih začina tiče, sva tri izvedena testa su dala konzistentne rezultate. Za uzor-ke koji imaju najviši sadržaj polifenolnih jedinjenja, do-kazana je i najveća antioksidativna aktivnost kroz sva tri testa, i obrnuto, što se može videti u Tabeli 2. Ovo ukazuje na činjenicu da je ukupni sadržaj polifenola u začinskim biljkama dobar prediktor i glavni nosilac nji-hove antioksidativne aktivnosti.

Kombinacijom rezultata antioksidativne aktivnosti merene kroz tri različita testa (DPPH, FRAP, ABTS) omo-gućeno je izračunavanje vrednosti antioksidativnog kompozitnog indeksa (ACI) svih analiziranih začina. S obzirom na to da pojedinačni testovi pokrivaju različite segmente antioksidativne aktivnosti, njihova kombina-cija ima određene prednosti. Integrisanjem vrednosti pojedinačnih testova u jednu ACI vrednost omogu-ćen je sveobuhvatniji uvid u antioksidativnu aktivnost eskperimentalnih uzoraka začina. Prema dostupnim li-teraturnim podacima, ACI vrednost korišćena je za pro-cenu antioksidativne aktivnosti pića bogatih polifenoli-ma u SAD [12], biljnih i voćnih čajeva [25] kao i različitih kakao proizvoda dostupnih na tržištu Srbije [7]. Ovakav način prikazivanja i izražavanja rezultata antioksida-tivne aktivnosti pruža mogućnost poređenja velikog broja različitih prehrambenih proizvoda i potencijalno može poslužiti za izradu popularnih tablica namirnica bogatih antioksidansima koje bi bile od izuzetne koristi kako istraživačima tako i krajnjim potrošačima.

ACI vrednosti analiziranih začina prikazane su na Grafi konu 1. Vrednost od 100% pripala je cimetu, dok je za vlašac izračunata čak dvanaest puta niža ACI vred-nost. Prilikom procene stepena korelacije između TPC i ACI vrednosti, dobijen je statistički značajan pozitivan Pearson-ov koefi cijent korelacije (r = 0,976, p<0,01), što ukazuje na snažnu povezanost između sadržaja uku-pnih polifenolnih jedinjenja i antioksidativne aktivno-sti začina. Nešto niža vrednost koefi cijenta korelacije (r = 0,877) dobijena je u proceni korelacije sadržaja ukupnih fl avonoida i vrednosti ACI, ali je ta povezanost takođe statistički značajna (p < 0,01). Dobijeni rezultati potvrđuju prethodno navedenu činjenicu da začinske biljke, pored fl avonoida, sadrže i druge komponente sa izraženim antioksidativnim potencijalom.

Dobijeni rezultati su u skladu sa prethodnim istraži-vanjem Zhenga i saradnika, u kom je takođe pokazana povezanost antioksidativnog potencijala začina i sadr-žaja polifenolnih jedinjenja [4].

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Međutim, neophodno je uzeti u obzir i bioiskoristlji-vost polifenolnih jedinjenja iz začinskih biljaka, poseb-no imajući u vidu da se začini najčešće koriste u kom-binaciji sa drugim namirnicama i da se one obično pre konzumiranja podvrgavaju termičkoj obradi [17].

ZAKLJUČAK

U pogledu sadržaja polifenola različitog začinskog bilja, posebno visoka vrednost TPC je određena u etanolnim ekstraktima cimeta, ruzmarina i origana. Slični rezultati su dobijeni i u pogledu TFC vrednosti, pri čemu je u ostalim analiziranim začinima sadržaj ovih jedinjenja znatno niži. Ispitivani ekstrakti pokazali su dobru, kon-centracijski zavisnu antioksidativnu aktivnost, u svim primenjenim antioksidativnim testovima.

Na osnovu dobijenih rezultata može se zaključiti da začinske biljke, sa visokim sadržajem polifenolnih jedinjenja, predstavljaju potencijalni izvor prirodnih antioksidanasa sa mogućom primenom u prehrambe-noj, farmaceutskoj i kozmetičkoj industriji. Ostavlja se prostor budućim istraživanjima u pravcu ispitivanja an-tiinfl amatornih, antikancerogenih i ostalih potencijalno pozitivnih zdravstvenih efekata, kao i detaljnijoj hemij-skoj analizi biološki aktivnih jedinjenja začinskog bilja.

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Grafi kon 1. Antioksidativni kompozitni indeks (ACI) analiziranih začina

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22. Llorent-Martinez EJ, Ortega-Barrales P, Zengin G, Uysal S, Ceylan R, Guler GO et al. Lathyrus aureus and Lathyrus pratensis: characterization of phytochemical profi les by liquid chromatography-mass spectrometry, and evalua-tion of their enzyme inhibitory and antioxidant activities. RSC Adv 2016; 6:88996–9006.

23. Dai J, Mumper RJ. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecu-les 2010; 15(10):7313–52.

24. Prior RL, Gu L. Occurrence and biological signifi cance of proanthocyanidins in the American diet. Phytochemistry 2005; 66(18):2264–80.

25. Veljkovic JN, Pavlovic A, Mitic S, Tosic S, Stojanovic G, Kali-canin BM et al. Evaluation of individual phenolic compo-unds and antioxidant properties of black, green, herbal and fruit tea infusions consumed in Serbia: Spectropho-tometrical and electrochemical approaches. J Food Nutr Res 2013; 52(1):12–24.

Antioxidant activity of selected spices from Serbian market

Abstract

Spices are aromatic parts of herbs with characteristic smell and taste. They are added to food in order to achieve the appropriate organoleptic proper-ties, as well as to extend their shelf life. Since they are sources of natural an-tioxidants, spices are considered as functional food ingredients. The main goal of this research was determination of total polyphenol and fl avonoid content in selected spices from our market and comparative assessment of their antioxidant activity. The study was conducted on ten diff erent commercial samples of herbs. After extraction with ethanol, total polyphe-nol content (TPC), and total fl avonoid content (TFC) were determined by spectrophotometric methods. Evaluation of antioxidant potential was carried out using three diff erent tests (FRAP, DPPH, ABTS). Based on these re-sults, values of antioxidant composite index (ACI) was determined by com-putation. The highest content of polyphenolic compounds was found for cinnamon (61.3±3.1 mg GAE/g), followed by rosemary (30.2±3.6 mg GAE/g) and oregano (21.0±0.8 mg GAE/g), while the lowest content showed shallot (4.3±0.0 mg GAE/g). The content of total fl avonoids ranged from 3.2 μmol CE/g (cumin) to 133.7 μmol CE/g (cinnamon). When it comes to antioxidant potential, all three tests showed consistent results. A statistically signifi cant correlation was fi gured out for total polyphenol content and ACI values (r = 0.976, p<0.01). Based on obtained results, it can be concluded that cinna-mon, rosemary and oregano are the rich sources of polyphenol compounds and have conspicuous antioxidant potential.Key words: spices, polyphenols, fl avonoids, antioxidant composite index

Vanja Todorović*, Anđelka Dančetović, Nevena Dabetić, Slađana Šobajić, Bojana VidovićDepartment of Bromatology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia

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UV OD

Mikotoksini su toksični sekundarni metaboliti mno-gih fi lamentoznih gljiva [1,2]. Sam naziv mikotoksin potiče od grčke reči mykes, što znači plesan i latinske reči toxicum, što znači otrov [2]. Sintetišu se usled ak-tivnosti različitih enzima u procesima kondenzacije, oksido-redukcije, alkiliranja i halogeniranja, od biohe-mijski jednostavnih međuprodukata primarnog meta-bolizma (kao što su: malonat, acetat, mavalonat, serin, fenilalanin, alanin, triptofan) [3]. Glavni biohemijski putevi nastajanja mikotoksina su: terpenski (pr. nasta-ju trihoteceni), aminokiselinski (pr. nastaju: ergotamin, gliotoksini, malformin C, sporidezmin, ksantocilin, ci-klohlorotin i ksantoascin), poliketidni (pr. nastaju: afl a-toksini, zearalenoni, patulin, sterigmatocistin, citrinin, ohratoksini) i put trikarbonskih kiselina (pr. nastaju: rubratoksini) [4–6].

Zbog sposobnosti da proizvode mikotoksine u hrani, gljive su privukle posebnu pažnju u poslednjih 50 godina. Prisustvo toksigenih gljiva i mikotoksina u namirnicama životinjskog i biljnog porekla, začinima, lekovitom bilju, kao i u hrani za životinje, dokumento-vano je od strane mnogih autora [6–14].

