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FOOD AND NUTRITION ПИЩА И ПИТАНИЕ LES ALIMENTS ET L’ALIMENTATION NAHRUNG UND ERNÄHRUNG

VOL. 56 BEOGRAD 2015. BROJ 2

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Marina Peruničić Lučić, Zorica Basić, Brižita Đorđević, Bojana Vidović• Determination of ethanol in dietetic supplements syrups type .................. 27

Bojan Čalija, Jelena Đuriš, Vladimir Dobričić, Bojana Vidović, Jela Milić, Slađana Šobajić• Monokomponentni dijetetski suplementi folne kiseline na tržištu Republike

Srbije – farmaceutsko-tehnološka ispitivanja i karakteristike ...................... 31

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Nada Pavičić, Ivan Velikinac, Tamara Miladinović• Identifikacija karakterističnih antocijanina u proizvodima dobijenim iz ploda

aronije tehnikom visokoefikasne tečne hromatografije (HPLC) ................. 37

Jelena Golijan, Мilovan Veličković• Nutritivni sastav organski i konvencionalno proizvedenih namirnica............ 43

Miloš Milošev, Ivana Đuričić, Dušan Mitić• Effects of additional fitness program on body composition and general

motor skills in overweight children of school age ..................................47

Obaveštenja o skupovima / Featured Meetings .................................................... 52

Uputstvo autorima / Instruction to Authors ............................................................ 53

VOL. 56 BEOGRAD, 2015. BROJ 2

UDK:613.2 CODENHRIS-A ISSN 0018-68727

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The Journal of Serbian Nutrition Society “Food and Nutrition” is a scientific-technical publication with basic policy to publish origi-nal scientific papers, communications, pro-fessional papers and literature reviews in the field of nutrition, food technology, agriculture, and related disciplines. Book reviews, congres reports, current news in the fields of food and nutrition, information on scientific and profes-sional meetings, and letters to the editor are also published.

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DRUŠTVO ZA ISHRANU SRBIJE, ČASOPIS ˝HRANA I ISHRANA˝ SERBIAN NUTRITION S0CIETY, JOURNAL ˝FOOD AND NUTRITION˝

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

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 27-30, 2015. Marina Peruničić Lučić et al.: Determination of ethanol in dietetic supplements syrups type 27

Determination of ethanol in dietetic supplements syrups type

AbstractDietary supplements are intended to supplement normal nutrition

and they are concentrated sources of vitamins, minerals and other substances with nutritional and physiological effects. Because of large use of dietary supplements, which often contain ethanol, we have to educate health workers, as well as consumers in this area, and hope that the use of dietary supplements will become safe, controlled and rational.

Ethanol is used for extraction, and it is also important as a stabilizer in a significant number of dietary products in the form of syrup. Because of that, a lot of dietary products contain ethanol, and often it is not declared. One more problem is the fact that users who read the declaration cannot conclude that ethanol is present in these products. Also, some of these syrups can be ordered through the internet without information of percentage of ethanol, which is worrying. Besides that, some syrup contains concentrations of ethanol in the amount that is the same as is in spirit drinks. We used liquid chromatography with a refractometer detector and carbohydrate column, temperature 90◦C, for the detection of ethanol in a dietary supplements syrup-type between 2007 to 2016. Then we ordered concentration of ethanol by external standard method. Content of ethanol was from <0,05% to 28,35%. We analyzed 76 syrups and 67% of them did not have ethanol on a declaration. 8% of syrups with ethanol declaration did not have the same amount of ethanol as remarked on the declaration. Also, 66% of all tested syrups had alcohol higher than 1,2% [12].

Syrups are traditionally used in our country, and often used in combination with some drugs, and that can lead to different adverse reaction.

Keywords: ethanol, HPLC, supplements, declaration

Marina Peruničić Lučić1*,Zorica Basić1,Brižita Đorđević2,Bojana Vidović2

1 Medical Military Academy, Crnotravska 17 , 11000 Belgrade, Serbia2Faculty of Pharmacy - University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia

Rad primljen: 04.06.2015.

Corresponding author:Marina Peruničić LučićMedical Military Academy 11000 Belgrade, Serbiae-mail: [email protected]. 063/85-86-872

UDK: 615.451.2.074:547.262

INTRODUCTION

Making spirits dates back 7000 years ago–from Asian nations (China), and then Egypt, Ancient Greece, the Roman Empire. Ethanol is used as a solvent in the pharmaceutical and cosmetic preparation (perfumes, aftershave lotions, water for rinsing the mouth…), in the industry, as well as an antidote for methanol and ethylene glycol poisoning. Alcoholic drinks have been produced and consumed by human for thousand years and have played an important role in religion, supplying nutrition and energy; providing medicinal, antiseptic and analgesic benefits; increasing the pleasure of eating; providing pharmacological benefits and generally enhancing the quality and pleasures of life [7]. Term “alcohol” was created by Paracelsus, a doctor and chemist (1493 - 1541), by using the Arabic word which means very fine powder (Walter P. 1982). Ethanol can be used as an extraction solvent in herbal pharmaceutical products. In this case, the use of ethanol is necessary for extraction of some constituents that are important for efficacy. The adverse reactions to alcohol are individual so it can be very different from person to person.

Many factors are responsible for the adverse effects of ethanol, such as age, gender, nutritional status, psychological state, and especially the initial amounts of alcohol. It should be pointed out that ethanol’s effects on the immune system are rather complex. Acute exposure to ethanol in vivo suppresses various inflammatory responses (e.g., leukocyte recruitment and endothelial cell activation) (Saeed et al. 2004) [15]. Allergy symptoms include palpitations, nasal congestion, headache, low blood pressure and abdominal pain. Because of all these symptoms it is very important to detect and warn about the content of ethanol in the products [4]. A moderate dose of alcohol had no effect on motor performance [1], but small amounts of alcohol can potentiate the effects of many of drugs the resulting hazard to patients is likely to be considerable. Using alcohol would indicate significant depression, with an inability to perform complicated tasks such as driving or operating machinery [5].

We used liquid chromatography for determina-tion of ethanol and retain analyze molecules on the carbohydrate column based on differences in the exchange equilibrium between the various sample


ions and the effluent ion [6]. In relation to retention time, and compared pick area of the sample to area of standards, we can find out the concentration of ethanol. Detection is based on the change of the re-fractive index. It is possible to measure differences in index of refraction to 1 part in 106 or even better. High sensitivity depends on the mobile phase and the temperature [2,14]. We validated this method in our laboratory. Selectivity factor (α), which is a measure for separation of the two components and it is believed that the α >> 1 achieved a satisfactory separation of the tested components [3].

The WHO (World Health Organization) proposes to limit the ethanol amount in OTC (over-the-coun-ter) products to less than 0,5% for children less than 6 years old, less than 5% for children 6-12 years old and less than 10% for children over 12 years. However, these limits do not take into consideration the actual dose given [11]. The FDA (Food and Drug Administration) also recommends not including ethanol in medicinal products intended for use in children. But if necessary, the amount of ethanol in OTC liquid preparations should not be more than 5% ethanol [10,13]. In Serbia we have only one limit for declaration of ethanol up to 1,2 % [12].

MATERIAL AND METHODS

Gas chromatography (GC) and high-perfor-mance liquid chromatography (HPLC) are common techniques to identify and quantify components of ethanol. Infrared spectroscopy (IR) is used for quality assurance of ethanol. Many extensive re-searches for ethanol analysis with HPLC have done (Sen et al., 1995, Yarita et al., 2002, Alcázar et al., 2006) [9]. Our choice was high performance liquid chromatography.

We analyzed 76 syrups and 69,74 % of them are herbal mixtures; 14,47% are vitamin type; 5,26 % marshmallow (Althaea radix); 3,95 % are primrose (Primula veris); 2,63 % ivy (Hederae helicis folium); 2,63 % plantain (Plantago lanceolata); 1,32 % medical type.

Calibrations of ethanol were done in five points at concentration of 0.05%; 0.1%; 0.5%; 1.0% and 2.0%. The working solution was 10% ethanol. Absolute ethanol needs to be rapidly dissolved in a small amount of water to avoid evaporation. Solutions can be shaken, but not heated to improve solubility. The correlation coefficient R2> 0,998, slice b is less than 2%, and that confirms the good linearity of our calibration curve.

The column temperature was 90◦C, a flow of a mobile phase was 1ml/min. The mobile phase is a pure deionized water which is also an advantage of this method because water is available, inexpensive and does not adversely affect health, unlike many organic solvents. Standard solutions were filtered and injected. The retention time of ethanol was at about the 12,6 minute. Every standard injected three times at least. We solved samples with water, then filtered and injected into device two times at least.

HPLC liquid chromatography system includes: injector (loop 20 ul), pump (Waters 600E System Controller Millipore, Millipore manufacturer 4883 Waters Chromatography Division USA), pre-column (filter 0, 2 to 0.5 um), column (CH Polyspher CA, type SC1011, size: 8,0mm x 300mmL, Machery Nagel), column heater (model: SFD, factory number 95030, manufacturer of Germany), refract metric detector (Waters 410, Millipore Corporation manufacturer of Waters, Chromatography USA, type M410); Standard of ethanol (99.9% purity Merck KgaA S4271, Darmstadt, Germany); filter for sample and standard ( CA 0.45 um Filter- lab).

RESULTS AND DISCUSSION

We analyzed 7 different types of syrups (Figure 1 and Figure 2). Content of ethanol was from <0,05% to 28,35%. We analyzed 76 syrups and 67% of them did not have ethanol on a declaration. 8% of syrups with ethanol declaration, did not have an amount of ethanol according to their declaration. Also 66% of all tested syrups had alcohol higher than 1,2% [12]. The European Union (EU) requires the declaration of the alcohol content of beverages containing more than 1,2% volume of alcohol. This limit is inadequate for many groups wishing to avoid alcohol: children, people of certain religious faiths, violence users, etc. [8].

The concentration of ethanol, in one herbal syrup, made by the same manufacturer, was analyzed between the 7-year long period 2007-2014., are presented in Table 1. It has been found that different concentrations of ethanol were present in different batches of the syrup despite using the same technology as illustrated in Figure 3 and Figure 4. According to this for each batch of ethanol we had to determine the concentration of ethanol, and declare it. The information given on the label is primarily allowed to a consumer. Labeling should allow consumer to judge a product’s contents, quality use characteristics and nutritional value [8].

Table 1. The concentration of ethanol in the one herbal syrup between 7 yearsYear Concentration of ethanol ( %)

2007 2.52008 0.62009 2.02012 1.272013 0.152014 0.95


Our results show that the concentration of ethanol in medical and Hedera helicis syrup type was not higher than 1,2 %. Also, we can say that herbal mix, Althaea officinalis and Primula veris syrup type, had a significant number of syrups, with the percentage of ethanol higher than 1,2 % in relation to all analyzed syrups. This can be seen below in Figure 5.

CONCLUSIONS

Through analysis of 76 dietary products by HPLC with refractometer detector and carbohydrate column we found the presence of ethanol in the

most of the analyzed syrup and 66% of them had a concentration of ethanol more than 1,2%. The effect of ethanol on the liver, CNS and its influence on the combination with other drugs, is well known, therefore it is necessary to declare its concentration-on the package of syrups. People that most often consume these products are older people and children who are more vulnerable and more prone to hypersensitivity reactions and other unwanted reactions. Also regulations about labeling and declaring in our country are to declare all ingredients of the product and it has been found in our study that 67% of the analyzed syrup did not have the amount of ethanol declared, which is a significant percentage.

69,74%

14,47%

5,26%3,95%2 ,63%

2,63% 1,32%

Herbal mixVitaminAlthaea o cinalisPrimula verisHederae helicisPlantago lanceolataMedical

Figure 1.The different types (%) of all analyzed syrups - the most of them 67% were herbal mixtures

Figure 2.The different types of analyzed syrups into years2007-Primula veris 6,67%, Althaea officinalis 6,67%,

Vitamin 33,33%, Herbal mix 53,33% 2008-Vitamin type 25%, Herbal mix 75%2012- Vitamin type 18,75%, Herbal mix 81,25%2014-Vitamin type 25%, Herbal mix 75%2015-Medical 5,55%, Primula veris 11,11%, Hederae

helicis 11,11%, Plantago lanceolata 11,11%, Althaea officinalis 16,67%, Herbal mix 44,44%

2009,2010,2013,2016-Herbal mix only

Figure 3. The Concentration of ethanol (%) in the one herbal syrup between 2007-2014.

Figure 5. The percentage of syrup that had concentration of ethanol > 1,2

Figure 4. The Concentration of ethanol (%) in the one herbal syrup between 2007-2014. -huge statistic difference in % of ethanol in different batches of the syrup

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

2007 2008 2009 2010 2012 2013 2014 2015 2016

Medical

Plantago lanceolata

Herbal

Hederae helicis

Vitamin

Primula veris

33%

8%27%

17%

2%13% 2007

2008

2009

2012

2013

2014

51,31%

2,63% 5,26% 3,95% 2,63%

05

1015202530354045

Herbal VitaminA lthaeao cinalis

Primula verisP lantagolanceolata

0

5

10

15

20

25

30

35

2007 2008 2009 2012 2013 2014

2,50,6

2 1,27 0,15 0,95

33%

8%

27%

17%

2%

13%


REFERENCES

1. Connors GJ, Maisto SA. Effects of alcohol in-structions and consumption rate on motor performance. J Stud Alcohol 1980; 41:509-17.

