FISHERIES AND MARINE SERVICE. Translation Series No The influence of antioxidant compounds on the stability of. medical fish-liver oil.

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1 FISHERIES AND MARINE SERVICE C.C1-11.VES Translation Series No The influence of antioxidant compounds on the stability of. medical fish-liver oil. by A. Fiebig, and J. Kozlowska Original title: Wplyw mieszanin przeciwut1eniaczy na trwalosc tranu leczniczego From: Ann. Acad. Med. Gedanensis (4): , 1974 Translated by the Translation Bureau(NWJ) Multilingual Services Division Department of the Secre.:ary of State of Canada Department of the Environment Fisheries and Marine Service Halifax Laboratory Halifax, N.S pages typescript

2 t 722 DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION ge etc i tv-rv jeef iz.iur Je CANA DA SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES TRANSLATED FROM - TRADUCTION DE Polish INTO - EN English AUTHOR - AUTEUR Fiebig Adolf, Kozlowska janina TITLE IN ENGLISH - TITRE ANGLAIS The influence of antioxidant compounds on the stability of medical fish-liver oil TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTLRES ROMAINS) Wplyw mieszanin przeciwutleniaczy na trwalosc tranu leczniczego REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE:FOREIGN CHARACTERS. RÉFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÈRES ROMAINS. Ann. Acad. Med. Gedan. REFERENCE IN ENGLISH - 'RÉFÉRENCE EN ANGLAIS PUBLISHER - ÉDITEUR Not given PLACE OF PUBLICATION LIEU DE PUBLICATION YEAR ANNÉE Poland 1974 DATE OF PUBLICATION DATE DE PUBLICATION VOLUME Not given ISSUE NO. NUMÉRO 4 PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS LORI GI NAL NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES 12 REQUESTING DEPARTMENT MINIST ẸRE.CLIENT BRANCH OR DIVISION DIRECTION OU DIVISION Environment Fisheries service Office of the Editor TRANSLATION BUREAU NO NOTRE DOSSIER N 0 TRANSLATOR (IN)TIA LS) TRADUCTEUR (INITIALES) N.W.J. PERSON REQUESTING DEMANDÉ PAR Dr. R.G. Ackman YOUR NUMBER VOTRE DOSSIER N 0 DATE OF REQUEST Sept. 26, DATE DE LA DEMANDE SOS E V. 2/ OS ) UNEDITFri TRANSLATION Co For infor, i..-zn enly TRADUCTION NON REV/SEE Informaiion seulement

3 -,-1b DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DiVISION CANADA SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES CLIENTS NO. DEPARTMENT DIVISION/BRANCH CITY NC) DU CLIENT MINISTÈRE DIVISION/DIRECTION VILLE Fisheries service Environment Office of the Editor Ottawa OUREAU NO. LANGUAGE nmslarm(imnals) NO DU BUREAU UNDUE TRADUCTEUR (INITIALES) NOV polish N W J.. Ann. Acad, Med. Gedan., 1974, No. 4, ( Poland ) THE INFLUENCE OF ANTIOXIDANT COMPOUNDS ON THE STABILITY OF MEDICAL FISH-LIVER OIL upeprup rramitaten ebfrieemefienvay By InâMeMION NON e.visee. Fiebig Adolf and Kozlowska Janina leemeionsintiermvnt DePartment cf Applied pharmacy, Institute of the Technology and Analysis of Drugs, medical Academy of Gdansk. Director of the Department: prof. dr. Adolf Fiebig u ni in a r y The ex-periments conducted for this study were concerned with determining the influence of antioxydant compounds with synergetical properties on the inhibition of oxidation in medical cod-liver oil. Their effectiveness was assessed by the accelerated ageing test. The length of time needed for the decomposition of Vitamin A was accepted as a criterion. The results obtained from the experiments indicate that a favourable effect can be gained by the utilization of phenol antioxydants in the form of compounds acting synergetically. The best preservative effect was shown by Tenox 2 and Antracine 20, i.e. solutions of antioxydant compounds such as butylohydroxyanisol, butylohydroxytoluene and gallates mixed with citric acid, dissolved in propylglycol. It 1.vas also established that the amount of antioxydant added to cod-liver oul requires a knowledge of the natural resistance of this oil t,o oxidation