Najčešći kontaminenti zrna žita i proizvoda od žita su vrste gljiva iz rodova Fusarium i Alternariа (takozva-ne “poljske gljive” [15]), one se, pored vrsta iz rodova Botrytis, Sclerotina, Rhizopus, Monillia, Mucor i Penicil-lium, mogu naći kao česti uzročnici oboljevanja voća i povrća u polju [16–18]. Česti kontaminenti mesa i mleka su vrste gljiva iz rodova Aspergillus, Penicillium, Cladosporium, Mucor, Geotrichum, Trichoderma i Spo-rotrichum [19–22]. Vrste rodova Penicillium, Aspergil-lus i Eurotium su takozvane „skladišne gljive“ koje se razvijaju na začinima [11], sušenom voću i povrću [23] i sličnim proizvodima (pr. kafa, kakao, seme susama i suncokreta, musli) [24–28].

Fungicidi su često prva odbrambena linija protiv mikotoksigenih gljiva. Međutim, neselektivna upotre-ba fungicida je dovela do pobune javnosti zbog njiho-vih štetnih efekata na životnu sredinu i zdravlje ljudi i životinja. Stoga se povećava javni pritisak za sigurniju i ekološku alternativu za kontrolu ovih organizama. U tom kontekstu, biološka kontrola koristi mikrobiološke antagoniste kao što su bakterije, gljivice i kvasci. Oni su se pokazali izvodljivom zamenom za smanjenje upo-trebe hemijskih sredstava [1]. Pored toga, danas se na-

Koliko smo upoznati sa osobinama i prisustvom

mikotoksina u hrani?

Kratak sažetak

Pojava mikotoksina u lancu ishrane je neizbežan i ozbiljan problem sa ko-jim se suočava svet. Zbog veoma vlažnih i toplih klimatskih uslova, može se očekivati da će Srbija ove i sledeće godine biti veoma pogodno tle za ra-zvoj toksigenih gljiva. Moramo biti upoznati sa osobinama mikotoksina, hemijskom strukturom i osnovnim mehanizmima delovanja pojedinačnih mikotoksina, kako bismo imali osnove za razvoj protokola ili metoda za efi kasno upravljanje problemima vezanim za mikotoksine, kao i da bi se ra-zumeli njihovi biološki efekti. Cilj rada je bio da se napravi analiza koliko su studenti upoznati sa problemom pojave mikotoksina u hrani. Istraživanje je sprovedeno pomoću anonimnog upitnika, koji je uključivao pitanja koja se tiču mikotoksina i mikotoksikoza. Anketirani studenti su odabrani nasu-mično, tj. studenti osnovnih studija na Institutu za prehrambenu tehnolo-giju i biohemiju, Poljoprivrednog fakulteta, Univerziteta u Beogradu imali su jednake šanse da budu izabrani za uzorak. Anketa je bila edukativna za anketirane studente i pokazala je da su u veoma visokom procentu upo-znati sa osnovnim karakteristikama mikotoksina, kao i da od predstavnika pojedinih grupa mikotoksina, najbolje poznaju karakteristike afl atoksina. Pored toga, studenti su pokazali relativno dobro poznavanje osnovnih ka-rakteristika i drugih predstavnika mikotoksina. Relativno dobro predznanje anketiranih studenta o osobinama mikotoksina, može biti odlična osnova za dalji rad i usavršavanje. Ključne reči: mikotoksini, mikotoksikoze, hrana, anketno istraživanje

Emilija Oreščanin1, Ivana Perić1, Mirjana Pešić2, Slađana Stanojević2*

1 Student II godine osnovnih studija Poljoprivrednog fakulteta u Zemunu – Univerziteta u Beogradu, modul: Mikrobiologija hrane; Institut za prehrambenu tehnologiju i biohemiju

2 Poljoprivredni fakultet u Zemunu – Univerziteta u Beogradu, vanredni profesor, Institut za prehrambenu tehnologiju i biohemiju

* Autor za korespodenciju: Slađana StanojevićPoljoprivredni fakultet, Univerzitet u Beogradu, Institut za prehrambenu tehnologiju i biohemiju; Nemanjina 6, 11080 Zemun; P.O.Box 14, Srbija. Tel./Fax: +381 112199711.E-mail adresa: [email protected]

UDK: 663/664:[615.918:528.28

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stoji da se primenom različitih tehnika u prehrambenoj tehnologiji smanji sadržaj mikotoksina u namirnicama (sortiranje, čišćenje, termički tretmani, konzerviranje, alkalno kuvanje, ekstruzija) [29,30].

Mikotoksini uzrokuju brojne i različite toksične efek-te (citoksičnost, hepatotoksičnost, neurotoksičnost, te-ratogenost, mutagenost). Akutna i hronična oštećenja zdravlja izazvana mikotoksinima nazivaju se mikotok-sikoze. Neki mikotoksini su kancerogeni, napadaju je-tru, bubrege ili imuni sistem čoveka [1–4]. Do danas je detektovano više od 400 mikotoksina i njihov broj se stalno povećava. Neki od najčešće prisutnih u hrani su: afl atoksini, ohratoksini, fuzariotoksini (pr. trihoteceni, zearalenoni, fumonizini), alternaria toksini, patulin, er-got alkaloidi, citrinin, sterigmatocistin, ciklopiazonska kiselina i deoksinivalenol.

Pojava mikotoksina u lancu ishrane je neizbežan i ozbiljan problem sa kojim se suočava svet. Može se očekivati da će Srbija ove i sledeće godine biti veoma pogodno tle za razvoj mikotoksičnih gljiva, zbog veoma vlažnih i toplih klimatskih uslova. Moramo biti upozna-ti sa osobinama mikotoksina da bi se mogli naznačiti toksični efekti povezani sa trovanjima mikotoksinima kod ljudi i životinja. Generalno, razumevanje hemij-ske strukture i osnovnog mehanizma delovanja poje-dinačnog mikotoksina može pružiti dovoljno osnova za razvoj protokola ili metoda za efi kasno upravljanje problemima vezanim za mikotoksine, kao i da se razu-meju njihovi biološki efekti.

Cilj ovog rada je bio da se napravi ana liza koliko su studenti druge godine osnovnih studija na Institutu za prehrambenu tehnologiju i biohemiju, Poljoprivrednog fakulteta, Univerziteta u Beogradu upoznati sa problemom pojave mikotoksina u hrani. Na taj način će biti moguće stvoriti sliku i o informisanosti prosečnih potrošača u Srbiji, s obzirom da će se ispitivani studenti ovom tematikom značajno baviti tek na višim god-inama studija i u trenutku anketiranja su ra-spolagali samo osnovnim saznanjima.

MATERIJAL I METODE

Istraživanje je sprovedeno pomoću ano-nimnog upitnika. Ukupno 100 studenata druge godine osnovnih studija na Institutu za prehrambenu tehnologiju i bio hemiju, Poljoprivrednog fakulteta, Univerziteta u Beogradu (68 ženskih i 32 muških ispitanika). Anketi-rani studenti su odabrani nasumično, tj. studenti svih pet modula osnovnih studija (Tehnologija animalnih proizvoda – 18 studenata, Tehnologija konzervisanja i vrenja – 19 studenata, Tehnologija ratarskih proizvo-da – 17 studenata, Mikrobiologija hrane – 33 studenta, Upravljanje bezbednošću i kvalitetom u proizvodnji hrane – 13 studenata) na Institutu za prehrambenu tehnologiju i biohemiju, Poljoprivrednog fakulteta, Univerziteta u Beogradu i imali su jednake šanse da

budu izabrani za uzorak. Od 100 ispitivanih studenata njih samo 34 su u trenutku ispitivanja bili položili sve ispite iz prethodne godine, tako da nisu bili optereće-ni zaostalim ispitima i bili su u mogućnosti da aktivno učestvuju u aktivnostima predmeta na drugoj godini studija; pre svega predmetima: Opšta mikrobiologija i Biohemija hrane, koji su veoma značajni za rezultate ove ankete. Upitnik je uključivao pitanja koja se tiču mikotoksina i mikotoksikoza.

Za obradu i analizu podataka korišćene su metode deskriptivne statistike.

REZULTATI I DISKUSIJA

Dobijeni rezultati ukazuju na to da su anketirani stu-denti druge godine osnovnih studija na Poljoprivred-nom fakultetu, na Institutu za prehrambenu tehnologi-ju i biohemiju, u veoma visokom procentu upoznati sa osnovnim karakteristikama mikotoksina. Naime, visok procenat studenata je znao da mikotoksine stvaraju plesni (71%), kojima pogoduje tropska i vlažna klima (74%), da se veoma lako razvijaju na usevima u polju (63%), kao i na različitim prehrambenim proizvodima (67%), pri visokim vrednostima aktivnosti vode (51%), kao i da su mikotoksikoze bolesti izazvane mikotoksi-nima (68%) (Slika 1). Ovakav rezultat se može objasniti dobrom opštom informisanošću studenata. Rezultati sličnih anketa sprovedenih u različitim delovima sveta ukazuju na to da su uglavnom ispitanici boljeg obra-zovnog i materijalnog statusa upoznati sa ovim opštim podacima o mikrobiološkoj bezbednosti hrane [31].