2. Anderson C. Ion Chromatography: A New Technique for Clinical Chemistry. Clin Chem 1976; 22(9):1424-6.

3. Мedenica M, Мalešev D. Hromatografija. U: Мedenica M, Мalešev D, ured. Eksperimentalna fizička hemija.Beograd: Farmaceutski fakultet, izdanje 1998: 239-52.

4. Karch SB. Drug abuse Handbook. San Francisco: CRC Press; 1998; 322-8.

5. Fagan D, Tiplady B, Scott DB. Effects of ethanol on psychomotor performance. Br J Anaesth 1987; 59(8): 961-5.

6. James SF, Douglas TGj. Ion Chromatography. In : James SF, Douglas TGj, eds. Ion Chromatography . Weinheim : WILEY-VCH Verlag GmbH & Co. KGaA, fourh edition 2009: 1-17.

7. Hanson DJ. Historical evolution of alcohol consumption in society. In: Boyle P, Boffeta P, Lowenfels AB, Burns H, Brawley O, Zatonski W et al., eds. Alcohol: Science, Policy and Public Health. Oxford: Oxford University Press,first edition 2013: 3-10.

8. Kjelkevik R. Food Labelling. Copenhagen: TemaNord; 1998; 9-42.

9. Onuki S, Koziel JA, Leeuwen J, Jenks WS.; Grewell DA; Cai L. Ethanol production, purification, and analysis techniques: a review. Agricultural and Biosystems Engineering Conference Proceedings and Presentations “Asabe Annual International Meeting”, Rhode Island 2008;68.

10. Over-the-Counter Drug Products intended for oral ingestion that contain alcohol. FDA. Fed Reg 1993;58:202.

11. Pharmaceutical excipients, an overview including considerations for paediatric dosing. WHO. Tech Rep Ser 2010:167.

12. Pravilnik o deklarisanju, označavanju i reklamiranju hrane. Službeni glasnik RS, Beograd 2013;8:3-4.

13. Pruitt AW, Anyan WR, Hill RM, Kauffman RE, Mofenson HC, Rumack BH et al. Ethanol in Liquid Preparations Intended for Children. Pediatrics 1984; 73:405-7.

14. Sharma BK. Liqiud chromatography. In: Sharma M, Sharma A,eds. Chromatography. India: Prakashan Media, first edition 1993: 68-95.

15. Shukla SD, Lim RW. Epigenetic Effects of Ethanol on the Liver and Gastrointestinal System. J NIAAA 2013; 35:47-55.

Određivanje etanola u dijetetskim suplementima tipa sirupa

Kratak sadržaj: Dijetetski suplementi su namenjeni dopuni normalne ishrane i predstavljaju koncentrovane izvore vitamina, minerala i drugih supstanci sa hranljivim i fiziološkim efektom. Zbog velike upotrebe dijetetskih preparata, koji često sadrže etanol, moraju se edukovati zdravstveni radnici, kao i potrošači koji ih konzumiraju, kako bi upotreba dijetetskih suplemenata postala sigurna, kontrolisana i racionalna. Etanol se koristi za ekstrakcije, i takodje je važan kao stabilizator u velikom broju dijetetskih suplemenata u obliku sirupa. Zbog toga, veliki broj dijetetskih proizvoda sadrži etanol, a često se dešava da nije ni deklarisan. Još jedan problem je i činjenica da potrošači koji čitaju deklaraciju ovih proizvoda ne mogu da zaključe da je etanol prisutan. Takođe, neki od ovih sirupa se mogu naručiti putem interneta, bez informacije o sadržaju etanola, što je zabrinjavajuće. Osim toga, neki sirupi sadrže istu koncentraciju etanola, kao što je to i u alkoholnim pićima. Koristili smo tečnu hromatografiju, refraktometrijski detektor, ugljeno-hidratnu kolonu, temperaturu 90°C, za detekciju etanola u dijetetskim suplementima, tipa sirupa, tokom 2007 do 2016.godine. Zatim smo određivali koncentraciju etanola eksternom standardnom metodom. Koncentracija etanola se kretala u opsegu od <0,05% do 28,35%. Od 76 analiziranih sirupa 67% nije imalo deklarisan etanol. 8% od sirupa sa deklarisanim etanolom, nije imalo istu koncentraciju kao što je deklarisano. Takodje, 66% od svih analiziranih sirupa je imalo alkohol veći od 1,2% [12].Sirupi se tradicionalno koriste u našoj zemlji, a često i u kombinaciji sa drugim lekovima, što može izazvati različite neželjene reakcije.

Ključne reči: etanol, HPLC, suplementi, deklarisanje

Marina Perunicic Lucic1*,Zorica Basic1,Brižita Đorđević2,Bojana Vidović2

1 Vojno medicinska Akademija (VMA), Crnotravska 17 , 11000 Beograd, Srbija

2Farmaceutski Fakultet – Univerzitet u Beogradu, Vojvode Stepe 450, 11000

Beograd, Srbija

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 31-36, 2015. Bojan Čalija et al.: Monokomponentni dijetetski suplementi folne kiseline na tržištu Republike Srbije – farmaceutsko-tehnološka ispitivanja i karakteristike 31

Monokomponentni dijetetski suplementi folne kiseline na tržištu Republike Srbije – farmaceutsko-tehnološka ispitivanja i karakteristike

Kratak sadržaj: Uvod. Za biološku raspoloživost folne kiseline iz čvrstih doziranih

oblika dijetetskih suplemenata, od posebnog značaja su raspadljivost i brzina rastvaranja folne kiseline. Međutim, trenutni zakonski propisi u Republici Srbiji, kao i u drugim evropskim zemljama, ne postavljaju zahteve za farmaceutsko-tehnološki kvalitet dijetetskih suplemenata, za razliku od Američke farmakopeje koja navodi zahteve za farmaceutsko-tehnološki kvalitet dijetetskih suplemenata u pogledu raspadljivosti, brzine rastvaranja i variranja mase.

Cilj. Cilj rada je bio da se ispitaju i uporede parametri od značaja za farmaceutsko-tehnološki kvalitet čvrstih doziranih oblika monokomponentnih dijetetskih suplemenata folne kiseline, na tržištu Republike Srbije, u odnosu na propise Američke farmakopeje za dijetetske suplemente i Evropske farmakopeje za čvrste dozirane oblike farmaceutskih proizvoda.

Metod rada. Sedam monokomponentnih dijetetskih suplemenata folne kiseline dostupnih na tržištu Republike Srbije, od čega jedan u obliku tvrdih kapsula, tri u obliku neobloženih i tri u formi obloženih tableta, podvrgnuto je, zavisno od vrste doziranog oblika aktivnog sastojka odgovarajućeg suplementa, ispitivanju variranja mase, frijabilnosti, čvrstoće, raspadljivosti i brzine rastvaranja folne kiseline.

Zaključak. Dobijeni rezultati su pokazali da od ukupno sedam ispitivanih monokomponentnih dijetetskih suplemenata folne kiseline, šest u potpunosti ispunjava zahteve Američke farmakopeje za farmaceutsko-tehnološki kvalitet dijetetskih suplemenata, a samo jedan ispitivani preparat (tvrde kapsule) nije odgovarao zahtevima i to samo u pogledu raspadljivosti.

Ključne reči: folna kiselina, dijetetski suplementi, farmaceutsko-tehnološki kvalitet, raspadljivost, brzina rastvaranja folne kiseline

Bojan Čalija1,Jelena Đuriš1,Vladimir Dobričić2,Bojana Vidović3,Jela Milić1,Slađana Šobajić3

1Katedra za farmaceutsku tehnologiju i kozmetologiju, Univerzitet u Beogradu – Farmaceutski fakultet, Vojvode Stepe 450, 11221 Beograd, Srbija2 Katedra za farmaceutsku hemiju, Univerzitet u Beogradu – Farmaceutski fakultet, Vojvode Stepe 450, 11221 Beograd, Srbija3 Katedra za bromatologiju, Univerzitet u Beogradu – Farmaceutski fakultet, Vojvode Stepe 450, 11221 Beograd, Srbija


Autor za korespondenciju:Bojan ČalijaVojvode Stepe 450, 11 221 [email protected]

UDK: 638.18:547.973

UVOD

Folati su hidrosolubilni vitamini B grupe, koji kroz proces prenosa grupa sa jednim ugljenikovim atomom učestvuju u sintezi proteina, neurotransmitera, fosfolipida i nukleotida [1]. S obzirom na to da se ne mogu sintetisati u organizmu, neophodno ih je unositi hranom, posebno tokom trudnoće kada postoji povećana potreba za ovim vitaminom [2,3]. Osim namirnicama biljnog i životinjskog porekla, povećan unos folata može se postići suplementacijom ili fortifikacijom pojedinih namirnica folnom kiselinom, sintetskim oblikom koji ima veću stabilnost i bioraspoloživost nego folati koji se prirodno nalaze u namirnicama [4]. S obzirom na to da ima esencijalnu ulogu u embrionalnom razvoju, u cilju prevencije poremećaja nervnog sistema (anencefalija, spina bifida), trudnicama se preporučuje dodatna suplementacija ishrane folnom kiselinom u količini od 400 µg/dan, 4-12 nedelje pre začeća pa sve do kraja prvog trimestra trudnoće [5].

Dijetetski suplementi folne kiseline su dominantno zastupljeni kao čvrsti dozirani oblici, prvenstveno tablete, ali i tvrde i meke želatinske

kapsule. Za biološku raspoloživost folne kiseline, iz čvrstih doziranih oblika dijetetskih suplemenata, od posebnog značaja su raspadljivost i brzina rastvaranja folne kiseline. Naime, apsorpcija folne kiseline, nakon ingestije je maksimalna u proksimalnim delovima tankog creva, što ukazuje da je poželjno kompletno oslobađanje i rastvaranje folne kiseline već u želucu [6,7]. U suprotnom, sporo raspadanje doziranog oblika i smanjena brzina rastvaranja folne kiseline mogu značajno umanjiti njenu biološku raspoloživost. Trenutni propisi u Republici Srbiji ne postavljaju zahteve za farmaceutsko-tehnološki kvalitet dijetetskih suplemenata [8]. Međutim, iako Američka farmakopeja (United States Pharmacopeia 39 – National Formulary 34, USP 39–NF 34), u poglavljima <2040> i <2091> navodi zahteve za farmaceutsko-tehnološki kvalitet dijetetskih suplemenata u pogledu raspadljivosti, brzine rastvaranja i variranja mase [9], dostupni literaturni podaci pokazuju da dijetetski suplementi folne kiseline često ne ispunjavaju ove zahteve [10-12].

Ispitivanje farmaceutsko-tehnološkog kvaliteta čvrstih doziranih oblika je značajno zbog toga što odstupanja od definisanih parametara

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 31-36, 2015. Bojan Čalija et al.: Monokomponentni dijetetski suplementi folne kiseline na tržištu Republike Srbije – farmaceutsko-tehnološka ispitivanja i karakteristike32

kvaliteta mogu da ukažu na probleme vezane za mehaničke karakteristike čvrstih doziranih oblika (uticaj na oblaganje, pakovanje, rukovanje), kao i na potencijalni uticaj na smanjenje biološke raspoloživosti aktivnog/aktivnih sastoj(a)ka.

Cilj ove studije je bio da se izvrši ispitivanje i komparativna analiza parametara od značaja za farmaceutsko-tehnološki kvalitet čvrstih doziranih oblika dijetetskih suplemenata folne kiseline, na tržištu Republike Srbije, u odnosu na propise Američke farmakopeje za dijetetske suplemente i Evropske farmakopeje [13] za čvrste dozirane oblike farmaceutskih proizvoda.

U tu svrhu, sedam monokomponentnih dijetetskih suplemenata folne kiseline od ukupno dvanaest dostupnih na tržištu Republike Srbije [14], podvrgnuto je, zavisno od vrste doziranog oblika aktivnog sastojka odgovarajućeg suplementa, ispitivanju variranja mase, frijabilnosti, čvrstoće, raspadljivosti i brzine rastvaranja folne kiseline.

MATERIJALI I METODE

MaterijaliIspitivano je sedam monokomponentnih

dijetetskih suplemenata folne kiseline, od čega jedan u obliku tvrdih kapsula (F1), tri u obliku neobloženih tableta (F2 – F4) i tri u obliku film tableta (F5 – F7). Deklarisani sadržaj folne kiseline u svim ispitivanim preparatima je bio 400 µg. U radu su korišćeni standard folne kiseline 89,6 % (Supelco, Bellefonte, SAD), amonijum-acetat (Centrohem, Beograd), glacijalna sirćetna kiselina (Sigma Aldrich, SAD) i prečišćena voda (Ph. Eur. 8.0).

MetodeIspitivanje variranja mase (<2091>,

Weight variation of dietary supplements, USP 39–NF 34)

Variranje mase tvrdih kapsula (F1) ispitivano je tako što je merena masa 20 intaktnih kapsula i izračunata prosečna vrednost je poređena sa zahtevom da mase svih kapsula budu u opsegu od 90-110 % u odnosu na prosečnu vrednost.