4 The pharmacological usefulness of medical fish- 91 * liver oil depends,in the first place,on its content of Vitamins A and D. For this reason, fish-liver oil is a highly valuable preparation in the prevention and elimination of diseases caused by a deficiency of these vitamins. Other components, especially polyunsaturated fatty acids, considerably increase the utilization of figh-liver oil in medicine. Among other adventages, it has been noted that fish-liver oil has a significant ability to decrease the cholesterol in blood ( 8, 15 ). Its antibacterial properties ( 18, 21 ) are also not without value. The interest in fish-liver oil has not decreased. in spite of an increased production of synthetic Vitamins A and D. Fish-liver oil is listed in all the latest pharmacopeic publications. Recent reports show that liver oil is still a most valued medicine to combat rickets ( 5 ). However, the greatest disadventage of fish-liver oil is its limited stability. As fish-liver oil ages, not only do its biologically active substances decrease but it develops 92 products of oxidation which can be harmful ( 2, 12, 18, 19 ). For this reason, the proper preservation of fish-liver oil is a frequent subject of research. Amongst other methods, some attempts at using antioxidants to inhibit the oxidation process in fish-liver oil Numbers in the right-hand margin indicate the corresponding pages in the original.

5 3 have been undertaken ( 1, 3, 4, 17 ). Views as to the effectiveness of fish-liver oil preservation by means of antioxidants are varied. some authors ( 6, 23 ) believe that the use of antioxidants to preserve fish-liver oil is unnecessary, because, when properly stored, the oil retains its value for at least one year. However, according to / Creac h's opinion ( 3 ), the quantity of natural antioxidants in fish-liver oil is not sufficient to preserve entirely the vitamins contained, especially axerophthol. For this reason, it is necessary, in his view, to introduce gynthetic antioxidants to fish-liver oils. Kaufman ( 14 ) and Schou ( 20 ) express similar opinions with respect to fats. Créacsh recommends chemical compounds of the di- and polyphenol groups as the most effective antioxidaats in the, preservation of fish-liver'oil. The English pharmaceutical codex also considers the possibility of using this method ( 1963 ). At present, the most recent publications of the British pharmacopoeia ( 1968 ) officially approves the use of esters of gallic acid, as a means to preserve fish-liver oil. The Swiss pharmacopoeia VI ( 1971 ) sanctions the use of the above-mentioned compounds to preserve lipophilic pharmaceutical preparations. In this study phenol antioxidants in the forai of synergistic compounds were used to preserve fish-liver oils. Citric, ascorbic and tartaric acids are component parts of

6 4 synergistic compounds. Since these acids are capable of forming compounds or chelates with heavy metals, they are of considerable significance to the product which is being stabilized. The effectiveness of the antioxidants used was determined by the accelerated aging test which was adapted to assess the stability of medical fish-liver oil ( 16 ). The length of time required for the decomposition of vitamin A served as a criterion. EXPERIMMZT Two cod-liver oils and one redfish oil, produced in Poland and obtained from the Gdynia Fish Factory, as well as three imported cod-liver oils e purchased in the Gdansk Administration of pharmacies,were used as the material for our experiment. The content of vitamin A in these oils was measured with a spectrophotometer ( 16 ); other indicators were determined according to the methods accepted by the Polish pharmacopoeia IV ( FPIV ).The resistance to oxidation was determined by the accelerated aging test ( 16 ). The results obtained are presented in Table I. The results are 98 mean values of the three notations obtained. The following antioxidants were added to the oils: 0.02 % butylo4ydroxyanisol ( BHA ), 0.02 % propyl gallate ( PG ), 0.01 % nordihydltuuaiaretic acid ( NDGA ) together with 0.01 % ascorbyl palmitate ( PA ), as well as synergistic compounds in the form of solutions with propylglycol, consisting

7 5 of 20 % BHA, 6 % PG, 4 % citric acid ( Tenox 2 ), and 10 % BHA, 10 5 BHT, 6 % DG ( dodecyl gailate ), 4 % citric acid ( Antracine 20 ) ( 7 ). These solutions were introduced at a concentration of 0.1 %. Diagrams 1-12 present the results obtained from the experiments. Table 1. Charakterystyka tran6w u±ytych do badah Properties of the assayed liver oils Tabela 1 No. Rodzaj tranu Sort of the oil 1 Dorszowy Cod-liver oil 2 Dorszowy Cod-liver oil Witamina A j.m./g Vitamin A i.u./g Liczba nad-i Licz-. tlenkowa 1 ba Licz- Wsp6lczyn- (Liczba Lea)' jodoba. k-wa ink zalama Peroxide wa ma sl,viatia MW3 value Iodi- Acid Refractive (Lea's num- ne index ber) value value Odpornoge na utlenianie w min P.esistance against oxidation (mm) 812.8, ,61 1, , ,79 1, Olej z karmazyna 540 I 12,5 123 Redfish oil 1,14 I 1, Dorszowy impor ,2. towany 163 1,48 1, Imported cod-liver oil ', 5 Dorszowy impor , ,38 1, towany Imported cod-liver oil 6 Dorszowy importowany 832 8, ,88 1, Imported cod-liver oil