Na pitanja koja su se odnosila na osobine poje-dinih mikotoksina studenti su pokazali niži stepen znanja. Naime, najbolje poznavanje karakteristika po-jedinih konkretnih mikotoksina studenti su pokazali na pitanja koja su se odnosila na afl atoksine, za koje smatraju da predstavljaju najveću potencijalnu pretnju po zdravlje ljudi (66%). Literaturni podaci ukazuju da, ogromna većina stanovništva, pre svega u nerazvije-nim delovima sveta, nije svesna prisustva afl atoksina, a naročito nisu upoznati sa njihovim štetnim zdravstve-

Slika 1. Procenat anketiranih studenata koji su na postavljena pitanja dali tačne odgovore.

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nim efektima [31,32]. Poražavajući su rezultati ankete koju su u Gani sproveli Awuah i saradnici [33] koji pokazuju da je svega 8% ispitanika čulo za afl atoksine. Ustanovljeno je da očigledni znaci kao što su: pro-mena boje, zaraženost insektima ili truljenje zrna bi mogli pomoći ve-ćini needukovanih potrošača da se identifi kuje gljivična kontaminacija u zrnu [31]. Studenti su bili upoznati (70%) da po EU pravilniku, za gotovo sve sirovine, maksimalno dozvoljena količina afl atoksina mora biti manja od 10 μg/kg, mada je manji broj an-ketiranih studenata (45%) znalo da je jetra glavna “meta” napada ovih toksina u organizmu čoveka. Poka-zali su različito poznavanje karakte-ristika pojedinih afl atoksina. Naime, relativno veliki procenat ispitanika (69%) je znalo da se afl atoksin M1

formira u organizmu krave i drugih sisara i da se može vezivati za proteine mleka, tako da može biti prisutan u različitim mlečnim proizvodima (siru, jogurtu). Dok je manji broj anketiranih studenata (41%) bio upoznat da je afl atoksin B1 snažan mutagen, koji može dovesti do promena na hromozomima u ćelijama biljnog, animal-nog i ljudskog tkiva. Istraživanja Ezekiel-a i saradnika [32] o svesti ispitanika o mogućoj zaraženosti kikirikija B1 afl atoksinom i mogućim štetnim efektima po njiho-vo zdravlje bila je poražavajuća. Naime, čak 85% ispi-tanika mlađeg reproduktivnog doba nije imalo svest o kontaminaciji B1 afl atoksinom i mogućim zdravstvenim rizicima povezanih sa njegovim unosom. Iznenađuje da su ispitani studenti pokazali najslabije poznavanje proizvoda na kojima se dominantno mogu sintetisati afl atoksini. Naime, samo 11% ispitanih studenata je navelo da se afl atoksini uglavnom javljaju na kikiriki-ju, pistaćima, orasima i ostalom koštunjavom voću i žitaricama, dok je velika većina (52%) smatrala meso i jaja, odnosno leguminoze (37%) kao glavne izvore ovih toksina u ishrani čoveka. Sa druge strane, velika većina ispitanika (91%) je upoznata da sirovo mleko i mleč-ni proizvodi (preko ishrane životinja stočnom hranom zaraženom plesnima roda Aspergillus) mogu biti izvori afl atoksina u ishrani ljudi. Pa sa tim u vezi, njih 35% is-pitanih zna da se za dekontaminaciju zrna za ishranu životinja uglavnom koristi termički tretman, dok njih 26% je upoznato da je najpovoljniji način detoksikacije proizvoda za animalnu ishranu tretman amonijakom. Značajan procenat ispitanih studenata (39%) smatra da se tretman UV zračenja može koristiti za dekontamina-ciju stočne hrane, iako se ova metoda zapravo može koristiti samo kao inicijalni test kontaminiranosti žita-rica i drugih proizvoda, obzirom da afl atoksini pod UV svetlosti fl uoresciraju (Slika 2).

Anketna pitanja su se odnosila i na karakteristike drugih mikotoksina koji se mogu naći u prehrambenim

proizvodima. O karakteristikama ohratoksina ispitanici su znali da se u prehrambenim proizvodima najčešće nalazi ohratoksin A (46%), da njegova prevencija i kon-trola zavise od načina na koji su usevi sušeni i od dobre prakse higijene tokom čuvanja (31%), kao i da se nje-gova toksičnost sastoji u inhibiciji sinteze RNA (69%), a da su na ove toksine najosetljiviji bubrezi (61%). Ta-kođe, 45% anketiranih je znalo da zakonski regulisana maksimalna količina zearalenona u žitaricama name-njenim za direktnu ishranu čoveka iznosi 75 μg/kg dok je manji broj ispitanika (30%) znao da je zearalenon mikotoksin sa luteotropnim, estrogenim i anaboličkim delovanjem kod domaćih životinja i da je odgovoran za reproduktivne poremećaje kod domaćih životinja, naročito kod svinja. Značajan broj ispitanika (61%) je bio upoznat da su fumonizini grupa od najmanje 15 mikotoksina, koji se najčešće javljaju na žitaricama i da su manje akutne toksičnosti od afl atoksina (46%), dok je manji broj ispitanika (25%) znao da je toksičnost fumonizina posledica negativnog uticaja na sintezu sfi ngolipida. Da mikotoksin patulin sintetišu plesni iz roda Penicillium, Aspergillus i Byssochlamys, a da je u hemijskom pogledu lakton znalo je 37% ispitanih, dok je 41% ispitanih znalo da je patulin stabilno jedinjenje koje podnosi temperature i do 100°C u kiseloj sredini i da uglavnom kontaminira voće i povrće. Najniži stepen znanja anketirani studenti su pokazali na pitanja veza-na za ergot-alkaloide. Naime, samo 19% ispitanih stu-denata je znalo da ovi toksini kod čoveka i animalnih organizama izazivaju ergotizam koji dovodi do gubitka ekstremiteta i gangrene, kao i da još uvek nisu zakonski regulisani nivoi maksimalnih dnevnih unosa ergot-al-kaloida kod proizvoda na bazi žitarica (35%). S obzirom na to da će u nastavnom programu studenti tek biti de-taljno upoznati sa mikotoksinima, anketirani studenti su pokazali relativno dobro poznavanje karakteristika pojedinih mikotoksina. Na primer, da je ciklopiazon-

Slika 2. Procenat anketiranih studenata koji su pokazali poznavanje karakteristika afl atoksina.

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ska kiselina toksična samo pri visokim koncentracija-ma (44%), da tenuazonsku kiselinu sintetiše plesan iz roda Alternaria i da inhibira biosintezu proteina (39%), da su trihotecenski mikotoksini sekundarni metaboliti čije raznovrsne derivate sintetišu različite plesni ali da su u osnovi svi seskviterpeni (52%), kao i da je steri-gmatocistin metabolit koji je strukturno i toksikološki vrlo sličan afl atoksinima i ujedno predstavlja prekursor njegove biosinteze (40%) (Slika 3).

Veoma je značajno da nijedan od ispitanih studena-ta nije imao zdravstvenih problema izazvanih mikotok-sinima, kao i da značajan broj ispitanika (93%) smatra da je ova anketa imala edukativni karakter. Rezultati dobijeni iz našeg istraživanja u saglasnosti su sa dostu-pnim literaturnim podacima, gde autori takođe ističu potrebu za edukacijom potrošača u pogledu sigurne prakse upravljanja hranom od trenutka kupovine do kuće, kao i unutar doma [34]. Moramo edukovati potro-šače kako bi se mogli nositi sa svim novitetima u oblasti bezbednosti hrane na dnevnom nivou [35]. Istraživanja ukazuju na to da televizija i novine trenutno dopiru do najvećeg broja ljudi i da su ovi mediji najbolji način ši-renja informacija o bezbednosti hrane, zdravoj ishrani i zaštiti potrošača. Kao i da angažovanja univerzitetskih profesora i specijalista iz ove oblasti u ovim programi-ma imaju značajan efekat na promenu svesti potrošača [36].