Variranje mase neobloženih (F2 – F4) i film tableta (F5 – F7) ispitivano je tako što je merena masa 20 pojedinačnih tableta i izračunata prosečna vrednost. Zahteva se da samo 2 mase smeju da odstupaju više od dozvoljenog % u odnosu na prosečnu masu, a nijedna ne sme odstupati više od dvostruke vrednosti dozvoljenog % odstupanja predstavljenog u Tabeli 1.

Ispitivanje čvrstine tableta (Ph. Eur. 8.0)Čvrstina tableta je određena na uređaju za

ispitivanje otpornosti tableta na lomljenje Erweka TBH 125 (Erweka GmbH, Nemačka). Ispitivano je po 10 pojedinačnih tableta svakog ispitivanog preparata. Rezultati su prikazani kao minimalna, maksimalna i srednja vrednost sile lomljenja izražene u N, sa standardnom devijacijom.

Ispitivanje frijabilnosti neobloženih tableta (Ph. Eur. 8.0)

Frijabilnost neobloženih tableta je određivana na uređaju za ispitivanje frijabilnosti Erweka AR 400 (Erweka GmbH, Nemačka). Ispitivanje je vršeno tako što je 10 ili 20 tableta (u zavisnosti od prosečne mase) precizno odmereno i preneto u bubanj za ispitivanje frijabilnosti koji je podešen tako da rotira 4 minuta brzinom od 25 obrtaja/minuti. Nakon izvođenja testa merena je ukupna masa tableta, a frijabilnost je računata kao procentualna razlika u ukupnoj masi tableta pre i posle ispitivanja.

Ispitivanje raspadljivosti (<2040>, Disintegration and dissolution of dietary supplements, USP 39–NF 34)

Ispitivanje raspadljivosti tableta (neobloženih i film) i tvrdih kapsula izvedeno je u aparaturi za ispitivanje raspadljivosti ZT 52 (Erweka GmbH, Nemačka), na 6 pojedinačnih uzoraka svakog preparata, korišćenjem prečišćene vode kao medijuma za ispitivanje (za neobložene i film tablete), odnosno 0.05 M acetatnog pufera (za tvrde kapsule), zagrejanih na temperaturu od 37 ± 0,5 ˚C. Prema zahtevima USP 39–NF 34, tablete i tvrde kapsule treba da se raspadnu za 30 minuta.

Ispitivanje brzine rastvaranja folne kiseline (<2040>, Disintegration and dissolution of dietary supplements, USP 39–NF 34)

Američka farmakopeja navodi opšte zahteve za ispitivanje brzine rastvaranja aktivnih sastojaka iz dijetetskih suplemenata u čvrstim doziranim oblicima, kao i specifične uslove za ispitivanje brzine rastvaranja folne kiseline iz tableta i tvrdih kapsula. Ispitivanje brzine rastvaranja folne kiseline iz dijetetskih suplemenata izvedeno je u aparaturi sa rotirajućim korpicama (za tvrde kapsule), odnosno rotirajućim lopaticama (za neobložene i film tablete) DHT 600 (Erweka, GmbH, Nemačka), na 6 pojedinačnih uzoraka, pri temperaturi medijuma od 37 ± 0,5 ˚C i brzini obrtanja korpica od 100 o/min za tvrde kapsule, odnosno brzini obrtanja lopatica od 75 o/min za neobložene i film tablete. Kao medijum korišćena je prečišćena voda u zapremini

Tabela 1. Dozvoljeni procenti odstupanja za variranje mase tableta (USP 39–NF 34)

Farmaceutski oblik Prosečna masa Procenat odstupanja

Tablete

(neobložene i film tablete)

130 mg i manje 10

> 130 mg ali < 324 mg 7,5

324 mg i više 5


od 900 ml. Nakon 15, 30, 45 i 60 min vršeno je uzorkovanje, a zatim određivan sadržaj rastvorene folne kiseline iz analiziranih uzoraka opisanom metodom visokoefikasne tečne hromatografije (HPLC). U skladu sa preporukom farmakopeje, zbog fotonestabilnosti folne kiseline ispitivanje je izvođeno u uslovima smanjene izloženosti svetlosti. Prema zahtevima USP 39–NF 34, najmanje 75 % folne kiseline treba da se rastvori nakon 60 min ispitivanja.

Određivanje količine rastvorene folne kiseline u uzorcima prikupljenim tokom ispitivanja brzine rastvaranja izvršeno je na Dionex Ultimate 3000 HPLC sistemu, opremljenim kvaternernom pumpom, autosemplerom i DAD detektorom. Korišćena je kolona Zorbax Eclipse Plus C8 (250 × 4,6 mm; 5 µm). Mobilnu fazu čine metanol i vodeni rastvor koji sadrži 11,16 g/L KH2PO4 i 5,50 g/L K2HPO4 (12:88 V/V). Temperatura kolone je iznosila 25 ˚C, a protok mobilne faze 1 ml/min. Talasna dužina detekcije je 280 nm.

Standardni rastvor folne kiseline je pripremljen rastvaranjem 2,5 mg standarda folne kiseline u 2 ml 0,1 M rastvora natrijum-hidroksida i dopunjavanjem destilovanom vodom do zapremine od 25 ml. Pripremljeni rastvor je razblažen prečišćenom vodom do konačne koncentracije od 0,00045 mg/ml.

REZULTATI I DISKUSIJA

Ispitivanje variranja mase Rezultati ispitivanja variranja mase prikazani su

u Tabeli 2. Na osnovu dobijenih rezultata može se zaključiti da svi ispitivani preparati odgovavaraju zahtevima Američke farmakopeje za variranje mase čvrstih doziranih oblika dijetetskih suplemenata. Najmanje variranje mase utvrđeno je za preparat F3, sa prosečnom masom tableta od 102,85 mg i standardnom devijacijom 1,6, a najveće za preparat F1, sa prosečnom masom kapsula od 368,90 mg i standardnom devijacijom 6,0. S obzirom da se radi o doziranim oblicima sa niskim sadržajem aktivnog sastojka (folne kiseline), od velikog je značaja ujednačena masa doziranih oblika jer se na taj način obezbeđuje i ujednačenost sadržaja aktivnog principa.

Ispitivanje čvrstine tableta Američka farmakopeja ne daje smernice za

ispitivanje čvrstine dijetetskih suplemenata u obliku tableta, te je ovo ispitivanje izvedeno prema zahtevima Evropske farmakopeje. S obzirom na to da postoje razlike u dimenzijama i mehaničkim karakteristikama tableta kao doziranih oblika, u Evropskoj farmakopeji se ne definiše vrednost čvrstine koju tablete treba da poseduju, ali se očekuje da čvrstina tableta bude ujednačena. Rezultati ispitivanja čvrstine tableta prikazani su u Tabeli 3. Prosečna čvrstina ispitivanih tableta varirala je od 45,9 N (preparat F6) do 130,9 N (preparat F2). Razlika između maksimalne i minimalne sile potrebne za lomljenje tableta predstavlja merilo ujednačenosti čvrstine tableta. Najmanje vrednosti ovog parametra iznosile su 13 N (preparati F6 i F7), dok je najveća razlika između sila, u iznosu od 32 N, izmerena za preparat F4. Varijacije u čvrstini tableta mogu da ukažu na potencijalne probleme prilikom rukovanja sa tabletama (habanje, lomljenje) kao i prilikom izvođenja tehnoloških operacija tokom proizvodnje (poput oblaganja tableta) ili tokom pakovanja u primarnu i sekundardnu ambalažu, itd.

Ispitivanje frijabilnosti neobloženih tableta

S obzirom na to da Američka farmakopeja ne definiše zahteve za ispitivanje frijabilnosti dijetetskih suplemenata u obliku neobloženih tableta, i ovo ispitivanje je izvedeno prema zahtevima Evropske farmakopeje. Rezultati ispitivanja frijabilnosti neobloženih tableta (preparati F2 – F4) prikazani su u Tabeli 3. Svi ispitivani preparati odgovaraju zahtevu Evropske farmakopeje po kome frijabilnost neobloženih tableta treba da bude manja od 1 %. Dobijene vrednosti za frijabilnost variraju od 0,23 % (preparat F2) do 0,48 % (preparat F4). Može se primetiti da preparat F4, za koji je zabeležena najveća neujednačenost u čvrstini tableta, ima i najveću vrednost za frijabilnost.

Ispitivanje raspadljivostiRezultati testa raspadljivosti prikazani su u Tabeli

3. Poređenjem prikazanih vremena raspadanja sa zahtevom Američke farmakopeje da se kapsule/tablete moraju raspasti u roku od 30 min, zaključeno je da 6 od ispitivanih 7 preparata odgovara zahtevu

Tabela 2. Rezultati ispitivanja variranja mase

Formulacija msr (mg) sd Donja granica odstupanja

(mg)Gornja granica

odstupanja (mg)Br. tableta čija

masa odstupa od definisanog opsega

Odgovara zahtevu USP

39–NF 34

F1 368,90 6,00 332,01 405,79 0 Da

F2 281,60 2,78 260,48 302,72 0 Da

F3 102,85 1,60 92,57 113,14 0 Da

F4 253,35 3,73 234,35 272,35 0 Da

F5 103,30 2,15 92,97 113,63 0 Da

F6 83,70 2,13 75,33 92,07 0 Da

F7 82,65 2,62 74,39 90,92 0 Da

msr – prosečna masa tablete/kapsulesd – standardna devijacija


u pogledu raspadljivosti. Preciznije, samo preparat F1 (tvrde kapsule), ne ispunjava zahtev USP 39–NF 34, jer se kapsule nisu raspale ni nakon 60 min. Sve ispitivane tablete su se raspale brzo, za manje od 2 min, a nije uočena značajna razlika u raspadljivosti između neobloženih i film tableta. Dobijeni rezultati ukazuju na značajne razlike u raspadljivosti tvrdih kapsula u odnosu na tablete kao najzastupljenije dozirane oblike folne kiseline.

Ispitivanje brzine rastvaranja folne kiseline

Profili brzine rastvaranja folne kiseline su predstavljeni na slici 1. Vrednosti rastvorene folne kiseline nakon 60 minuta su se kretale u rasponu od 80,92 % ± 4,37 do 112,47 % ± 3,76, računato u odnosu na deklarisani sadržaj. Dobijeni rezultati su u skladu sa zahtevom USP 39–NF 34, da se najmanje 75 % folne kiseline rastvori iz tvrdih kapsula, neobloženih i film tableta nakon 60 minuta ispitivanja. Potrebno je istaći da, iako ne odgovara zahtevima u pogledu raspadljivosti, preparat F1 ispunjava zahteve u pogledu brzine rastvaranja folne kiseline. Preciznije, rastvoreno je 80,92 % folne kiseline iz ovog preparata nakon 60 minuta eksperimenta. Međutim, rastvaranje folne kiseline iz ovog preparata je bilo značajno sporije, u odnosu na ostale ispitivane preparata, što se upravo može pripisati znatno sporijem raspadanju doziranog oblika. Više od 75 % folne kiseline se oslobodilo iz ostalih ispitivanih preparata već nakon prvih 15 minuta testa pri čemu nije bilo značajnijih razlika između profila brzine rastvaranja folne kiseline iz ovih preparata.

ZAKLJUČAK

Ispitivani monokomponentni dijetetski suplementi folne kiseline su u skladu sa zahtevima Američke famakopeje za farmaceutsko-tehnološki kvalitet dijetetskih suplemenata, izuzev tvrdih kapsula koje ne ispunjavaju zahtev za ispitivanje raspadljivosti (preparat F1).

S obzirom na to da je folna kiselina na tržištu dostupna i u obliku multikomponentnih dijetetskih suplemenata, koji su kompleksnijeg sastava u odnosu na monokomponentne preparate obuhvaćene ovim istraživanjem, planirano je ispitivanje farmaceutsko-tehnološkog kvaliteta i ovih preparata.

Tabela 3. Rezultati ispitivanja čvrstine, frijabilnosti i raspadljivosti

Formulacija

Čvrstina Frijabilnost Raspadljivost

fsr (N) sd (N) fmin(N) fmax(N) Frijabilnost (%)

Odgovara zahtevuPh.

Eur. 8.0Vreme raspadanja Odgovara zahtevu

USP 39–NF 34

F1 n/a n/a n/a n/a n/a n/a >60 min Ne

F2 111,4 5,1 102 120 0,23 Da 30 sekundi Da

F3 130,9 6,1 122 138 0,44 Da 1 minut 40 sekundi Da

F4 55,8 10,8 42 74 0,48 Da 32 sekunde Da

F5 74,6 8,9 67 93 n/a n/a 38 sekundi Da

F6 45,9 4,6 40 53 n/a n/a 52 sekunde Da

F7 63,3 4,4 57 70 n/a n/a 35 sekundi Da

fsr – prosečna sila koja dovodi do lomljenja tabletesd – standardna devijacijafmin – minimalna sila koja dovodi do lomljenja tabletefmax – maksimalna sila koja dovodi do lomljenja tablete

Slika 1. Profili brzine rastvaranja folne kiseline iz ispitivanih preparata (F1 – F7)


LITERATURA

1. Stover P. Folic acid. In: Ross AC, Caballero B, Cousins R, Tucker K, Ziegler T, eds. Modern Nutrition in Health and Disease. Baltimore: Lippincott Williams & Wilkins, eleventh edition 2014: 358-68.

2. Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Institute of Medicine. Food and Nutrition Board. 1998:1-592.

3. Optimal serum and red blood cell folate concentrations in women of reproductive age for prevention of neural tube defects. WHO. 2005:1-44.

4. Ohrvik VE, Witthoft CM. Human Folate Bioavailability. Nutrients 2011;3(4):475-90.

5. Gomes S, Lopes C, Pinto E. Folate and folic acid in the periconceptional period: recommendations from official health organizations in thirty-six countries worldwide and who. Public Health Nutr 2016;19(1):176-89.

6. Butterworth CE, Baugh CM, Krumdieck C. A study of folate absorption and metabolism in man utilizing carbon-14-labeled polyglutamates synthesized by the solid phase method. J Clin Invest 1969;48(6):1131-42.

7. Hepner GW, Booth CC, Cowan J, Hoffbrand AV, Mollin DL. Absorption of crystalline folic acid in man. Lancet 1968;292(7653):302-6.

8. Pravilnik o zdravstvenoj ispravnosti dijetetskih proizvoda. Službeni glasnik RS, Beograd br. 45/2010, 27/2011, 50/2012, 21/2015, 75/2015

9. The United States Pharmacopeia. United States Pharmacopeial Convention, Rockville, USA, 2016.

10. Giebe K, Counts C. Comparison of prenate advance™ with other prescription prenatal vitamins: a folic acid dissolution study. Adv Ther 2000;17(4):179-83.

11. Hoag SW, Ramachandruni H, Shangraw RF. Failure of prescription prenatal vitamin products to meet USP standards for folic acid dissolution. J Am Pharm Assoc 1996; NS37(4):397-400.

12. Sculthorpe NF, Davies B, Ashton T, Allison S, McGuire DN, Malhi JS. Commercially available folic acid supplements and their compliance with the British Pharmacopoeia test for dissolution. J Public Health 2001;23(3):195-7.

13. The European Pharmacopoeia 8th Edition, Council of Europe, Strasbourg, France, 2013.

14. Registar dijetetskih proizvoda [Internet]. Beograd: Ministarsvo zdravlja Republike Srbije; 2016 [cited 2016 Feb 22]. Available from: http://www.zdravlje.gov.rs/downloads/2016/Februar/Februar2016RegDP.pdf.


Monocomponent folic acid dietary supplements marketed in serbia – pharmaceutical technical investigation and characteristics

Abstract:Introduction. Disintegration of the solid dosage form of dietary supplements and folic acid dissolution rate are of particular importance for its bioavailability. However, current legislation in the Republic of Serbia does not set criteria for the pharmaceutical technical quality of dietary supplements. The United States Pharmacopoeia, on the other hand, gives testing standards for disintegration, dissolution and weight variation of dietary supplements. Aim. The objective of this study was to investigate and compare pharmaceutical technical quality of solid dosage forms of dietary supplements marketed in Serbia, with regard to the United States Pharmacopoeia standards for dietary supplements and European Pharmacopoeia standards for solid dosage forms of pharmaceutical preparations.Methods. Seven monocomponent folic acid dietary supplements commercially available in Serbia, from which three uncoated tablets, three coated tablets and one hard capsules, were subjected, depending on the dosage form, to the weight variation, friability, hardness, disintegration and dissolution testing.Conclusion. The obtained results have shown that six out of seven investigated products complies with United States Pharmacopoeia standards for pharmaceutical technical quality of dietary supplements, while only one (hard capsules) failed to meet the requirements for disintegration.

Key words: folic acid, dietary supplements, pharmaceutical technical quality, disintegration, folic acid dissolution

Bojan Čalija1,Jelena Đuriš1,Vladimir Dobričić2,Bojana Vidović3,Jela Milić1,Slađana Šobajić3

1Katedra za farmaceutsku tehnologiju i kozmetologiju, Univerzitet u Beogradu –

Farmaceutski fakultet, Vojvode Stepe 450, 11221 Beograd, Srbija

2Katedra za farmaceutsku hemiju, Univerzitet u Beogradu – Farmaceutski

fakultet, Vojvode Stepe 450, 11221 Beograd, Srbija

3 Katedra za bromatologiju, Univerzitet u Beogradu – Farmaceutski fakultet, Vojvode Stepe 450, 11221 Beograd,

Srbija

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 37-42, 2015. Nada Pavičić et al.: Identifikacija karakterističnih antocijanina u proizvodima dobijenim iz ploda aronije tehnikom visokoefikasne tečne hromatografije (HPLC) 37

Identifikacija karakterističnih antocijanina u proizvodima dobijenim iz ploda aronije tehnikom visokoefikasne tečne hromatografije (HPLC)

Kratak sadržajPlod aronije ima široku primenu kao boja i korigens ukusa u

prehrambenoj industriji, zbog svojih adstringentnih osobina i tamnoljubičaste boje. Ova biljka ima dugogodišnju primenu u tradicionalnoj medicini u Evropi i Severnoj Americi, i postoje brojne naučne studije o njenim farmakološkim efektima. Plodovi su bogati fenolnim sastojcima (procijanidinima, antocijaninima i fenolnim kiselinama). Poznato je da ovi sastojci imaju osobine antioksidanasa, i stoga imaju primenu u prevenciji srčanih, inflamatornih i malignih bolesti. Antocijanini su biljni pigmenti široko rasprostranjeni u obojenom voću i cveću. Količina i sastav u bobicama se razlikuju u odnosu na biljnu vrstu, način uzgajanja i obrade. U poređenju sa ostalim bobicama i voćem, aronija ima jednostavan sastav antocijanina,, koji uglavnom čine cijanidin glikozidi. Cilj ovog rada je bio identifikacija karakterističnih antocijanina u proizvodima od aronije i razlikovanje aronije od drugih biljnih izvora sličnog sastava. Za dobijanje karakterističnog hromatografskog fingerprinta korišćena je HPLC/PDA tehnika. Na hromatogramu rastvora uzorka aronije, karakteristična su četiri pika, koji odgovaraju pikovima glavnih antocijanina aronije (cijanidin-arabinozid, cijanidin-glukozid, cijanidin-galaktozid i cijanidin-ksilozid). Na hromatogramima dobijenim iz rastvora drugih uzoraka, razlikuje se distribucija pikova. Ima više pikova, na različitim retencionim vremenima, i nema pikova karakterističnih antocijanina aronije. Ovo ukazuje da se navedna metoda može primeniti za razlikovanje preparata aronije od mogućih falsifikata.

Ključne reči: Aronia melanocarpa, antocijanini, hromatografija

Nada Pavičić1,Ivan Velikinac1, Tamara Miladinović1

1Kontrola kvaliteta, Pharmanova d.o.o, Industrijska 8, Obrenovac, Srbija


Autor za korespondenciju:Nada Pavičiće-mail:[email protected]

UDK: 613.73-053.5572.512-053.5

UVODBiljke iz roda Aronia, familija Rosaceae potiču

iz istočnih predela Severne Amerike i Kanade,u Evropu je doneta početkom XX veka. Aronia melanocarpa je žbunasta biljka, raste 2-3 m u visinu, cveta od maja do juna. Štitaste cvasti sa 30-tak sitnih belih cvetova, sazrevaju u crvene do ljubičasto-crne bobice. Plodovi se sakupljaju u avgustu i septembru [1]. Danas se gaji u zemljama Istočne Evrope i Nemačkoj, uglavnom vrsta Aronia melanocarpa. Biljka ne zahteva poseban tretman prilikom uzgoja:štetočine je retko ugrožavaju, upotreba mineralnih đubriva povećava prinos, ali ne i sadržaj farmakološki aktivnih jedinjenja [2]. Koristi se u prehrambenoj industriji kao boja i aroma, a poslednjih decenija postaje atraktivna i zbog svojih lekovitih osobina.

Antocijanini su biljni pigmenti široko rasprostranjeni u obojenim plodovima i cvetovima biljaka. Ispoljavaju značajnu antioksidantnu aktivnost i sposobnost da vezuju slobodne radikale,čime se može objasniti njihov doprinos u prevenciji kardiovaskularnih, malignih i inflamatornih bolesti. Plodovi(bobice) aronije, borovnice, crne ribizle, zove, sadrže antocijanine u visokom procentu. Zato se ovo voće često koristi kao biološki izvor aktivnih principa u sastavu biljnih lekovitih proizvoda i dodataka ishrani.

Sastav i odnos pojedinih antocijanina specifičan je za različite biljne vrste, a može zavisiti od uslova kultivacije i obrade biljnog materijala [3].

Nema dostupnih podataka o farmakološkoj aktivnosti aronije iz kliničkih studija na ljudima, ali su dostupni izveštaji iz studija na in vitro i animalnim modelima. Jedna skorašnja studija pokazala je da sveži plodovi aronije imaju najveću sposobnost za neutralisanje slobodnih radikala u poređenju sa plodovima drugih biljnih vrsta [1]. Mehanizam kojim se ostvaruje aktivnost može se objasniti prisustvom o-dihidroksifenil grupe u molekulima procijanidina i antocijanidina, koji mogu da grade helatne komplekse sa jonima teških metala (Fe, Cu) [4]. Izveštaji iz različitih in vitro studija ukazuju da antocijanini ispoljavaju sledeća dejstva: inhibiraju lipidnu peroksidaciju, aktiviraju enzime uključene u antioksidativni sistem, suzbijaju proliferaciju i stimulišu apoptozu malignih ćelija, deluju antimutageno, hepatoprotektivno, zaštitno na endotelijalne ćelije srca.

U plodovima aronije suva materija čini 17-29%, od toga 5-10% u vodi nerastvorne supstance. Glavni sastojci ploda su dijetetska vlakna (celuloza, pektini, lignini) oko5%, organske kiseline (jabučna i limunska kiselina)1-1,5%, redukujući šećeri (glukoza i fruktoza) 16-18%, lipidi, proteini, minerali (K i Zn) i vitamini [1].

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 37-42, 2015. Nada Pavičić et al.: Identifikacija karakterističnih antocijanina u proizvodima dobijenim iz ploda aronije tehnikom visokoefikasne tečne hromatografije (HPLC)38

Najvažniji sastojci ploda aronije, odgovorni za njenu farmakološku aktivnost, su fenolna jedinjenja: procijanidini (epikatehini),fenolne kiseline (hlorogenska i neohlorogenska), antocijanini (cijanidin-3-arabinozid, cijanidin-3-galaktozoid, cijanidin-3-glukozid, cijanidin-3-ksilozid) i flavonoli (derivati kvercetina: kvercetin-3-galaktozid, kvercetin-3-glukozid, kvercetin-3-rutinozid) [3]. Ne postoji opšteprihvaćen zahtev za sadržaj polifenola u oficijelnoj literaturi. Takođe, u literaturi se pominju različite tehnike kojima se mogu ispitivati (spektrofotometrijski, HPLC/DAD, HPLC/ESI-MS), koje daju različite i često neuporedive rezultate.

Antocijanidini (cijanidin, delfinidin, petunidin) su klasa flavonoida od kojih potiče boja plodova. U biljkama su prisutni u vidu heterozida - antocijanina, koji za aglikon imaju vezanu šećernu komponentu (glukozu, galaktozu, arabinozu) [5]. Aronia melanocarpa je jedan od najbogatijih izvora antocijanina i oni predstavljaju glavnu karakteristiku kvaliteta kojoj se teži tokom kultivacije i prerade biljnog materijala [2]. Čine oko 25% ukupnih polifenola, a najviše je zastupljen cijanidin-3-galaktozid (Tabela 1). Kvalitativni i kvantitativni sastav ovih jedinjenja isti je i u proizvodima od ploda aronije.

Sastav i međusobni odnos antocijanina u aroniji i sličnim plodovima specifičan je za svaku

biljnu vrstu. Npr. ekstrakti ploda dve vrste borovnice,Vaccinium myrtillus iVaccinium ashei, sadrže trinaest identičnih antocijanina, ali u različitom odnosu. Crna ribizla (Ribes nigrum) ima šest, a zova (Sambucus nigra) dva karakteristična antocijanina [3].

Tema ovog rada je primena metode zasnovane na visoko-efikasnoj tečnoj hromatografiji za ispitivanje kvalitativnog i kvantitativnog sastava karakterističnih antocijanina u proizvodima dobijenim iz ploda aronije (sok, ekstrakt, sirup). Razmotrena je mogućnost primene hromatografskog fingerprinta za njihovo razlikovanje od eventualnih falsifikata, proizvoda dobijenih iz drugih botaničkih izvora sličnog sastava.

MATERIJALI I METODE

Za ispitivanje uzoraka korišćena je HLPC/DAD metoda objavljena u monografiji za ekstrakt borovnice (Powdered Bilbery Extract) USP36/NF31.

Uslovi hromatografskog sistemaGradijentna eluacija sa mobilnim fazamaA -

mravlja kiselina/voda (1/9) iB - acetonitril/metanol/mravlja kiselina/voda (45/45/20/80),po programu datom u Tabeli 3.