8 6 94 1,/ii A NTRAN 1 COD-L1VER OIL! Lien tenm TRAN I COD-LIVER OIL f o so m no m 150 mn Ryc. 1 Fig. 1 Ryc. 2 Fig. 2 Ma A TRAN 2 COD-LIVER OIL 2 4.1e0 tees» TRAN 2 COD-LIVER OIL I à 5:0 à Rye. 3 Fig.3 Ryc. 4 Fig.4

9 7 95 A TRAN 3 REDFISi4 OIL LLea 100'5» TRAN 3 REDPISII OIL i f 2 cs ce nrieb IS 60 is S'10 i05 fec ijs t.:93 Rye. 5 Fig. 5 Ryc. 6 Fig tt TRAN 4 COD-IIVER OIL 4 TPAN 4 COD -LIVER OIL 4 7:0 T IS 30 --r---r le rrun fe m.,) Ryc. 7 FI g. 7 Ryc. 8 Fig. 8

10 TRAN 5 COD-LIVER OIL 5 TRAN S COD-LI ver 01L5 2,04bri Rye. 9 Fige 9 Rye. 10 Fig. 10 TRAN 6 COD-L1VER OIL 6 riban6 COD - L 1 V,E2 OIL 6 me& Q 43 enfflio eommmi, 1 [ ;5 910 ' Rye. 11 Fig. 11 Rye. 12 Fig. 12

11 9 97 Rye Wyniki badafi testem przygpieszonego starzenia tran6w dorszowych, produkcji krajowej. Zrniana czasu rozkladu witaminy A w zale±nosci od zastosowanego przeciwutleniacza (ryc. 1, 3). Zmiana wskainika nadlenkow w zaleinoci od zastosowanego przeciwutieniacza (ryc. 2, 4). 1 tran bez przeciwutleniacza, 2 - tran + 0,09% BHA, 3 - tran+ 0,02% PG, 4 - tran + 0,01% NDGA + 0,01% PA, 5 - tran + 0,1% Teno k 2, 6 - tran + 0,1% Antracine 20 Fig Results of the accelerated ageing test on Polish cod-liver oils. Variation of the time needed for the determination of vitamin A according to antioxydant used (Fig. 1, 3). Variation of the peroxide values according to antioxydant used (Fig. 2, 4). 1-cod-liver oil without antioxydant, 2- cod-liver oil+ 0.02% BHA, 3-cod- -liver oil+ 0,02% PG, 4 - cod-liver oil+ 0,01% NDGA + 0,01 0/0 PA, 5 - cod-liver oil+ 0,1 0/0 Tenox 2, 6 - cod-liver oil+ 0,1% Antracine 20 Ryc Wyniki badari testem przygpieszonego starzenia oleju z karmazyna. Zmiana czasu rozkladu witaminy A w zaleinaci od zastosowanego przeciwutleniacza (ryc. 5). Zmiana wskainika nadtlenk6w w zaleinogci od zastosowanego przeciwutleniacza (ryc. 6). 1 - olej bez przeciwutleniacza, 2 - olej + 0,02% BHA, 3 - olej + 0,02% PG, 4 - olej + 0,01% NDGA+ 0,01 0/0 PA, 5 - olej +0,1 0/0 Tenox 2, 6 - olej + 0,1./o Antracine 20 Fig Results of the accelerated ageing test on redfish oil. Variation of the time needed for the deterioration of vitamin A according to antioxydant used (Fig. 5). Variation of the peroxide values according to antioxydant used (Fig. 6). 1 - oil without antioxydant, 2 - oil + 0,02 '0 BHA, 3 - oil + 0,02% PG, 4 - oil + 0,010/, NDGA + 0,01 0/, PA, 5 - oil+ 0,1% Tenox 2, 6 - oil+ 0,1% Antracine 20 Ryc Wyniki badan testern przygpieszonego starzenia tran6w dorszowych importowanych. Zmiana czasu rozkladu witaminy A w zaleinoaci od zastosowanego przeciwutleniacza (ryc. 7, 9. 11). Zmiana wskainika nadtlenk6w w zaleinosci od zastosowanego przeciwutleniacza (ryc. 8, 10, 12). 1 - tran bez przeciwutleniacza, 2 - tran + 0,G2 0.0 BHA. 3 - tran + 0,02 0 '0 PG, 4 - tran + 0,01% NDGA + 0,01% PA, 5 - tran + 0,1% Tenox 2, 6 - trait + 0,1% Antracine 20, 7 - tran + 0,2% Tenox 2. Fig Results of the accelerated ageing test on imported cod-liver oils. Variation of the time needed for the deterioration of vitamin A according to antioxydant used (Fig. 7, 9, 11). Variation of the peroxide values according to antioxydant used (Fig. 8, 10, 12). 1- cod-liver oil without antioxydant, 2 - cod-liver oil + 0,02% BHA, 3 - cod- -liver oil + 0,02% P.G, 4 - cod-liver + 0,01% NGDA + 0,01% PA, 5 - cod-liver oil + 0,10/0 Tenox 2, e - cod-liver oil + 0,1% Antracine 20, 7 - cod-liver oil + + 0,2 Tenox 2 DISCUSSION OF. THE RESULTS The study as a whole has shown that the utilized antioxidants had favourable, though different, influences on the inhibition of oxidizing processes in the examined oils. In all cases it was observed that the inductive period became longer ( Fig. 2, 4, 6, 8, 12 ) and the decrease of Vitamin A level became slower ( Fig. 1, 3, 5, 7, 9, 11 ). The diagrams