ZAKLJUČAK

Sprovedena anketa je bila edukativna za anketirane studente i dala je uvid u to koliko su studenti druge godine osnovnih studija na Institutu za prehrambenu tehnologiju i biohemiju, Poljoprivrednog fakulteta, Uni-verziteta u Beogradu upoznati sa problemom pojave mikotoksina u hrani. Rezultati ukazuju na to da su an-ketirani studenti u veoma visokom procentu upoznati sa osnovnim karakteristikama mikotoksina, kao i da od

predstavnika pojedinih grupa mikotoksina najbolje poznaju karakteristike afl atoksina. Pored toga, studenti su pokazali relativno do-bro poznavanje osnovnih karakteristika i dru-gih predstavnika mikotoksina iako još uvek nisu u nastavnom programu svojih modula detaljno obrađivali ovu tematiku. Tako da se rezultat ove ankete može objasniti dobrom opštom informisanošću studenata. General-no, može se zaključiti da anketirani studenti raspolažu dobrim predznanjem o osobinama mikotoksina, koje može biti odlična osnova za dalji rad i usavršavanje.

Zahvalnica: Autori se zahvaljuju kolegama studentima druge godine osnovnih studija na Institutu za prehrambenu tehnologiju i biohe-miju, Poljoprivrednog fakulteta, Univerziteta u Beogradu, na učešću u ovom istraživanju.

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Oreščanin i sar.: Koliko smo upoznati sa osobinama i prisustvom mikotoksina u hrani?

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How much we know about properties and the presence of mycotoxins in the food?

Abstract

The occurrence of mycotoxins in the food chain is an inevitable and serious problem that the world faces. Due to the very humid and warm climate, it can be expected that Serbia this and next year will be a very fertile ground for the development of toxigenic fungi. We need to be familiar with the pro-perties of mycotoxins, the chemical structure and the basic mechanisms of the action of mycotoxins. So we can have the basics for developing proto-cols or methods for effi ciently managing problems related to mycotoxins, as well as to understand their biological eff ects. The aim of the paper was to analyze how many students are familiar with the problem of mycotoxins in food. The research was carried out using anonymous questionnaire, which included questions about mycotoxins and mycotoxicosis. The surveyed students were selected randomly, and they had an equal chance of being selected for the sample. These were students of bachelor studies at the Insti-tute of Food Technology and Biochemistry, Faculty of Agriculture, University of Belgrade. The survey was educational for interviewed students and they showed that they were in a very high percentage informed with basic cha-racteristics of mycotoxins. As well as, they showed the best know afl atoxin characteristics. In addition, students showed relatively good knowledge of basic characteristics and other representatives of mycotoxins. The relatively good knowledge of the student’s questionnaire about mycotoxin features can be a great basis for further work and improvement.Key words: mycotoxins, mycotoxicosis, food, survey research

Emilia Oreščanin1, Ivana Perić1, Mirjana Pešić 2, Slađana Stanojević2

1 Student II year of the bachelor studies of the Faculty of Agriculture in Zemun, University of Belgrade, module: Microbiology of food; Institute of Food Technology and Biochemistry2 Faculty of Agriculture in Zemun, University of Belgrade, associate professor, Institute of Food Technology and Biochemistry

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Iz prirode u susret Nobelovoj nagradi

Ove godine je Nobelova nagrada za fi ziologiju i me-dicinu dodeljena Džejmsu Alisonu i Tasuku Honjo za otkrića na polju imunoterapije malignih bolesti. Usva-jajući strategiju koju organizam koristi u ograničavanju imunskog odgovora onda kada bi on postao štetan za domaćina, tumor onesposobljava ćelije imunskog sistema, čineći to upravo kroz dominaciju inhibitornih nad aktivacionim signalima. Početkom devedesetih godina ovi istraživači su nezavisno jedan od drugog otkrili postojanje molekula CTLA-4 (engl. cytotoxic T-lymphocyte–associated antigen 4) i PD1 (engl. pro-

grammed death 1) na T limfocitima (T Li) koji deluju kao kočnice imunskog odgovora. Otkriće inhibitornih receptora je dovelo do razvoja takozvanih blokatora imunskih “kontrolnih punktova” koji su ostvarili izve-sni učinak u lečenju pacijenata obolelih od melanoma, kancera pluća i bubrega i limfoma. Nažalost, iako im-presivno, ovo otkriće ima ozbiljna ograničenja. Može-mo li korekcijom ishrane ili suplementacijom simulirati ili poboljšati efekte opisanih terapija? Postoje brojne studije koje ukazuju da bi određeni konstituenti prisut-ni u hrani, začinima ili čajevima mogli u značajnoj meri uticati na ekspresiju molekula odgovornih za gašenje antitumorskog imunskog odgovora oslobađajući ga supresije. Tako je pokazano da fenoli, epigalokatehin 3-galat prisutan u zelenom čaju, kurkumin iz kurkume, alkaloid berberin iz divljeg šimšira i zlatne repe, vitamin E, vitamin D, ekstrakt crne američke maline, resveratrol prisutan u grožđu, mogu na različite načine slabiti in-hibitorne signale.

Jasno je da nutrijenti i aktivne supstance u hrani mogu doprineti eliminisanju imunosupresivne aktiv-nosti tumora, osnažiti antitumorski potencijal ćelija imunskog sistema i potencirati efekte specifi čnih tera-pija poput pomenutih inhibitora. Konačno, otkrića ova dva velika naučnika će pomoći da sa pravilnim razu-mevanjem koristimo ogromne resurse koje nam nudi priroda i koncipirajući ishranu i suplementaciju pomo-gnemo proces izlečenja.

Iz prirode u susret Nobelovoj nagradi

Sanja Mijatović, Danijela Maksimović-IvanićOdeljenje za imunologiju, Institut za biološka istraživanja “Siniša Stanković“, Univerzitet u Beogradu

Džejms Alison Tasuku Honjo

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Prikaz: Letnja škola, Beograd, 2018. godine

U Beogradu je od 20–24. avgusta 2018. godine održana prva Letnja škola u organizaciji Federation of European Nutrition Societies (FENS), Društva za ishranu Srbije i Farmaceutskog fakulteta Univerziteta u Beogradu. Na jednonedeljnom kursu pod nazivom “Translating sci-entifi c fi ndings into nutritional recommendations“ obra-đene su aktuelne teme iz oblasti ishrane sa ciljem da se olakša umrežavanje mladih istraživača i naučnika. Kroz 15 inspirativnih predavanja i 7 radionica polazni-cima su predstavljeni dostupni epidemiološki podaci i osnovni koraci u formiranju dijetarnih preporuka.

Školu je pohađalo 55 studenata master i doktor-skih studija i mladih istraživača iz 18 zemalja sa 3 kon-tinenta. Ishrana, kao primarna oblast istraživanja, bila je predmet brojnih diskusija sa različitih aspekata, kako između polaznika tako i između predavača i polaznika. Profesor Slađana Šobajić, glavni organizator, okupila je eksperte i vodeće stručnjake iz Srbije i Evrope koji su polaznicima približili način generisanja rezultata i meta-analiza u dijetarne preporuke.

Na ovaj način FENS letnja škola podržala je profe-sionalnu i ličnu izgradnju svih polaznika, u prijatnoj atmosferi koja je dovela do proširenja i ažuriranja po-stojećih znanja iz oblasti ishrane. Kroz interaktivni rad ostvareni su brojni kontakti između mladih istraživača koji predstavljaju sigurnu osnovu za formiranje mreža neophodnih za rad na interdisciplinarnim projektima i formiranja detaljnijih dijetarnih vodiča.

Obrađene teme uključile su: generisanje dokaza – od eksperimentalne nauke do humanih eksperimental-nih studija; dobru praksu u dijetarnim intervencijama; ishranu i javno zdravlje - mogućnost doprinosa prepo-rukama; Nordijsko iskustvo u formiranju regionalnih dijetarnih preporuka; prevođenje naučnih podataka u potrošačko orijentisane informacije; razvoj dijetarnih preporuka za različite populacione grupe.

Profesor Slađana Šobajićdekan Farmaceutskog fakulteta Univerziteta u Beogradu

Translating scientifi c fi ndings into nutritional recommendations

Letnja škola, Beograd, 2018. godine

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Intervention studies are considered to be the ‘gold standard’ for testing a hypothesis and represent th e most reliable assessment tool for establishing causal relationships between drug, device, diet and health and disease risks in human population. Intervention is any kind of manipulation made in subject’s life that could have biomedical consequences. An intervention study is basicaly a clinical trial. World Health Organi-zation gives defi nition of clinical trial as „any research study that prospectively assigns human participants or groups of humans to one or more health‐related inter-ventions to evaluate the eff ects on health outcomes“. Dietary intervention studies are conducted in exactly the same way as clinical trials involving pharmaceuti-cal drugs, but with fewer legislative requirements as di-etary intervention studies use food or food ingredients. Because of these diff erences several guidelines were developed specifi cally for dietary intervention studies, such as: • Best practices for food-based clinical trials,

Agriculture and Agri-Food Canada;• Guidelines for the design, conduct and reporting

of human intervention studies to evaluate the health benefi ts of foods (Queen’s University, Belfast, Northern Irland);

• EFSA guidelines for health claim substantiation for diff erent organs and systems.