Razdvajanje se vrši na koloni C18, dimenzija 4,6x250mm, sa veličinom čestica 5µm, temperiranoj

Tabela 1: Sadržaj antocijanina u plodu aronije(1)Antocijanin Sadržaj (%)

cijanidin-3-O-galaktozoid 68,9

cijanidin-3-O-arabinozid 27,5

cijanidin-3-O-glukozid 1,3

cijanidin-3-O-ksilozid 2,3

Tabela 2: Sastav antocijanina u plodovima različitih biljnih vrsta dobijen HPLC/PDA/ESI-MS tehnikom (rastući intenzitet pikova označen je sa + < ++ < +++ < ++++ < +++++) (3)Komponenta Borovnica Crna ribizla Aronija Zova

cianidin 3,5-diglukozid ++

delfinidin 3-galaktozid ++++

delfinidin 3-glukozid +++ ++

delfinidin 3-rutinozid ++++

cianidin 3-galaktosid +++ +++++

delfinidin 3-arabinozid +++

cianidin 3-sambukozid +++++

cianidin 3-glukosid +++ + +

petunidin 3-galaktozid ++

cianidin 3-rutinozid ++++

cianidin 3-arabinozid +++ ++++

petunidin 3-glukozid ++

peonidin 3-galaktozid +

petunidin 3-arabinozid +

peonidin 3-glukozid ++

cianidin 3-ksilozid +

peonidin 3-arabinozid ++

malvidin 3-arabinozid +


na 30ºC. Uzorci se rastvore u smeši metanola i hloridne kiseline (49/1), a zatim razblaže smešom fosforne kiseline 85% i vode (1/9);i do injektovanja čuvaju na temperaturi od 4 ºC. Hromatogrami se snimaju pomoću UV/VIS detektora na 535nm[6].

Pripremana su dva uzorka: − koncentrovani sok dobijen iz ploda aronije

(ceđeni sok plodova aronije sa vitaminom C i fruktozom, komercijalni proizvod sa tržišta)

− tečni ekstrakt nepoznatatog botaničkog izvora - predmet međulaboratorijskog uporednog ispitivanja (PT šeme),za potvrdu identiteta aronije (Black chokeberry, Phyto Identity Confirmation), čiji je rezultat bio negativan.

Referentni rastvor pripreman je od cijanidin -3-O-galaktozida, sertifikovane referentne supstance (proizvođač European Directorate for the Quality of Medicinea and HeathCare, EDQM).

REZULTATI I DISKUSIJA

Primenom navedene hromatografske metode moguće je razdvojiti pikove različitih antocijanina u analiziranim uzorcima. Na hromatogramu uzorka dobijenog iz ploda aronije nalaze se četiri pika, koji po rasporedu i veličini odgovaraju antocijaninima prisutnim u plodu aronije: cijanidin-galaktozid, cijanidin-glukozid, cijanidin-arabinozid i cijanidin-ksilozid (Slika 1). Cijanidin –galaktozid se eluira prvi i daje najveći pik. Identifikovan je na osnovu retencionog vremena, u poređenju sa hromatogramom referentnog rastvora (Slika 3). Ostali pikovi identifikovani su na osnovu relativnih retencionih vremena dostupnih u literaturi [6]. Na hromatogramu nepoznatog uzorka raspored pikova je drugačiji: veliki broj pikova sa retencionim vremenima različitim od onih na kojima se nalaze antocijanini karakteristični za plod aronije (Slika 2). Dobijeni rezultati ukazuju da se metoda može koristiti za dobijanje karakterističnog fingerprinta i identifikaciju proizvoda dobijenih iz plodova aronije.

Fingerprint podrazumeva sveobuhvatnu analizu sastojaka složenih biljnih droga i njihovih proizvoda primenom pogodnog analitičkog postupka. Ova tehnika prihvaćena je od strane WHO kao racionalan pristup za identifikaciju i kontrolu kvaliteta biljnih lekovitih proizvoda. Kineska SFDA (State Food and Drug Administration) od 2000. godine razvija fingerprinting kao standard kontrole kvaliteta tradicionalnih biljnih lekova[7,8]. U literaturi su dostupni naučni radovi čija je tema razvoj i validacija hromatografskih metoda (HPLC/PDA,

MS) za generisanje karakterističnog fingerprinta antocijanina u različitim biljnim vrstama [3,9].Uspostavljanje opšteprihvaćenog standardnog fingerprinta omogućilo bi poređenje gotovih proizvoda sa originalnim biljnim materijalom dobrog kvaliteta. Ovo može biti korisno za praćenje stabilnosti proizvoda, održivosti kvaliteta od serije do serije ili između proizvođača, otkrivanje produkata razgradnje zbog neadekvatnog čuvanja, prisustva stranih materija ili falsifikata.

ZAKLJUČAK

Obojeno voće, bobice različitih biljnih vrsta, bogato je u sadržaju fenolnih jedinjenja. Zbog potencijalnih pozitivnih uticaja na ljudsko zdravlje široko se upotrebljavaju u prehrambenoj i farmaceutskoj indutriji, a u novije vreme raste i broj naučnih studija u kojima se ispituju hemijske i farmakološke karakteristike njihovih sastojaka.

Fenolna jedinjenja od kojih potiče boja plodova i za koje se smatra da su nosioci farmakološke aktivnosti su antocijanini. Kvalitativni i kvantitativni sastav antocijanina različit je zavisno od biljne vrste [3]. Aronija melanocarpa i proizvodi dobijeni od njenih plodova mogu se identifikovati na osnovu karakterističnog fingerprinta primenom pogodne hromatografske metode.

Značaj ove metode u kontroli kvaliteta hrane i dodataka ishrani je u potvrđivanju prisustva ploda aronije kao prirodne boje i korigensa ukusa, odnosno u otkrivanju upotrebe falsifikata, npr.veštačkih boja.

Značaj ove metode u kontroli kvaliteta u farmaceutskoj industriji je u identifikaciji proizvoda dobijenih iz ploda aronije kao aktivnih sastojaka u biljnim lekovitim proizvodima.

Tabela 3: Progam gradijentne eluacije Vreme Protok Mobilna faza A (%) Mobilna faza B (%)

0 min

1 ml/min

93 7

35 min 75 25

45min 35 65

46 min 0 100

50 min 0 100

51 min 93 7

60 min 93 7


Slika 1. Hromatogram uzorka koncentrovanog soka aronije

Slika 2. Hromatogram nepoznatog uzorka


LITERATURA

1. Kulling S., Rawel H., Chokeberry (Aronia melanocarpa)- A Review on the Characteristic Components and Potential Health Effects, Planta Medica 2008; 74:1625-1634

2. Skupien K., Oszmianski J., The effect of mineral fertilization on nutritive value and biologival activity of hokeberry fruit, Agricultural and Food Science 2007; 16:46-45

3. Nakajima J, Tanaka I, Seo S, Yamazaki M, Saito K, LC/PDA/ESI-MS Profiling and Radical Scavenging Activity of Anthocyanins in Various Berries, J Biomed Biotechnol 2004; 2004(6):241-247.

4. Jakobek L,Šeruga M,Novak I,Medvidović-Kosanović M,Šeruga B, DPPH radical inhibition kinetic and antiradical activity of polyphones from chokeberry and elderberry fruits, P Croatica, 2008; 14(2): 101-117.

5. Bondre S, Patil P, Kulkarni A, Pillai M,Study on isolation and purification of anthocyanins and

its application as pH indicator, International Journal of Advanced Biotechnology and Research, 2012; 3(3): 698-702

6. U.S.Pharmacopoeia, Dietary Supplements: Official Monograph Powdered Bilberry Extract, USP36/NF31, 2013; Vol1:13501352.

7. Xie P, Chen S, Liang Y, Wang X, Tian R, Upton R, Chomatographic fingerprint analysis-a rational approach for quality assessment of traditional Chinese herbal medicine, Journal of chromatography A, 2006, 1112(1-2):171-180

8. Tang D, Yang D, Tang A, GaoY, Jiang X, Mou J, Yin X, Simultaneous chemical fingerprint and quantitative analysis of Ginkgo biloba extract by HPLC-DAD, Analytical and Bioanalytical Chemistry, 2010, 396:3087-3095

9. Chandra A, Rana J, Li Y, Separation, Identification, Quantification, and Method Validation of Anthocyanins in Botanical Supplement Raw Materials by HPLC and HPLC-MS, Journel of Agricultural and Food Chemistry, 2001, 49:3515-3521

Slika 3. Hromatogram referentnog rastvora


Identification of Characteristic Anthocyanins in the Chokeberry Fruit Products by HPLC

Abstract:Aronia melanocarpa (black chokeberry) fruits are commonly used in food industry as colouring and flavouring agent, due to bitter, astringent taste and deep purple colour. This plant has a long tradition in European and North American folk medicine, and there is a growing number of scientific studies that examine its pharmacological properties. Fruits are rich in phenolic constituents (procyanidins, anthocyanins and phenolic acids). These structures are known to exhibit antioxidant activities and therefore may contribute to the prevention of heart disease, cancer and inflammatory diseases. Anthocyanins are plant pigments widely distributed in coloured fruits and flowers. The amount and distribution in berries differ depending on their plant species, cultivating and processing. In comparison with other berries and fruits, which are rich in various anthocyanins, black chokeberries have a simple anthocyanin spectrum, mainly containing cyanidin glycosides. The aim of this study was to identify characteristic anthocyanins in the Aronia products (juice, extract, syrup) and to distinguish chokeberry from other plant sources with similar composition. The HPLC/PDA technique has been used to obtain characteristic chromatographic fingerprint. On the chromatograms obtained from Aronia samples, four peaks with the same distribution appear, corresponding to four main chokeberry anthocyanins (cyanidin-arabinoside, cyanidin-glucoside, cyanidin-galactoside and cyanidin-xyloside). Chromatograms obtained from unknown samples have different peak distribution. There are more peaks with other retention times and lack of peaks characteristic for chokeberry anthocyanins. This indicates that the applied method can be useful in distinguish the chokeberry products from possible counterfeits.

Key words: black chokeberry, anthocyanins, chromatography

Nada Pavičić1, Ivan Velikinac1, Tamara Miladinović1

1Department of Quality Control, Pharmanova, Industrijska 8, 11500

Obrenovac, Serbia

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 43-46, 2015. Jelena Golijan, Мilovan Veličković: Nutritivni sastav organski i konvencionalno proizvedenih namirnica 43

Nutritivni sastav organski i konvencionalno proizvedenih namirnica

Kratak sadržaj: S obzirom na to da je činjenica da nutritivna vrednost, bezbednost i zdravstveno stanje namirnica proizvedenih konvencionalnom poljoprivredom variraju širom sveta, sistem organske proizvodnje se nameće kao sistem kojim se postiže održiva proizvodnja zdrave i bezbedne hrane bez ostataka pesticida. Ovim radom su prikazani podaci da organska hrana u poređenju sa konvencionalnom sadrži znatno manje nitrata, a više suve materije i minerala (naročito Fe, Mg i P), vitamina C, esencijalnih aminokiselina i ukupnih šećera, a zatim i fenolnih jedinjenja, dok namirnice životinjskog porekla sadrže i više nezasićenih masnih kiselina. Što se tiče nivoa proteina, on je uglavnom niži u organskim namirnicama. Dosadašnji naučni podaci ukazuju da postoji nedovoljan broj studija vezanih za ispitivanje nivoa vitamina, naročito β-karotena i vitamina B grupe. Organski proizvedeno voće i povrće (naročito jabuka, višnja, ribizla, šećerna repa, cvekla, šargarepa, spanać, kelj i krompir) sadrže viši nivo saharoze, kao i svih ukupnih šećera. Kada su u pitanju animalni proizvodi, dosadašnji dostupni podaci su nedovoljno dokumentovani usled različitog sistema uzgoja na farmama. Ipak, istraživanja dokazuju da kokošja jaja i kravlje mleko ne pokazuju značajne razlike u sadržaju proteina u odnosu na konvencionalno proizvedene, organski kravlji proizvodi sadrže manje lipida, dok meso organskih pilića ima znatno viši nivo gvožđa. Ipak, strogo propisanim pravilima sistem organske proizvodnje obezbeđuje proizvodnju hrane koja zadovoljava visoke standarde kvaliteta.

Ključne reči: organska hrana, vitamini, proteini, minerali, aminokiseline, šećeri, lipidi

1Jelena Golijan,1Мilovan Veličković1 Poljoprivredni fakultet, Univerzitet u Beo-gradu, Nemanjina 6, 11080 Zemun


Autor za korespondenciju:Jelena Golijan, Poljoprivredni fakultet, Univer-zitet u Beogradu, Nemanjina 6, 11080 Zemun, SrbijaTel.: 063 1501988E-mail: [email protected]

UDK: 664/664:631.147

UVOD

Usled povećanja zagađenja i drugih negativnih efekata tehnologije, društvo je počelo da ograničava upotrebu pesticida u konvencionalnom sistemu proizvodnje [1], nasuprot čemu se javlja organska proizvodnja kao sistem kojim se štiti priroda od zagađenja, a svakako i čovek, tako da se od 1990-ih godina sve veći broj poljoprivrednih proizvođača usmerava ka organskoj proizvodnji. Organska poljoprivreda koja zabranjuje upotrebu sintetičkih pesticida i ograničava upotrebu dozvoljenih prirodnih pesticida, nudi hranu bez ostataka pesticida, koju žele potrošači organske hrane. Organske namirnice su proizvedene u skladu sa propisanim standardima, i što je veoma bitno sa minimalnim uticajem na životnu sredinu. Iz godine u godinu, iako smo svedoci svetske ekonomske krize, potražnja za organskom hranom beleži stalan rast, tako da prema dosadašnjim podacima globalno tržište organske hrane vredi oko 72 milijarde evra [2]. Mnoga mišljenja sugerišu da je nutritivna vrednost organske hrane veća u poređenju sa namirnicama dobijenim konvencionalnim sistemom proizvodnje, što ima mnogo nedoslednosti [3], te su neophodna dalja istraživanja.