12 10 show that the addition of BHA or pg alone, has considerably less influence'upon the inhibition of the oxidizing processes than the use of Tenox 2 which is a solution of both these oxidants and citric acid as a.synergist. 98 Similarly, a good stabilizing effect was obtained from the use of Antracine 20, a solution of a slightly different composition. In both cases the entire decomposition of Vitamin A in all the examined oils took place mach later than in the oils treated with the other antioxidants. Since Tenox 2 introduced at a level of 0.1 % had a rather insignificant preservative effect in cod-liver oil No. 6 ( Fig. 11, 12 ) its content was increased to 0.2%. Figure 11 shows that the accelerated aging test has proved that this cod-liver oil has a very limited natural resistance to oxidation ( 30 minutes ). Therefore, it is obvious why Tenox 2, introduced at a concentration of 0.1 %, did not bring the expected effect. Some authors ( 11 ) do stress the nece. sity to increase the antioxdant concentration in the fats whose oxidation processes are more advanced. Nordihydroguaiaretic acid ( NDGA ) ( 7, 10, 17 ) is another antioxidant strongly recommended for the preservation of animal fats and preparations containing Vitamin A. It is stressed in the literature on this subject that when PA is applied together with the NDGA, this considerably intensifies its stabilizing effect ( 9, 22 ). The results of

13 11 of our study ( Fig ) show that the preserving powers of this solution are much weaker than those of Tenox 2 and Antracine 20. Its effect is rather similar to the results obtained by the use of PG in the examined fish-liver oils. The fact that the rish-liver oils exhibited different degrees or chemical stability when stabilized by the same antioxj:dants can be explained by the different content of natural antioxidants in them. The higher or lower amount of natural antioxidants in the examined fish-liver oils may depend on several factors such as, e.g. the initial material, the type of technology used for production, and also the duration of storage. Our research as a whole shows that by treating fish-liver oils with phenol antioxidants in the form of synergistic solutions such as Tenox 2 and Antracine 20 the oxidation processes in them can be considerably hindered and the Vitamin A content can be held for a longer time at its initial level. CONCLUSIONS 1. In the preservation of fish-liver oils,synergistic solutions proved to have more favourable effects than other antioxidants. 2. The best results were obtained from the use of Tenox 2 and Antracine The quantity of antioxidant to be added to fish-liver 99 oil depends on the natural resistance of the oil to oxidation.

14 12 BIBLIOGRAPHY 1. Byczkowski St., Vitamin A in cod,liver oils produced in poland. Polish Pharm. 1951, 7, Doboszynska B., Polish fish oils as a valuable source of Vitamin D. Polish pharm , Kaszubkiewicz Cz., Wartenberg L. Zwierzchowski J. ) Cases of the poisoning of silver foxes by rancid fish-liver oil. Vet. Med.,. 1957, 13, Kozlowska J., Characteristics of medical fish-liver oil in relation to the change of certain constants and to the decrease of vitamin A. Part I. Accelerated aging test as a quick method to evaluate the stability of medical fish-liver oil. Acta pol. Pharm., 1966, 23, stechnij 1., studies on the antibacterial activity of fish-liver oil. Acta pol. pharm. 1967, 24, 525.