In each clinical trial, including dietary interventions, two main goals should be achieved:• The rights, safety and well-being of trial subjects

have to be protected;• Trials should be scientifi cally sound.

Elements and steps in clinical trial preparation and conductiong important for the two main goals are: ethical considerations; adequate informing of partici-pants including information about risks and potential benefi ts; reporting of adverse eff ects; medical protec-tion; privacy protection and confi dentiality; defi nition of study hypothesis and objective; evaluation of the scientifi c evidence; selection of the adequate primary outcomes; defi nition of study population, exclusion and inclusion criteria, sample size; adequate random-ization process; choice of appropriate study design.According to the World Medical Association Declara-tion of Helsinki ethical principles for medical research involving human subjects should be implemeted in order to promote and ensure respect for all human subjects and to protect their health and rights. Also, every precaution must be taken to protect the priva-cy of research subjects and the confi dentiality of their personal information. Subjects can not be enrolled in the study before giving informed and (preferably) writ-ten consent.

In last decade there is a growing interest of research groups, scientifi c journals and overall scientifi c com-munity to register clinical trials because registration of all interventional trials is considered to be a scien-tifi c, ethical and moral responsibility. There are several clinical trial registers, such as EU Clinical Trial Register; International Clinical Trial Registry Platform.

Good practice for human dietary intervention studies

Slađana ŠobajićFaculty of Pharmacy, University of Belgrade, Serbia

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Dietary intake refers to the daily eating patterns of an individual, including specifi c foods and calories con-sumed and relative quantities, whereas nutrition status refers to the availability of nutrients and calories in the individual's diet compared to nutrition recommenda-tions for the individual's age group and overall health status. Measurement of dietary intake is essential to understand the role of diet in causing and prevent-ing chronic diseases, especially in non-communicable diseases (NCDs). Accordingly, choice of appropriate methods for assessment depends primarily on the purpose for which it is needed. Of these methods, typ-ical include prospective (food records/diaries and the duplicate portion method) and retrospective (24-hour dietary recalls, dietary history, food frequency/propen-sity questionnaires). Nationally representative, individ-ual-level surveys are carried out in many countries in order to examine and monitor the population’s dietary habits, and to bring good evidence for public health recommendations. Despite their inherent strengths, methodological instruments and techniques applied for such purposes are fraught with challenges and limitations. Consequently, accuracy of dietary intake measurement may be aff ected. Self-reported dietary intake is assessed by methods of real-time recording and methods of recall. Nevertheless, sources of error, which include the participants’ inability to fully and accurately recall their intakes as well as limitations in-herent in the food composition databases applied to

convert the reported food consumption to energy and nutrient intakes, may limit the validity of the generated information. The use of dietary biomarkers is recom-mended to overcome such errors, in order to capture intra-individual variability in intake; however, it has its own limitations. In addition to non-modifi able (individ-ual’s gender and age) and modifi able factors (tobacco smoking, alcohol consumption, medication, and phys-ical activity) that can aff ect the measurement, inter-actions between dietary components, the type and handling of the biological samples (e.g. conditions re-lated to blood drawing or urine collections and sample transport and storage), and the characteristics of the laboratory methods used (precision, detection limits, and inter-laboratory variations) may contribute to vari-ations in biomarker levels. Thus, the use of a biomark-er at the population level should be preceded by an assessment of its validity, reproducibility, ability to de-tect changes, and suitability for the population under study. Understanding potential constrains in assessing dietary intake and reducing errors of self-reported data is of great importance, particularly for research aimed at the level of population. Innovative technologies may lead to improvement of the dietary intake measure-ment, but evidence-based intake must remain crucial research outcome. In line with this, future research pri-orities have to be defi ned, dietary recommendations have to be refi ned and dietary interventions have to be applied.

Assessment of dietary intake at the population level – methods,

signifi cance, application

Dragana JovićInstitute of Public Health of Serbia, Belgrade, Serbia

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Researchers in the fi eld of nutrition have yet to establish clear criteria and guidelines for how best to study the eff ects of nutrients in humans, and then how to evalu-ate these fi ndings as constitutes of evidence-based nu-trition. Evidence based on clinical studies set specifi c barriers in the fi eld of nutrition. The nutritional epide-miologic study designs, namely ecologic, cross-section, case-control, cohort, and clinical trials, have been used to examine the relationship between dietary expo-sures to health and disease risk. Levels of evidence are assigned to clinical studies based on the methodolog-ical quality of their design, validity, and applicability to patient care. These decisions gives the ”grade (or strength) of recommendation“. The pyramid of associ-ations suggested that science evidence should be eval-uated with predefi ned hierarchical schema based on study type. In most evidence hierarchies current, well designed systematic reviews and meta-analyses are at the top of the pyramid. In research designs hierarchy results of randomized placebo-controlled trials (RCTs) considered the highest levels of evidence. RCTs involv-ing whole foods or diets are diffi cult to conduct, for the following reasons: lack of compliance, no true placebo group, and the impossibility of having a control group. Factors aff ecting the level of certainty of evidence (Blumberg, Nutr Rev 2010) in RCT studies are: control group (or period) with suffi ciently low intake, accuracy of intake assessment, minimal losses of sampling units, replication, adherence/compliance, optimization/con-trol of conutrient intakes and estimates of eff ect size are large. Prospective cohort studies provided critically important information for identifying diet-disease re-lations. In cohort design studies factors aff ecting the

level of certainty of evidence are: low intake control group, intake estimate validation, correct temporal sequence, dose-response relationship, replication/multiplicity of studies, low between-subject variance, biological plausibility, adequate control for conutrient intake, adequate control for other confounding factors, large eff ect size. The strongest nutrition recommenda-tion should be reserved for fi led in which observation-al studies and RCT results align. Example of alignment between observational studies and RCT are Mediter-ranean dietary patterns and risk for coronary heart disease. The clinical nutrition case study represents an important step in evidence based nutrition and the regular validation of clinical nutrition practice. An ad-vantage of study design was to provide direct evidence of a cause and eff ects relationship with ability to con-trol confounding eff ectively. A disadvantage of study design was that blinding was not always possible, eth-ical consideration, size, complexity, duration and cost and asses only one or two factors at time. Tools to help in reporting study evidence was CONSORT statement (RCT), TREND statement (non RCT), STROBE statement (observational), PRISMA (systematical reviews of trials). Nutrition research needs biomarkers for both, expo-sure and outcome. Nutrient such as vitamin D is ex-ample of nutrient with functional dose-response indi-cator of status. It is necessary to point that diffi culties are encountered in the assessment of dietary intake. An understanding of the strengths and weaknesses of nutritional epidemiologic and clinical studies and par-ticular dietary studies is essential for translating them to evidence based-public health practice and their in-terpretation for the purposes of setting public policy.

Clinical study and evidence

Danijela Ristic-MedicCentre of Research Excellence in Nutrition and Metabolism, Institute for Medical Research, University of Belgrade, Serbia

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Prikaz: Letnja škola, Beograd, 2018. godine

Population-level salt reduction is one of the most im-portant public health interventions with enormous public health gains. A “30% relative reduction in mean population intake of salt/sodium” is one of nine key targets for the reduction of chronic diseases, set by the World Health Organization (WHO) Global Action Plan for the Prevention and Control of Non-Commu-nicable Diseases 2013–2020. This shift from previous recommendations defi ned by absolute level (<5 g salt) to a relative reduction represents more feasible target for most populations. A gradual reduction in popula-tion sodium intake is likely to be more achievable for countries, hence the focus on percentage reduction is a more pragmatic approach. Current daily salt con-sumption is in a range of 8–12 g/day, and was higher than WHO recommended levels in almost all countries. Essential for reporting on this target and monitoring of the eff ectiveness of salt reduction interventions is a monitoring of salt intake. High-income countries already have established high quality systems for nu-trients intake surveillance. Other countries, with limit-ed resources, fi nd salt intake monitoring challenging. Measuring population sodium intake has to obtain a valid estimate of the range and frequency of salt dietary intake across the population, and must provide a valid estimate of mean population level intake in the rep-resentative population sample. Therefore, it is import-ant to select method relevant with research interests, profi le of the respondents and their environment, as well as with available resources. A number of diff erent methods of dietary sodium intake estimation are cur-rently available, including dietary and urinary assess-ments. Dietary assessment consists of food frequency questionnaire, 24-hour dietary recall, dietary records, weighed diet records (food consumption records), as well as dietary history assessment. Dietary assessment