Cilj ovog rada je da predstavi pregled studija u kojima je prikazana hemijska analiza namirnica dobijenih organskim i konvencionalnim metodama proizvodnje u cilju razjašnjenja nedoumica povodom veće prednosti organske hrane.

Poređenje nutritivnog sastava organske i konvencionalne hrane

Dangour i sar. [4] su istaživali sadržaj hranljivih materija organski i konvencionalno proizvedene hrane (u 100 različitih namirnica) na osnovu tri različite studije (poljske parcele, farme i hrana u maloprodajnim objektima). Pri analizama je ustanovljeno da ne postoji razlika u 8 od 11 hranljivih kategorija (vitamin C, fenolna jedinjenja, Mg, K, Zn, Cu i ukupno rastvorljive suve materije) (Tab. 1). Sadržaj azota je značajno veći kod konvencionalnih useva, dok je sadržaj fosfora i titrirani aciditet značajno veći kod organskih useva, dok ne postoji razlika između proizvodnog metoda kada su u pitanju nespecifične masti ili pepeo u stočarskim proizvodima (Tab.1).

Istraživanja ukazuju da su otkrivene razlike u usevima biološki prihvatljive i rezultat razlika u upotrebi đubriva (azotnih i fosfornih) kao i vremena žetve. U organskim usevima je dokazan veći sadržaj vitamina C, Fe, Mg, P i znatno manje nitrata u odnosu na konvencionalne [5, 6, 7, 8]. Ipak, izvori navode da postoji heterogenost i slab kvalitet istraživanja u ovoj oblasti [2]. Dodatu poteškoću u ovim analizama predstavlja to što laboratorijske metode imaju različite nivoe osetljivosti, metode proizvodnje (one koje regulišu upotrebu hemijskih đubriva i pesticida) mogu uticati na hemijski sastav, kao i to što organska sertifikaciona tela imaju

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 43-46, 2015. Jelena Golijan, Мilovan Veličković: Nutritivni sastav organski i konvencionalno proizvedenih namirnica44

različitu regulaciju proizvodnje u različitim zemljama (navesti izvor podatka)

Prema dosadašnjim procenama organski usevi sadrže 21% više Fe i 29% više Mg od konvencionalnih useva [8]. Kada je u pitanju sadržaj suve materije, najviše dostupnih literaturnih podataka postoji za voće i povrće. Istraživanja pokazuju da u sistemu organske proizvodnje kod lisnatog povrća postoji viši sadržaj suve materije u poređenju sa voćem [7, 9]. Kada je u pitanju voće, a naročito jabuke, dostupni podaci ukazuju da se njihov mineralni sastav gotovo neprimetno menja. Povrće kao što je paradajz, luk, praziluk, celer, repa, kelj, zelena salata, cvekla, krompir i šargarepa, sadrži više nivo magnezijuma i gvožđa u poređenju sa ovim povrćem dobijenim konvencionalnim sistemom proizvodnje [9].

Nivo proteina je generalno niži kod organskih proizvoda, tako npr. organski gajene žitarice, naročito pšenica, mogu imati sličan nivo sa konvencionalno gajenim. Takođe, u organski proizvedenoj pšenici dokazano je povećanje lizina za 25-30% [10]. Ispitivanja mineralnog sastava žitarica, u kojima su uzeti kao najznačajniji elementi P, K, Ca, Mg, Mn, Zn, Fe, Cu i Cr, ukazala su da đubrenje ne utiče značajno na režim rodnosti, dok je jedino primećen nešto viši nivo Ca, Cu i Zn kod organskog ječma [9].

Na osnovu obavljenih istraživanja, maslinovo ulje dobijeno sistemom organske proizvodnje sadrži viši nivo oleinske kiseline i vitamina E [11]. Fenolni metaboliti su posebno značajni zbog

svoje antioksidativne, lekovite i antikancerogene aktivnosti. Usled ove činjenice, bitna prednost organskih namirnica je to što one u 85% istraživanja imaju u proseku oko 30% viši nivo u poređenju sa konvencionalno dobijenom hranom [8]. Sa druge strane, veoma malo je istraživanja vezanih za vitamine, kao što su β-karoten (u povrću nema bitnih razlika u sadržaju ovog vitamina između ova dva sistema proizvodnje, dok ipak značajno viši nivo postoji u organskom paradajzu), B1 i B2, a dodatan problem predstavlja činjenica da su rezultati kontradiktorni, usled čega ne postoji jasan zaključak o ovoj grupi vitamina. Najveći broj studija vezan je za proučavanje vitamina C, naročito u krompiru, paradajzu i kelju, te je tim putem dokazan viši nivo ovog vitamina u sistemu organske proizvodnje, dok kod cvekle, praziluka, šargarepe i jabuke nije detektovana nikakva razlika [8].

Veći sadržaj šećera u biljnim kulturama ne samo da poboljšava ukus, već je i važna komponenta njihovog tehnološkog kvaliteta, kao što je slučaj kod šećerne repe. Dosadašnji podaci ukazuju da organski proizvedeno voće i povrće kao što je šećerna repa, cvekla, šargarepa, spanać, kelj, krompir, jabuka, ribizla i višnja sadrže više ukupnih šećera, naročito saharoze [8, 12, 13].

U studijama u kojima su ispitivana kokošja jaja i kravlje mleko nije dokazana značajna razlika u proteinskom sadržaju [14]. Proizvodi dobijeni od krava uzgajanih po sistemu organske proizvodnje

Tabela 1. Poređenje sadržaja hranljivih materija i drugih nutritivno bitnih supstanci u organski i konvencionalno proizvedenim usevima i stočarskim proizvodima (masti i pepeo)

Nutritivna kategorija

Broj studija Broj poređenjaRezultati analize Viši nivoi u organskim ili

konvencionalnim usevima?

Standardna devijacija

P

%

Azot 17 64 6.7 ± 1.9 0.003 Konvencionalni

Vitamin C 14 65 2.7 ± 5.9 0.84 Nema razlike

Fenolna jedinjenja13 80 3.4 ± 6.1 0.60 Nema razlike

Magnezijum 13 35 4.2 ± 2.3 0.10 Nema razlike

Kalcijum 13 37 3.7 ± 4.8 0.45 Nema razlike

Fosfor 12 35 8.1 ± 2.6 0.009 Organski

Kalijum 12 34 2.7 ± 2.4 0.28 Nema razlike

Cink 11 30 10.1 ± 5.6 0.11 Nema razlike

Ukupno rastvorljive suve materije

11 29 0.4 ± 4.0 0.92 Nema razlike

Bakar 11 30 8.6 ± 11.5 0.47 Nema razlike

Titrirani aciditet10 29 6.8 ± 2.1 0.01 Organski

Masti (nespecifične) 6 13 13.0 ±14.6 0.42 Nema razlike

Pepeo 4 8 13.7 ± 7.8 0.18 Nema razlike

Izvor: Dangour i sar. (2009)

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 43-46, 2015. Jelena Golijan, Мilovan Veličković: Nutritivni sastav organski i konvencionalno proizvedenih namirnica 45

imaju manji sadržaj ukupnih lipida, za razliku od svinjskih proizvoda [15]. Što se tiče svih animalnih proizvoda, informacije su vrlo ograničene, zbog različitog sistema uzgoja. Međutim, ipak je dokazano da sastav mesa pilića gajenih organskim sistemom sadrži viši nivo gvožđa [9].

ZAKLJUČAK

Dosadašnje analize ukazuju na to da postoji mali broj razlika u sadržaju hranljivih sastojaka između organski i konvencionalno proizvedenih namirnica. Sve dok su ove razlike biološki prihvatljive, one verovatno neće biti od velike važnosti kada je u pitanju zdravlje ljudske populacije. Evidentno je da će dalja naučna istraživanja u ovoj oblasti imati znatno veće koristi ne samo u naučnom pogledu, već i u boljem razumevanju različitih faktora, koji pored režima proizvodnje određuju sadržaj hranljivih elemenata u namirnicama.

Literatura

1. Veličković, M., Golijan, J. Koncept integralne zaštite jabuke i kruške. Journal of Agricultural sciens, 2015; 60 (4), 381-393.

2. The World of Organic Agriculture 2015: The global statistic almanach in it’s 16th edition. Available from: http://www.fibl.org/fileadmin/_processed_/csm_1663_01_a4e0c91930.gif

3. Winter CK, Davis, SF. Organic foods. Journal of Food Science; 2006; 71(9); 117-124.

4. Dangour AD., Dodhia SK., Hayter A, Allen E, Lock K, Uauy R. Nutritional quality of organic foods: a systematic review. The American journal of clinical nutrition, 2009; 90(3): 680-685.

5. Magkos F, Arvaniti F, Zampelas A. Organic food: nutritious food or food for thought? A review of the evidence. International journal of food sciences and nutrition, 2003; 54(5): 357-371.

6. Worthington, V. (2001): Nutritional quality of organic versus conventional fruits, vegetables, and grains. The Journal of Alternative & Complementary Medicine, 2001; 7(2): 161-173.

7. Woese K, Lange D, Boess C, Bögl KW. A Comparison of Organically and Conventionally Grown Foods--Results of a Review of the Relevant Literature. Journal of the Science of Food and Agriculture; 1997; 74(3): 281-293..

8. Rembiałkowska E. Quality of plant products from organic agriculture. Journal of the Science of Food and Agriculture 2007; 87.15: 2757-2762.

9. Lairon D. Nutritional quality and safety of organic food. In: Sustainable Agriculture Volume 2. Springer Netherlands; 2011; 99-110.

10. Brandt K, Mølgaard JP. Organic agriculture: does it enhance or reduce the nutritional value of plant foods?. Journal of the Science of Food and Agriculture, 2001; 81.9 : 924-931.

11. Gutiérrez F, Arnaud T, Albi M. Influence of ecological cultivation on virgin olive oil quality. Journal of the American Oil Chemists’ Society, 1999; 76(5): 617-621.

12. Veličković, M. Jagodasto voće kao biološki i ekološki vredna hrana. Ecologica, 1999; 21 (1): 35-40.

13. Veličković, M. Jezgraste vrste voćaka kao izvor lekovitih biljnih sirovina i biološki vrednije hrane. Ecologica, 1998; 20 (4): 36-39.

14. Toledo P, Andrén A, Björck L. Composition of raw milk from sustainable production systems. International dairy journal, 2002; 12(1): 75-80.

15. Hansson I, Hamilton C, Ekman T, Forslund K. Carcass quality in certified organic production compared with conventional livestock production. Journal of Veterinary Medicine, Series B, 2000; 47.2: 111-12.

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 43-46, 2015. Jelena Golijan, Мilovan Veličković: Nutritivni sastav organski i konvencionalno proizvedenih namirnica46

Nutritional composition of conventional and organically produced food

Abstract:Given the fact that the nutritional value, safety and health status of food produced by conventional agriculture vary around the world, the system of organic farming is emerging as a system that achieves a sustainable production of healthy and safe food, free of pesticide residues. This paper presents evidence that organic foods compared to conventional contain significantly less nitrates, a higher level of dry substance and minerals (especially Fe, Mg and P), vitamin C, essential amino acids and total sugars, then the phenolic compounds, while foods of animal origin contain more unsaturated fatty acids. As for the level of protein, it is generally lower in organic foods. The current scientific data indicate that there is an insufficient number of studies on vitamin levels, in particular β-carotene and vitamin B group. Organically produced fruits and vegetables (in particular apple, cherry, currants, sugar beet, beetroot, carrot, spinach, kale and potatoes) contain higher levels of sucrose and total all sugars. When it comes to animal products, the current available data are insufficiently documented due to different farming systems on farms. However, studies show that chicken eggs and cow’s milk do not show significant differences in protein content compared to conventionally produced, organic cow’s milk products contain less lipids, while organic chicken meat has a much higher level of iron. However, strict rules laid down by the system of organic production provides food production that meets high quality standards.

Key words: organic foods, vitamins, proteins, minerals, amino acids, sugars, lipids

Jelena Golijan1,Мilovan Veličković1

1Faculty of Agriculture-University of Belgrade, Nemanjina 6,11080 Zemun-Belgrade

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 47-50, 2015. Miloš Milošev et al.: Effects of additional fitness program on body composition and general motor skills in overweight children of school age 47

Effects of additional fitness program on body composition and general motor skills in overweight children of school age

AbstractPhysical activity, health and quality of life are closely interconnected.

The human body was designed to move and therefore needs regular physical activity in order to function optimally and avoid illness.