methods require adequate food composition databas-es. Dietary assessment is very technically demanding process, which is often inaccurate, since the process of quantifi cation of the amount of sodium in diff erent foods cannot always be estimated accurately. The same is true for discretionary salt intake, which results in un-der-reporting of the amounts of salt consumed. Nev-ertheless, the aforementioned method makes possible recognition of essential origins of sodium in diet. This fact is crucial for the establishment of the potent and suitable mechanisms for salt intake reduction. Urinary assessment by 24h urine collections is widely regard-ed as “gold standard” and the most precise method to measure individual’s mean intake, but also gives valid refl ection of the population mean sodium intake. It is expected that approximately 90% of ingested sodium is excreted in urine over 24h period. Completeness of 24h urine sample is the prerequisite for accurate salt intake assessment, often limited by lack of suitable and standardized methodology to identify incomplete samples. This method does not provide information on dietary sources of sodium intake. Spot urine (sin-gle spot urine or repeat spot urine samples) is increas-ingly used as a potentially convenient and aff ordable alternative, and represents sodium intake over a short period (few hours only). It is not suitable method to estimate mean sodium intake of the population. Uri-nary methods require adequate laboratory equipment, too. It is of outmost importance to conclude that, no matter which method is chosen, it has to provide val-id assessment of population sodium intake and to make possible consistent monitoring over given time period. However, it is still a gargantuan challenge for researchers to establish most effi cient dietary assess-ment method that would make salt intake monitoring possible, especially in low-income countries.

Approaches to monitor population dietary sodium/salt intake

Milka PopovićFaculty of Medicine, University of Novi Sad, SerbiaInstitute of Public Health of Vojvodina, Novi Sad, Serbia

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IN MEMORIAM

PROFESORKA PETRICA RUŽIĆ

(1939–2018)

Profesorka Petrica Ružić nas je napustila 06. oktobra 2018. godine. Rođena je 1939. godine u Imotskom, gde je završila osnovnu školu i gimnaziju. Diplomirala je 1966. godine na Tehnološkom odseku Poljoprivred-nog fakulteta Univerziteta u Beogradu, a titulu doktora nauka stekla na Veterinarskom fakultetu Univerziteta u Beogradu. U svojoj karijeri objavila je preko 150 nauč-nih i stručnih radova, a svoje rezultate prezentovala na brojnim domaćim i svetskim kongresima.

P ored svog osnovnog profesionalnog opredeljenja da se bavi obrazovanjem stručnjaka iz oblasti ishrane na Visokoj zdravstvenoj školi strukovnih studija u Ze-munu (raniji naziv Viša medicinska škola), na odseku Zdravstveni dijetetičar-nutricionista, profesorka Petrica Ružić veliki deo svog profesionalnog i životnog anga-žmana posvetila je Društvu za ishranu Srbije.

Profesorka Ružić, od 1978. godine kao član Jugo-slovenskog društva za ishranu (današnje Društvo za ishranu Srbije), daje nemerljiv doprinos učestvujući u svim aktivnostima, posebno u organizaciji kongresa i naučno-stručnih seminara. „Susreti nutricionista“ su bili nezamislivi bez energije i ličnog pečata profesorke Ru-žić, bilo kao predavača ili glavnog organizatora, a do danas ih je bilo 24.

Društvo za ishranu Srbije je bilo deo života profe-sorke Ružić. Prva asocijacija u radu Društva je bila ta njena posvećenost i ljubav, što smo svi poštovali i ce-

nili. Kao rezultat toga na izbornoj skupštini Društva za ishranu Srbije 1993. godine profesorka Ružić izabrana je za generalnog sekretara, a od 1997. do 2008. godi-ne bila je predsednik Društva za ishranu Srbije. Ceneći ukupni doprinos profesorke Ružić, sama po sebi se na-metnula činjenica da bude izabrana za počasnu pred-sednicu Društva.

Bilo kao član Jugoslovenskog društva za ishranu ili Društva za ishranu Srbije, aktivno je doprinosila ugledu i povezivanju sa međunarodnim naučnim asocijacija-ma, posebno International Union on Nutrition Sciencies i Federation of European Nutrition Societies. Zahvaljuju-ći njenom pregalaštvu i naučnoj reputaciji Društvo za ishranu Srbije je i u najtežim trenucima održavalo me-đunarodni kontinuitet kao član Federation of European Nutrition Societies.

Profesorka Petrica Ružić je držala do tradicije, ne-govala doprinos radu svih članova Društva za ishranu Srbije, brižljivo, sve do svoje smrti sakupljala i čuvala arhivu Društva.

Na isti način ophodila se i radila na kontinuitetu i održanju kvaliteta časopisa Društva za ishranu Srbije – Hrana i ishrana, čiji je prvi broj publikovan 1960. go-dine. Svojim posebnim smislom za okupljanje prove-renih stručnjaka iz zemlje i inostranstva, kao i mladih naučnih saradnika, profesorka Petrica Ružić obezbedila je redovnost publikovanja časopisa. Funkciju glavnog urednika časopisa obavljala je u periodu 2008–2016. godine, a od 2016–2018. godine bila je zamenica glavnog urednika časopisa.

Sve ovo za profesorku Ružić nije bila obaveza, po-sao, već ljubav ka ostvarenju viših ciljeva, podizanju prosvećenosti stanovništva Srbije kada su u pitanju svi aspekti ishrane, od zdravog života do visokih naučnih standarda. Radeći stručno i ozbiljno, profesorka Petrica Ružić je tiho i nenametljivo prenosila svoju energiju i znanje na sve nas, posebno na mlađe saradnike koji danas jesu taj kontinuitet i okosnica Društva za ishranu Srbije.

Time je profesorka Petrica Ružić ostvarila i svoj i naš san, da smrt nije kraj, da pečat koji ostane posle ovoze-maljskog, nastavlja da živi kroz taj rad.

S velikim poštovanjem i zahvalnošću,Profesor Ljiljana Trajković-PavlovićPredsednik Društva za ishranu Srbije

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Uputstvo autorima / Instructions to Authors

Saradnici se mole da detaljno i pažljivo pročitaju pred-ložena uputstva za pripremu radova pre predaje ruko-pisa za štampanje. Izuzetno je važno da saradnici/autori pripreme radove prema ustanovljenim principima jer je to izuzetno značajno za klasifi kaciju naučnih časopisa.

“Hrana i ishrana” je časopis Društva za ishranu Srbije osnovanog 1956. godine, u kome se objavljuju radovi članova Društva za ishranu Srbije i članova društava drugih srodnih struka. Objavljuju se originalni radovi, saopštenja, pregledni radovi, izveštaji sa kongresa i stručnih sastanaka, stručne vesti, prilozi, prikazi knjiga, pisma uredništvu i dopisi „U spomen”. Uz rukopis član-ka treba priložiti potvrdu s potpisima svih autora da članak nije objavljen, kao i da nije u toku razmatranje za objavljivanje. Prispeli članak Uređivački odbor upućuje recenzentima radi stručne recenzije (2 recenzenta). Ako recenzenti predlože izmene ili dopune, kopija recenzije, bez imena recenzenata, dostavlja se autoru radi njego-ve konačne odluke. Radovi se ne honorišu. Rukopisi se ne vraćaju.

Neophodno je da se celokupni materijal (rad) pošalje i elektronskom poštom glavnom uredniku na e-mail: [email protected].

Opšta pravila

Rukopis članka i svi prilozi treba da budu jasni i napi-sani na engleskom ili srpskom jeziku, a za izradu rada koristiti isključivo tekst-procesor Microsoft Word. Ruko-pis treba da je pripremljen na formatu A4. Sve margine treba da budu 2,5 cm. Stranice je potrebno numerisati. Koristiti tip slova (font) Times New Roman, veličine 12. Radove treba kucati proredom 1,5.

U rukopisu članka obeležiti mesta za slike, sheme, gra-fi kone, tabele i ne ostavljati prazan prostor u tekstu. Literaturni podaci u tekstu se označavaju arapskih bro-jevima u zagradama redosledom kojim se pojavljuju u tekstu, na primer [1,2].

Skraćenice upotrebljavati samo izuzetno, i to u sluča-jevima kada se navode veoma duga imena hemijskih supstancija ili veoma poznate skraćenice (npr., DNK) ali je preporučljivo dati objašnjenje.

Merne jedinice: dužina, visina, težina i zapremina ozna-čavaju se u metričkim jedinicama (metar – m; kilogram – kg; litar -l) ili podjedinice. Temperatura se izražava u stepenima Celzijusa (0C), koncentracije u molima; ure-đaji se označavaju trgovačkim nazivima, a naziv i mesto

Contributors are strongly encouraged to read the in-structions carefully before preparing the manuscript for submission and to check the manuscript for com-pliance with the terms before submitting it for publica-tion. It is essential for the authors to prepare the manu-scripts according to the established specifi cations.

“Food and Nutrition” is the Journal of the Serbian Nu-trition Society founded in 1956. Articles supplied by the members of the Serbian Nutrition Society are pub-lished, as well as articles by members of other associa-tions in the fi eld of Public Health Nutrition (PHN) and related fi elds. The Journal publishes original articles, communications, case reports, review articles, congress and scientifi c meeting reports, professional news, book reviews, obituaries, as well as comments and letters to the Editorial Board in relation to the published papers.

The manuscript should be accompanied by signed confi rmation of all contributors that the paper has not been previously published and not submitted for pub-lication elsewhere.

The papers are forwarded to the expert evaluation and an anonymous copy of the evaluation containing sug-gested changes is mailed to the authors for their fi nal consent.

The authors are not rewarded and the manuscripts are not returned.

The manuscripts should be forwarded to the following address: Uredništvo “Hrane i ishrane” Savska 9/II 11000 Beograd, Serbia in duplicate. All papers also have to be submitted to the Editor-in-Chief as an electronic ver-sion: [email protected].

General demands on manuscripts

Manuscripts should be written in clear concise Serbi-an or English language, using MS WinWord program in short and clear sentences. Manuscripts should be on A4 format, all margins 2.5 cm, pages numbered. Rec-ommended font is Times New Roman 12. Papers should be typed 1.5 spaced. The author(s) should indicate in the text where fi gures and tables fi t in. All references should be numbered in sequence as they appear in the text and indicated with Arabic numbers in parentheses – example [1,2].Abbreviations should be avoided, only to be used if ap-propriate, for very long names of chemical compounds, or as well-known abbreviations (such as DNA).Units of measure: Length, height, weight and volume should be expressed in metric units (meter – m, kilo-

INSTRUCTIONS TO AUTHORSUPUTSTVO AUTORIMA

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Uputstvo autorima / Instructions to Authors

proizvođača su u zagradama. Svi rezultati kliničkih i bi-ohemijskih istraživanja izražavaju se u jedinicama me-đunarodnog sistema mera – SI.

Autorstvo. Svi autori treba da budu odgovorni za autor-stvo. Svaki autor treba da aktivno učestvuje u pisanju članka da bi bio odgovoran za rad u celosti. Autorstvo se bazira samo na višeslojnom spoju koncepcije rada, dobijenih rezultata/analize kao i interpretacije dobije-nih rezultata. Konačna verzija nakon stručne obrade priprema se za štampu.

Sastav/struktura rukopisa

Rukopisi treba da sadrže sledeća poglavlja: naslov, autore, ustanove, kratak sadržaj na srpskom jeziku sa ključnim rečima, uvod, eksperimentalni deo (materi-jal, metode), rezultate, diskusiju, zahvalnica, literaturu i kratak sadržaj (Abstract) na engleskom jeziku tako-đe sa ključnim rečima. Pregledni članak sadrži sledeća poglavlja: uvod, pregled slučaja, zaključak i literaturu. Pregledni članak mora da sadrži u literaturi navedena najmanje 4 autocitata autora.

Sva poglavlja se pišu velikim slovima koristeći oznaku „bold”.

Naslov rada. Na posebnoj stranici navesti naslov članka, bez skraćenica, velikim slovima, a ispod naslova navesti imena autora indeksirana brojkama koje odgovaraju onima pod kojima se nalaze nazivi i adrese ustanova u kojima autori rade. Pri dnu ove stranice otkucati ime i prezime autora odgovornog za dalji kontakt, punu adresu, broj telefona, faksa ili e-mail adresu.

Kratak sadržaj. Uz originalni rad, saopštenje ili pregled iz literature treba priložiti na posebnoj stranici kratak sadržaj, koji sadrži naslov rada, prezimena, inicijale ime-na autora, nazive ustanova i mesta (iz kojih su autori), zatim sadržaj članaka u ne više od 200 do 300 reči. Za naslove kratkog sadržaja koriste se oznake italic i bold a za tekst sadržaja samo italic.

Kratak sadržaj ne treba da sadrži literaturne podatke. U njemu se navode, bez opisivanja, bitne činjenice, kra-tak prikaz problema i osnovni zaključak. U originalnom članku kratak sadržaj treba da sadrži sledeća poglavlja: uvod, cilj, metod rada i zaključak. U saopštenju/pregle-du kratak sadržaj sadrži sledeća poglavlja: uvod, pre-gled slučaja i zaključak.

Radovi na engleskom jeziku moraju da sadrže Kratak sadržaj i Ključne reči na srpskom, sa svim navedenim elementima.

Ključne reči. Na kraju apstrakta/kratkog sadržaja dodaju se ključne reči ne više od 8, koje su bitne za brzu identi-fi kaciju i klasifi kaciju sadržaja članka.

Uvod rada se piše jasno, sažeto, uz navođenje suštine

gram – kg, and liter -l) or their sub-units. Temperature should be given in Celsius degrees (0C). Concentrations are given in moles, proprietary names of instruments with factory name and place of manufacture in paren-thesis. All results of clinical and biochemical measure-ments should be expressed in the metric system ac-cording to the International System of Units – SI.

Authorship

All individuals listed as authors should be qualifi ed for authorship. Every author should have participated suf-fi ciently in writing the article in order to take responsi-bility for the whole article and results presented in the text. Authorship is based only on crucial contribution to the article conception, obtaining of results or analysis and interpretation of results, and fi nal revision of the manuscript being prepared for publication.

Structure of the manuscripts

The manuscript has to be arranged as follows: The ti-tle, Authors, Institutions, Abstract, Introduction, Exper-imental part, Results, Discussion, Acknowledgements, and References.

Review articles include Introduction, corresponding section heading, Conclusions and References. The re-view article may be published only by authors who may cite at least four auto-citations (references in which they are either authors or co-authors).

Title page. The title should be short, clear and without abbreviations, typed on the separate sheet. Names and family names of authors should be written under the title, as well as full names of their institutions indicated by corresponding Arabic numbers if there is more than one institution. The address of corresponding author, with the telephone, fax number and e-mail address should be added at the bottom of this page.

Abstract. Original articles, communications, case re-ports, review articles and book reviews; the abstract not exceeding 200–300 words should be typed on a separate sheet of paper. (Srp. Arh) The abstract should not contain any references.

Key words. Key words – four to eight, relevant for rap-id identifi cation should be typed below the abstract in English. In original articles the abstract should have the following structure: introduction, objective, meth-od, results and conclusion. In case reports the abstract should consist of the following: introduction, case out-line and conclusion.

Introduction should be clear, concise, pointing to the essence of the problem and with the purpose of the study. References related to the problem should be cit-ed.

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Uputstvo autorima / Instructions to Authors

materije i radova koji su u vezi sa problematikom, kao i ciljem istraživanja.

Eksperimentalni deo opisuje materijale i metode, bez posebnih detalja ako su već opisani u literaturi (navesti literaturni podatak), a detaljno opisati ako je metodolo-gija nova ili modifi kovana. Potrebno je navesti metode izračunavanja parametara i statističke analize rezultata. Ukoliko se upotrebljavaju skraćenice, pri prvom navo-đenju u tekstu treba napisati i njihov pun naziv.

Rezultate prikazati jasno i pregledno, sa odgovaraju-ćom statističkom obradom.

Diskusija obuhvata interpretaciju dobijenih rezultata i njihovo upoređenje sa literaturnim podacima. Rezultati i diskusija mogu se objediniti.

Zaključak se daje na kraju teksta jasno i koncizno kao rezultat istraživanja u vidu opšteg zaključka ili više po-jedinačnih označenih numerički (arapskim brojevima).

Originalni članci sadrže sledeća poglavlja: uvod, cilj rada, metod, rezultate, diskusiju, zaključak i literaturu.

Kratak sadržaj (Abstract) na engleskom jeziku treba da bude otkucan na posebnoj stranici i treba da sadrži sve elemente kao i kratak sadržaj na srpskom jeziku.

Obim rukopisa

Ceo tekst rukopisa: naslovna strana, kratak sadržaj, uvod, eksperimentalni deo (materijal, metode), rezul-tati, diskusija, zahvalnica, literatura uključujući legende (tabele, fotografi je, grafi kone, sheme itd.) mogu imati 5.000 reči za originalne članke; za saopštenja i pregled-ne radove 2.000 reči; za stručne izveštaje 1.500 reči a za ostale preglede 1.000 reči.