The aim of this research was to explore the differences in body composition and motor abilities after applying additional fitness program during 8 consecutive weeks and appropriate diet regime in overweight children of school age. A total of 12 boys were enrolled into the study. They were overweight basketball players with average body mass index 26.6 ± 4.2 and age 11.4 ± 0.5. Body composition was tested by bioelectrical impedance, while basic motor tests were used for physical abilities (Cooper test, strength tests and bent over test). All measures were done before and after 8 weeks of fitness program.

Additional program of training and diet regime influenced statistically significant differences in all measured anthropometric parameters (body height, body weight, body mass index, amount of fat, amount of muscles, p < 0.05), except in basal metabolic rate.

After two months of training, subjects also showed statistically significant improvement in all tested motor abilities (p<0.05).

Keywords: body composition, motor abilities, obese, fitness program, healthy diet

Miloš Milošev1,Ivana Đuričić2,Dušan Mitić1

1Faculty of sport and physical education, University of Belgrade, Blagoja Parovića 156, 11030 Belgrade, Serbia2School of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia


UDK: 615.3:577.164.1

INTRODUCTION

According to the World Health Organization (WHO) [1], every year at least 1.9 million people worldwide die from lack of physical activity. Lack of physical activity is closely associated with the emergence of non-communicable diseases such as diabetes, cardiovascular diseases and obesity. Hypokinesia and obesity are the diseases of modern world, and persons under stress, adolescents, children and older people are particularly vulnerable [2]. Obesity could be defined as an excessive accumulation of fat, which may lead to serious health problems and a series of complications [3]. In accordance with the guidance documents of the WHO, the European Union and its Member States recommend a minimum of 60 minutes of daily moderate-intensity physical activity for children and young people [4].

The aim of this research was to apply additional fitness programs and appropriate diet in overweight and obese school children in order to reduce the negative phenomena of obesity and improve their body composition and sports performance. The training program was focused on losing fat, increasing muscle mass and development of general motor skills for a period of 8 weeks.

MATERIAL AND METHODS

This research was experimental study without a control group. More specifically, it was quantitative research which used descriptive (basic indicators

measuring morphological characteristics and motor abilities of subjects) and comparative methods (detection of differences in anthropometric parameters and motor ability level by using T-test).

A total of 12 boys were enrolled into the study. They were overweight (n=10) and obese (n=2) basketball players with average body mass index 26.6 ± 4.2 and age 11.4 ± 0.5. Subjects were tested by bioelectrical impedance [3,5,6,7,8] (body’s composition parameters) and basic battery of tests for physical abilities (Cooper test, strength tests and bent over test)[9].After the initial tests, training program was performed during 8 consecutive weeks. It was aimed to improve body composition of subjects, and the basic physical skills such as endurance, strength and flexibility. Also, dietary advice was given to the children and their parents toward adjusted diet with reduced intake of energy, sugar and saturated fats, and increased intake of protein and complex carbohydrates. Recommended calorie intake was based on data from basal and rest metabolic rates. Carbohydrates were recommended in morning hours, intake of protein almost right after training sessions, and meals after 10 am were avoided. Except protein meals after trainings, any other food was avoided in next several hours. Trainings were in aerobic intensity zone, which included running and walking. Main part of the training was in zone between 55% and 70% of maximum heart rate. Also, training program contained strength and exercise for increasing muscle mass, improvements of flexibility and speedy recovery. The children were tested again after completing the fitness program.

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 47-50, 2015. Miloš Milošev et al.: Effects of additional fitness program on body composition and general motor skills in overweight children of school age48

All tests were done in special laboratory for anthropometric measurements at Faculty of sport and physical education in Belgrade and several gym centers.

The results are presented as means ± SD. Value p< 0.05 was considered significant. The T-test was used to determine whether differences in the body composition and physical abilities before and after training program were significant.

RESULTS AND DISCUSSION

Based on the data in Table 1, the average age of subjects was 11.4, the average height was 157.6 cm, while the average weight was 58.7 kg.

Table 1. Characteristics of the study group at baseline

Parameters Means ± SD Range CV (%)

Age (y) 11.4 ± 0.5 10.8 - 12.3 4.4

Body weight (kg) 58.7 ± 11.9 40.5 - 79 20.4

Height (cm) 157.6 ± 7.8 147.7 - 175.6 4.9

BMI (kg/m2) 26.6 ± 4.2 18.3 - 31.3 15.7

Body fat (kg) 21 ± 6.3 9.2 - 29.6 29.8

Body fat (%) 36.3 ± 7.1 21 - 46 23.7

Muscle mass (kg) 20.3 ± 4 15.2 - 30.1 19.8

Basal metabolism (kcal) 1322 ± 217.3 1045 - 1658 17.2

It was also noted that the average value of the body mass index was 26.6, the amount of fat 21 kg, while the average percentage of fat was 36.3. Average value of muscle mass was 20.3 kg, while the average of basal metabolic rate was 1322 kcal. The maximum value of fat content was 46%, and the lowest 21% of total body weight. It was shown that the greatest amount of muscle mass was 30.1 kg, and the lowest one was 15.2 kg. The value of the basal metabolic rate was in the range of 1045 to 1658 kcal. Coefficient of variation (CV) for all parameters was below 30%, which indicates that the group was relatively homogenous.

Table 2 shows parameters of physical abilities before the beginning of applied fitness program.

Table 2. Results of physical abilities in tested group before fitness program

Assays Means ± SD Range CV (%)

Cooper test (m) 1800 ± 88.6 1075 - 2034 4.9

Push-ups on bench 8.3 ± 2.4 6 - 13 28.2

Pull-up hold (seconds) 5.4 ± 3.6 1.9 - 14.3 67.4

Crunches (30 seconds) 11.7 ± 1.4 10 - 14 11.6

Back extensions (30 seconds) 16.4 ± 1.9 13 - 19 11.5

Long jump (cm) 114 ± 17.4 95 - 145 15.3

Bent-over (cm) 6 ± 9.1 1 - 31 151.8

The average distance traveled meters in a Cooper test was 1800, while the range of values was between 1705 and 2034 meters.The result for coefficient of variation (4.9%) confirms very good homogeneity of group. On the other hand, assay for flexibility extensors (Bent-over), showed that

tested boys were very different dynamically, i.e. mobile. The average value was 6 cm, ranged from 1 to 31. Therefore, the variability of results was extremely high (coefficient of variation was 151.8), which showed that the group was not homogeneous with respect to this physical ability test. On the holding endurance test (Pull-up hold), coefficient of variation was also high (67.4%), and group was not homogeneous. Coefficient of variation for all other assays was below 30%.

After two months of training program, all subjects were retested. Data is shown in Table 3.

As far as body mass index is concerned, the average value after two months of fitness program decreased by 14.3%. The average value for body fat also decreased (by 7%) at the end of the study. Mean value for the amount of muscles or lean body mass was2% higher comparing to previous result. The values for basal metabolism were in the range of 1045 to 1574 kcal. Values for coefficient of variation showed that all parameters were fewer than 30, which means that group was relatively homogeneous across all measured categories.

Re-test of physical abilities were done only a few days after the measurement of body composition. The same tests were determined as in a previous stage: the assessment of basic endurance (Cooper test), tests to assess the strength (holding endurance, lying-sit in 30 seconds, stretching back for 30 seconds, jump out of the place), and test to assess flexibility (deep forward bent test). Data are shown in Table 4.

The values for Cooper test and Long jump assay increased by 12% during the 8 weeks of fitness program. The average values for the push-ups on bench and pull up hold tests increased by 54% and 52%. Higher values were also found for Crunches

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 47-50, 2015. Miloš Milošev et al.: Effects of additional fitness program on body composition and general motor skills in overweight children of school age 49

Table 3. Results of anthropometric measure after 2 months of fitness program

Parameters Means ± SD Range CV (%)

Age (y) 11.5 ± 0.5 10.8 - 12.3 4.4

Body weight (kg) 57.4 ± 12 40.5 - 79.3 20.9

Height (cm) 157.9 ± 8.2 147.7 - 177.1 5.2

BMI (kg/m2) 22.8 ± 2.8 17.9 – 26.8 12.5

Body fat (kg) 19.5 ± 5.5 9.2 - 25.8 28

Body fat (%) 34 ± 7.4 18.8 - 44.2 21.9

Muscle mass (kg) 20.7 ± 4.2 16.1 - 30.7 20.4

Basal metabolism (kcal) 1262 ± 152.6 1045 - 1574 12.1

Table 4. Results of physical abilities in tested group after 2 months of fitness program

Assays Means ± SD Range CV (%)

Cooper test (m) 2020.5 ± 201.7 1780 - 2456 10

Push-ups on bench 12.8 ± 5.5 7 - 28 43.2

Pull-up hold (seconds) 8.2 ± 6.8 2.4 – 12.6 82.1

Crunches (30 seconds) 14.4 ± 1.5 12 - 16 10.4

Back extensions (30 seconds) 17.5 ± 2 14 - 20 11.6

Long jump (cm) 128.1 ± 23 101 - 162 18

Bent-over (cm) 19.4 ± 9.8 2 - 33 50.2

and Back extensions after the intervention period (23% and 7%, respectively). At the end of the study, the highest increase was measured for bent-over assay (323%).

It was shown that 2 months of fitness program were useful in improving anthropometric parameters and physical performance (Table 5).

Table 5. The estimates of change between values before and after fitness program

Parameters Difference P value

Body weight (kg) -1.5 <0.01**

Height (cm) 0.4 <0.05*

BMI (kg/m2) -2.1 <0.01**

Body fat (kg) -1.5 <0.001***

Body fat (%) -2.3 <0.001***

Muscle mass (kg) 0.6 <0.001***

Basal metabolism (kcal) -60.6 0.07

Assays

Cooper test (m) 220 <0.001***

Push-ups on bench 5 <0.001***

Pull-up hold 2.8 <0.05*

Crunches (30 seconds) 3 <0.001***

Back extensions (30 seconds) 1 0.05*

Long jump (cm) 13 <0.001***

Bent-over (cm) 5 <0.01***Novo statističke značajnosti

Additional program of training and diet regime influenced statistically significant differences in all measured anthropometric parameters (body height, body weight, BMI, amount of fat, percentage of fat, amount of muscles parameters, p < 0.05), except in basal metabolic rate.

After two months of training, subjects also showed statistical significant improvement in all tested motor abilities (p<0.05).

Regular, well-designed and planned training sessions, based on the general laws of development of the aforementioned physical abilities that are important for public health (and thus the control of body weight), manifested their influence on basic endurance, explosive and repetitive force and passive flexibility. Running in an aerobic intensity zone created on the basis of recommendations of a doctor Kenneth Cooper, resulted in a general improvement of aerobic endurance[10].Additional work in a recovery intensity zone, contributed above

HRANA I ISHRANA (BEOGRAD), VOL. 56. No. 2., 47-50, 2015. Miloš Milošev et al.: Effects of additional fitness program on body composition and general motor skills in overweight children of school age50

all to fat burning (impact on body composition). Strength endurance exercises, its isometric contractions influenced the development of static muscle strength, and thus the improvement of results in the holding endurance test. Additional exercises, such as squats and lunges, influenced the development of repetitive and explosive strength of muscles. This is the best demonstrated in the tests of crunches for 30 seconds, back extensions, push-ups on the bench and long jump.

The application of flexibility exercises, influenced the development of passive and general flexibility (flexibility of the muscles of the lower back and hamstrings). In this way, improved results in bent-over re-test (increased mobility of the lower part of body), were achieved by additional training program.

All subjects during the program performed major part of physical exercise in an aerobic intensity zone, primarily in the range of 55% to 70% of maximum heart rate, which is recommended as an excellent area for using fat as an energy source [9]. Post-exercise in this intensity zone with appropriate enhanced duration also contribute to fat burning. Then, the endurance exercise contributed to strengthen the muscles of the body, or increasing muscle mass in subjects. Increased muscle mass associates to the increased basal metabolic rate. On the other hand, the fat loss and increase in muscle mass can be directly linked to the implementation of appropriate exercise, improving body composition in a whole. Correction of dietary regime related to reduced intake of fat and simple carbohydrates, contributed to rapid weight loss, while increased intake of proteins in diet contributed to the increased muscle mass. Statistically insignificant changes of basal metabolic rate can be explained by duration of training period and relatively small, although significant, increase in muscle mass.

CONCLUSION

In conclusion, the results of the present study indicate that the additional fitness program if applied even for a moderate period of time can be used for improvement of the body composition and general motor skills in overweight children of school age.

REFERENCES

1. Jovanović R, Nikolovski D, Radulović O, Novak S. Influence of physical activity on nutritional status of preschool children. Med J . 2010; 47 (2): 62-68.

2. Mitić D, Stojiljković S. Fitness index and maximal oxygen uptake among people with active lifestyle in Serbia.10th Annual congress“European College of Sport Science”. Belgrade. Serbia. 2005; 370-371.

3. Оstojić S, Stojanović M, Stojanović V, Маrić Ј, Njaradi N. Correlation between Fitness and Fatness in 6-14-year Old Serbian School Chil-dren. Journal of Health, Population & Nutrition. 2011; 1 (29): 53.

4. WorldHealthOrganization. “Globalrecommen-dations onphysicalactivity for health”, 2010.

5. Morrow JR, Jackson AW, Disch, JG, Mood DP. Measurment and Evaluation in Human Perfomance. Human Kinetics 2005; Champaign. IL.