Broj tabela, slika, shema, crteža, grafi kona (zajedno) može biti najviše do polovine broja kucanih stranica rukopisa.

Tabele, slike, crteži, sheme, grafi koni

Svaka tabela se kuca na posebnoj stranici proredom 1,5, uključujući naslov, zaglavlja kolona i redova. Tabele se označavaju arapskim brojevima po redosledu navo-đenja u tekstu. Naslov tabele prikazuje sadržaj tabele. Upotrebu skraćenica u tabeli obavezno objasniti u le-gendi tabele. Fotografi je moraju biti isključivo crno-be-le, oštrih kontura. Tekst (opis) slike kuca se na poseb-nom listu hartije. Crteže (sheme i grafi kone) priložiti na posebnom listu (sa precizno unetim vrednostima na apscisi i ordinati).

Zahvalnica se kuca na kraju teksta a sadrži podatke ili izraze zahvalnosti autora na pomoći: naučnoj, stručnoj, tehničkoj ili fi nansijskoj.

The Experimental part should include description of materials (subjects) and methods used. If methods are widely known and described in the literature, only ref-erence(s) should be cited. New or modifi ed methodol-ogies should be fully described. Methods used for pa-rameters calculation and statistical analyses should be indicated. All abbreviations have to be explained in the manuscript when used for the fi rst time.

Results should be clear and precise, with corresponding statistical analysis.

Discussion encompasses interpretation of the results and their comparison to the references data. The last two parts can be given together as Results and Discus-sion.

At the end of this part conclusions obtained from the research should be reported.

All section headings should be in capital letters using bold lettering.

Original articles shall have the following section head-ings: Introduction, Objective, Method, Results, Discus-sion, Conclusion and References.

Case reports should consist of introduction, case out-line, discussion, references.

Length of the manuscripts

The entire text of the manuscript: title page, abstract, the whole text, list of references and captions to fi g-ures and tables should have maximum 5 000 words for original articles, 2 000 words for communications and review articles, 1 500 words for case reports and up to 1000 words in the section ‘’other’’.

The total number of fi gures and tables should not ex-ceed the half of the number of typed pages of the man-uscript.

Tables, fi gures (graphs, charts, photographs, and

illustrations)

Tables are typed on a separate sheet of paper 1,5 spaced, including title, subtitle, headings of lines and columns. They must be identifi ed by Arabic number in order or appearance with a shot description of the title, abbreviation should be explained. Photographs should be explained. Photographs should be black and white and good sharpness. First author’s name, title of the manuscript, number of the illustration and arrow indi-cating the top of the fi gure are given on the back with lead pencil. The legends are given on separate sheet. Drawings (schematic drawing and graphs) are supplied on separate sheets with lead precise identifi cation of abscissa and ordinate.

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Uputstvo autorima / Instructions to Authors

Literatura

Literatura se kuca na posebnim stranicama jednostru-kim proredom, a dvostrukim između pojedinih refe-renci, s rednim arapskim brojevima prema redosledu navođenja u tekstu.

Broj referenci u literaturi ne prelazi 30, osim za pregled iz literature gde je prihvatljivo i do 50 jedinica. Referen-ce se navode po ugledu na Vancouver sistem, koji se zasniva na principima National Library of Medicine i In-dex Medicus (Srp Arh Celok Lek). Citiranje literature uz poštovanje određenih standarda izuzetno je značajno za klasifi kaciju naučnih časopisa.

Za članke u časopisu:

1. Josselson J, Kyser BA, Weir MR, Sadler JH. Hepatitis B surface antigenemia in a chronic hemodialysis pro-gram: lack of infl uence on morbidity and mortality. Am J Kidney Dis 1987; 9(6):456–61.

(U zagradama je naveden broj sveske, a ispred je broj volumena). Navode se imena najviše šest autora; ako ih je više, iza šestog se dodaje: i sar.

Knjige:

2. Weinstein L, Swartz MN. Pathologic properties of in-vading microorganisms. Philadelphia: Saunders; 1974; 457–72.

Poglavlja u knjigama:

3. Clayton D, Gill C. Covariate measurement errors in nutritional epidemiology: eff ects and remedies. In: Mar-getts BM, Nelson M, eds. Design Concepts in Nutritional Epidemiology. Oxford: Oxford University Press, second edition 1997: 87–106.

Za članke sa kongresa ili sastanaka:

4. Marković P, Živković L. Uticaj zračenja na pojavu reci-diva. Zbornik radova “II kongres lekara”, Vrnjačka Banja 1975;315–6.

Stručna izdanja:

5. Medical Assessment of Nutritional Status. WHO. Tech Rep Ser 1993:298.

Javni/državni izveštaji (zakoni, pravilnici, direktive, izja-ve):

6. Pravilnik o normativu društvene ishrane dece u ustanovama za decu. Službeni glasnik RS, Beograd 1994;50:1643–9.

Citat internet stranice:

7. Complementary/Integrative Medicine [Internet]. Ho-uston: University of Texas, M. D. Anderson Cancer Cen-ter; c2007 [cited 2007 Feb 21]. Available from: http://www.mdanderson.org/departments/CIMER/.

Acknowledgements (sources of funding, confl ict of in-terest declaration, and authorship responsibilities): this should be included at the end of the text.

References

References should be supplied on a separate sheet, sin-gle spaced, with double space between each reference, Arabic numbers indicating the sequence of appear-ance.

The number of references should not exceed 30, except in literature reviews with maximum 50 is acceptable. References are cited according to the so-called Vancou-ver style, based on formats being used by the National Library of Medicine and Index Medicus (Srp Arh Celok Lek). In citation of references the defi ned standards should be strictly followed because it is the essential factor of indexing and classifi cation of scientifi c jour-nals.The following rules should be applied:

Journals:1. Josselson J, Kyser BA, Weir MR, Sadler JH. Hepatitis B surface antigenemia in a chronic hemodialysis pro-gram: lack of infl uence on morbidity and mortality. Am J Kidney Dis 1987; 9(6):456–61. (The number of the vol-ume is given in parentheses, the preceding number in-dicating the issue). Only up to six names of the authors are quoted, if more than six “et al “ is added.

Books and contributions to books:2. Weinstein L, Swartz MN. Pathologic properties of in-vading microorganisms. Philadelphia: Saunders, 1974; 457–72.

Book chapter:3. Clayton D, Gill C. Covariate measurement errors in nutritional epidemiology: eff ects and remedies. In: Mar-getts BM, Nelson M, eds. Design Concepts in Nutritional Epidemiology. Oxford: Oxford University Press, second edition 1997: 87–106.

Congress articles:4. Marković P, Živković L. Uticaj zračenja na pojavu reci-diva. Zbornik radova “II kongres lekara”, Vrnjačka Banja 1975; 315–6.

Other:5. Medical Assessment of Nutritional Status. WHO. Tech. Rep. Ser. 1993:298.

Legislation:6. Pravilnik o normativu društvene ishrane dece u usta-novama za decu. Službeni glasnik RS, Beograd 1994; 50: 1643–9.

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Citat internet stranice sa autorima:

8. Hooper JF. Psychiatry & the Law: Forensic Psychiatric Resource Page [Internet]. Tuscaloosa (AL): University of Alabama, Department of Psychiatry and Neurology; 1999 Jan 1 [updated 2006 Jul 8; cited 2007 Feb 23]. Ava-ilable from: http://bama.ua.edu/~jhooper/.

Standard citation of links:7. Complementary/Integrative Medicine [Internet]. Houston: University of Texas, M. D. Anderson Can-cer Center; c2007 [cited 2007 Feb 21]. Available from: http://www.mdanderson.org/departments/CIMER/.

Links with author(s):8. Hooper JF. Psychiatry & the Law: Forensic Psychiat-ric Resource Page [Internet]. Tuscaloosa (AL): University of Alabama, Department of Psychiatry and Neurology; 1999 Jan 1 [updated 2006 Jul 8; cited 2007 Feb 23]. Available from: http://bama.ua.edu/~jhooper/.

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CIP – Каталогизација у публикацији Народна библиотека Србије, Београд

613.2

HRANA i ishrana = Food and nutrition = Пища и питание = Les aliments et l’alimentation = Nährung und Ernährung : časopis društva za ishranu Srbije = the Journal of Serbian nutrition society / glavni i odgovorni urednik Bato Korać. – God. 1, br. 1 (1960)– . – Beograd : Društvo za ishranu Srbije, 1960– (Beograd : Ton Plus). – 30 cm

Dva puta godišnje. – Tekst na srp. i engl. jeziku. – Drugo izdanje na drugom medijumu: Hrana i ishrana (Online) = ISSN 2560-452X

ISSN 0018-6872 = Hrana i ishrana

COBISS.SR-ID 3531010

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