6. Steven B, Heymsfield Wang. Human body composition advances in models and methods. Obesity Research Center Department of Medicine. St. Luke’s-Roosevelt Hospital Center. Columbia University College of Physicians and Surgeons New York. New York. 1997; 1 (17):527–58.

7. Forbes GB. Simon W, Amatruda JM.Is bio-impedance a good predictor of body somposi-tion change? Am. J. Clin. Nutr. 1992; 1 (56): 4-6.

8. Wang M, Pierson R, Heymsfield Steven.Five level model: new approach to organizing body composition research.Obesity Research Center, Department of Medicine, St. Luke’s-Roosevelt Hospital Center, Columbia University College of Physicians and Surgeons. New York. New York 1992; 13 (1): 58-65.

9. Stojiljković S, Mitić D. Promene telesnog sastava rekreativaca nakon osmonedeljnog treninga aerobne izdržljivosti. Zbornik sažetaka“Prvi srpski kongres pedagoga fizičke culture & 2nd FIEP European congress”. Vrnjačka Banja. Srbija. 2004; 204-205.

10. Cooper, KH. Aerobics Program For Total Well-Being. Exercise, Diet, And EmotionalBalance. Bantam. 2013.

52

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Serbian Nutrition Society website:www.hrana-ishrana.org

60 godina Društva za ishranu Srbije

Before August 31st After August 31st

Before August 31st After August 31st

Members of Serbian Nutrition Society 50 euros / 6300 RSD 75 euros / 9400 RSD

Students 30 euros / 3750 RSD 50 euros / 6300 RSD

Other 75 euros / 9400 RSD 100 euros / 12500 RSD

Students 60 euros 80 euros

Other 100 euros 150 euros

UPUTSTVO AUTORIMASaradnici se mole da detaljno i pažljivo pročitaju

predložena uputstva za pripremu radova pre predaje rukopisa za štampanje. Izuzetno je važno da sarad-nici/autori pripreme radove prema ustanovljenim principima jer je to izuzetno značajno za klasifikaciju naučnih časopisa.

“Hrana i ishrana” je časopis Društva za ishranu Srbije osnovanog 1956. godine, u kome se objavlju-ju radovi članova Društva za ishranu Srbije i članova društava drugih srodnih struka. Objavljuju se origi-nalni radovi, saopštenja, pregledni radovi, izveštaji sa kongresa i stručnih sastanaka, stručne vesti, prilozi, prikazi knjiga, pisma uredništvu i dopisi “U spomen”. Uz rukopis članka 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 njegove konačne odluke. Radovi se ne honorišu. Rukopisi se ne vraćaju.

Rukopis, u dva primerka slati poštom preporučeno na adresu: Uredništvo “Hrane i ishrane”, Savska 9/II, 11000 Beograd. Neophodno je da se celokup-ni materijal (rad) pošalje i elektronskom poštom glavnom uredniku na e-mail: petrica @eunet.rs .

Opšta pravilaRukopis članka i svi prilozi treba da budu jasni i

napisani na engleskom ili srpskom jeziku, a za iz-radu rada koristiti isključivo tekst-procesor Microsoft Word. Rukopis 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 pro-redom 1,5.

U rukopisu članka obeležiti mesta za slike, sh-eme, grafikone, tabele i ne ostavljati prazan prostor u tekstu. Literaturni podaci u tekstu se označavaju arapskih brojevima u zagradama redosledom kojim se pojavljuju u tekstu, na primer [1,2].

Skraćenice upotrebljavati samo izuzetno, i to u slučajevima 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 označavaju se u metričkim jedinicama (metar - m; kilogram - kg; litar -l) ili podjedinice. Temperatura se izražava u stepenima Celzijusa (°C), koncentracije u molima; uređaji se označavaju trgovačkim nazivima, a naziv i mesto proizvođača su u zagradama. Svi re-zultati kliničkih i biohemijskih istraživanja izražavaju se u jedinicama međunarodnog sistema mera - SI.

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

Sastav/struktura rukopisaRukopisi treba da sadrže sledeća poglavlja:

naslov, autore, ustanove, kratak sadržaj na srpskom jeziku sa ključnim rečima, uvod, eksperimentalni deo (materijal, metode), rezultate, diskusiju, zah-valnica, literaturu i kratak sadržaj (Abstract) na en-gleskom jeziku takođe sa ključnim rečima. Pregled-ni članak sadrži sledeća poglavlja: uvod, pregled slučaja, zaključak i literaturu. Pregledni članak mora

INSTRUCTIONS TO AUTHORSContributors are strongly encouraged to read the

instructions 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 man-uscripts according to the established specifications.

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 published, as well as articles by members of other associations in the field of Public Health Nutri-tion (PHN) and related fields. The Journal publishes original articles, communications, case reports, review articles, congress and scientific meeting reports, pro-fessional 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 confirmation 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 suggested changes is mailed to the authors for their final consent.

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

The manuscripts should be forwarded to the follow-ing 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 elec-tronic version: petrica @eunet.rs

General demands on manuscriptsManuscripts should be written in clear concise Ser-

bian 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. Recommended font is Tames New Roman 12. Papers should be typed 1,5 spaced. The author(s) should in-dicate in the text where figures and tables fit 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 appropriate, for very long names of chemical com-pounds, or as well-known abbreviations (such as DNA).

Units of measure: Length, height, weight and vol-ume should be expressed in metric units (meter - m, kilogram - kg, and liter -l) or their sub-units. Tempera-ture should be given in Celsius degrees (°C). Concen-trations are given in moles, proprietary names of instru-ments with factory name and place of manufacture in parenthesis. All results of clinical and biochemical mea-surements should be expressed in the metric system according to the International System of Units – SI.

Authorship All individuals listed as authors should be qualified

for authorship. Every author should have participated sufficiently in writing the article in order to take re-sponsibility for the whole article and results presented in the text. Authorship is based only on crucial contri-bution to the article conception, obtaining of results or analysis and interpretation of results, and final revision of the manuscript being prepared for publication.

Structure of the manuscriptsThe manuscript has to be arranged as follows: The

title, Authors, Institutions, Abstract, Introduction, Ex-perimental part, Results, Discussion, Acknowledge-

ments, and References. Review articles include Introduction, correspond-

ing section heading, Conclusions and References. The review 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 with-out 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 indi-cated by corresponding Arabic numbers if there is more than one institution. The address of corresponding au-thor, with the telephone, fax number and e-mail ad-dress should be added at the bottom of this page.

Abstract.Original articles, communications, case reports, 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 rapid identification should be typed below the ab-stract in English. In original articles the abstract should have the following structure: introduction, objective, method, results and conclusion. In case reports the abstract should consist of the following: introduction, case outline 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 cited.

The Experimental part should include description of materials (subjects) and methods used. If methods are widely known and described in the literature, only reference(s) should be cited. New or modified meth-odologies should be fully described. Methods used for parameters calculation and statistical analyses should be indicated. All abbreviations have to be explained in the manuscript when used for the first time.

Results should be clear and precise, with corre-sponding statistical analysis.

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

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

All section headings should be in capital letters us-ing bold lettering.

Original articles shall have the following section headings: Introduction, Objective, Method, Results, Discussion, Conclusion and References

Case reports should consist of introduction, case outline, discussion, references.

Length of the manuscriptsThe entire text of the manuscript: title page, ab-

stract, the whole text, list of references and captions to figures 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 1 000words in the section ‘’other’’.

The total number of figures and tables should not exceed the half of the number of typed pages of the manuscript.

Tables, figures (graphs, charts, pho-tographs, 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 identified 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

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 naslo-va navesti imena autora indeksirana brojkama koje od-govaraju 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 kon-takt, punu adresu, broj telefona, faksa ili e-mail adresu.

Kratak sadržaj. Uz originalni rad, saopštenje ili pregled iz literature treba priložiti na posebnoj strani-ci kratak sadržaj, koji sadrži naslov rada, prezimena, inicijale imena 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 po-datke. U njemu se navode, bez opisivanja, bitne činjenice, kratak prikaz problema i osnovni zaključak. U originalnom članku kratak sadržaj treba da sadrži sledeća poglavlja: uvod, cilj, metod rada i zaključak. U saopštenju/pregledu kratak sadržaj sadrži sledeća po-glavlja: uvod, pregled slučaja i zaključak.

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

Ključne reči. Na kraju apstrakta/kratkog sadržaja dodaju se ključne reči ne više od 8, koje su bitne za brzu identifikaciju i klasifikaciju sadržaja članka.

Uvod rada se piše jasno, sažeto, uz navođenje suštine materije i radova koji su u vezi sa problema-tikom, 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 metodologija nova ili modifikovana. Potrebno je navesti metode izračunavanja parametara i statističke analize re-zultata. Ukoliko se upotrebljavaju skraćenice, pri prvom navođenju u tekstu treba napisati i njihov pun naziv.

Rezultate prikazati jasno i pregledno, sa odgovarajućom statističkom obradom.

Diskusija obuhvata interpretaciju dobijenih rezul-tata i njihovo upoređenje sa literaturnim podacima. Re-zultati i diskusija mogu se objediniti.

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

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

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

Obim rukopisaCeo tekst rukopisa: naslovna strana, kratak sadržaj,

uvod, eksperimentalni deo (materijal, metode), rezul-tati, diskusija, zahvalnost, literatura uključujući leg-ende (tabele, fotografije, grafikone, sheme itd.) mogu imati 5.000 reči za originalne članke; za saopštenja i pregledne radove 2.000 reči; za stručne izveštaje 1.500 reči a za ostale preglede 1.000 reči.

Broj tabela, slika, shema, crteža, grafikona (zajed-no) može biti najviše do polovine broja kucanih stranica rukopisa.

Tabele, slike, crteži, sheme, grafikoniSvaka tabela se kuca na posebnoj stranici prore-

dom 1,5, uključujući naslov, zaglavlje kolone i retka. Tabele se označavaju arapskim brojevima po redosledu navođenja u tekstu. Naslov tabele prikazuje sadržaj ta-bele. Upotrebu skraćenica u tabeli obavezno objasniti

u legendi tabele. Fotografije moraju biti isključivo crno-bele, oštrih kontura. Tekst (opis) slike kuca se na posebnom listu hartije. Crteže (sheme i grafikone) 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 finansijskoj.

LiteraturaLiteratura se kuca na posebnim stranicama jed-

nostrukim proredom, a dvostrukim između poje-dinih referenci, 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 jedi-nica. Reference se navode po ugledu na Vancouver sistem, koji se zasniva na principima

National Library of Medicine i Index 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 hemo-dialysis program: lack of influence 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 au-tora; ako ih je više, iza šestog se dodaje: i sar.

Knjige: 2.Weinstein L, Swartz MN. Pathologic properties

of invading microorganisms. Philadelphia: Saun-ders; 1974; 457-72.

Poglavlja u knjigama:3.Clayton D, Gill C. Covariate measurement er-

rors in nutritional epidemiology: effects and rem-edies. In: Margetts BM, Nelson M, eds. Design Con-cepts 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 po-

javu recidiva. 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, izjave):

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].

Houston: University of Texas, M. D. Anderson Cancer Center; c2007 [cited 2007 Feb 21]. Available from: http://www.mdanderson.org/departments/CIMER/.

Citat internet stranice sa autorima:8.Hooper JF. Psychiatry & the Law: Forensic Psy-

chiatric 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/.

the manuscript, number of the illustration and arrow indicating the top of the figure 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.

Acknowledgements (sources of funding, conflict of interest declaration, and authorship responsibilities): this should be included at the end of the text.

ReferencesReferences should be supplied on a separate sheet,

single spaced, with double space between each refer-ence, Arabic numbers indicating the sequence of ap-pearance.

The number of references should not exceed 30, except in literature reviews with maximum 50 is ac-ceptable. References are cited according to the so-called Vancouver style, based on formats being used by the National Library of Medicine and Index Medicus. (Srp Arh Celok Lek)

In citation of references the defined standards should be strictly followed because it is the essential factor of indexing and classification of scientific journals.

The following rules should be applied:Journals:1.Josselson J, Kyser BA, Weir MR, Sadler JH. Hepa-

titis B surface antigenemia in a chronic hemodialysis program: lack of influence on morbidity and mortality. Am J Kidney Dis 1987; 9(6):456-61. (The number of the volume is given in parentheses, the preceding num-ber indicating 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 invading microorganisms. Philadelphia: Saunders, 1974; 457-72.

Book chapter:3.Clayton D, Gill C. Covariate measurement errors

in nutritional epidemiology: effects and remedies. In: Margetts BM, Nelson M, eds. Design Concepts in Nutri-tional Epidemiology. Oxford: Oxford University Press, second edition 1997: 87-106.

Congress articles:4.Marković P, Živković L. Uticaj zračenja na pojavu

recidiva. 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

ustanovama za decu. Službeni glasnik RS, Beograd 1994; 50: 1643-9.

Standard citation of links:7.Complementary/Integrative Medicine [Internet].

Houston: University of Texas, M. D. Anderson Cancer 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 Psy-

chiatric 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/.

Documents

ČASOPIS DRUŠTVA ZA ISHRANU SRBIJE THE JOURNAL OF …hrana-ishrana.org/wp-content/uploads/2016/11/HRANA-I... · 2016-11-30 · za engleski jezik: Danica Pavlović ... Kolor oglas