Vegetable Oil Concentrates as Antioxidants for Vitamin A in Fish Liver Oils L.O.BGXTOS
I
.\utior~al Oil Prorlurts Conzpnny, I n r . , Hurrison. S. J .
Stability data on antioxidant concentrates, prepared from crude wheat germ, corn germ, and soybean oils by solvent extraction and tested in crude and carbon-treated soup-fin shark liver and halibut liver oils, establish that such concentrates are highly effective antioxidants for inhibiting peroxidation and vitamin .idestruction in such type oils. In all cases, arid for the same percentage of vitamin A destruction, much lower peroxide numbers w-ere observed in the oils containing the vegetable oil antioxidant concentrates than in the blank crude fish liver oils or carbon-treated oils. The crude vegetable oils, w-hen tested under similar conditions, displayed only minor powers to inhibit peroxidation arid ritamin -4destruction. The solvent-insoluble residual fractions, e\ en though exhibiting no antioxidant activity for inhibiting vitamin h destruction in the carbon-treated halibut liver oil, displayed greater activity for inhibiting peroxidation than the crude vegetable oils themselves.
GCH \Tork has appeared in the literature relating to the
1 Moxidative instability of glyceride-type oils and to the use of
cot tonreed and soybean oils and iiiiwd hydrogenated vegetable oils contain alkali-labile antioxidants other thaii the tocopherols, prohably chroman-5,B-i~uiiioncs. I n a latcr article Golumbic (8) states that the antioxygenic chrommS,6-quinonee which appear during the inductioti periorl in vegetable fats, but never during the oxidation of animal fats. retard the accumulation of fat peroxides in vegetable fats after the complete disappearance of the i,ocopherols. T h w r references are only a few of the many relating to the antiositl:mt qualities of vegetable oil materials which have appeared i r i the literature in recent years. This investigarion reports the antioxidant effect of crudc v~tgrtable oil materials for inhibiting vitamin d destruction aiid pvroxidp formation in samples of crude and carbon-treated soup-fin shark and halibut liver oils. S o attempt, has been made t u include antioxidant effectiveness data on solvent extracts prep:tred from all types of crude vegetable oils under different experinic~ntal conditions, or with all possible types of solvents or solvent niixtures. The only data included are on antioxidant concentrates prepared froni the more common crude vegei.able oils and with 01"2 aqueous isopropanol a t a low temperature.
synthetic, as well as natural-type antioxidants for inhibiting such CRUDE OILS undesirable deteriorative changes. S o attempt has been made t o rvvien. all of that work in this report. Previous studies if I from FISHLIVEROILS. T h r same lots of crude soup-fin shark antl this laboratory established that the antioxidants occurring naturhalibut liver oil -1from a previous study (2') w r e used in thia ally in crude fish liver oils were either removed or destroyed when irivrbtigation. The oils xere carbon-treated i r i exac the oils were treated with activated carbon in the presence of an nianiier as described previously. A partial :ma organic solvent for the oil. Furthermore, the addition of a-tocrude as i d 1 :is thr carbon-treated oils W:LS inrludid copherol or a-tocopherol plus lecithin to such carbon-mated fiqh article ( 2 ) . liver oils, or to the untreated oilb, failed to inhibit peroxid CRUDE VEGETABLE OILS. The n-heat germ oil and corii gcriii oil cffcctively in such oils, even though that type of antoisidants were commercial grades of solvent-extracted oils, obt:tinecl from markedly decreased the rate of vitamin A destruction (21. the VioBin Corporation. d commwrial lot 01' expeller soybcan .Ittempts to isolate and identify the individual, naturally o['oil was ohtained from A. E. Sta1c.y 3f:tnufactnring Company. curring antioxidant principles of crude veget,ablr and fish 1iyr.i The three oils were fresh and :vercl fi1tr:rod tidore use to i'rci. tliem oils have been only partially completed. 3Iost natural oils CIJJIfrom foreign ant1 .:mpended matter. \ W l e antioxidant solvent tain more than one active antioxidant component, and i n vegeextract concentrares from many ot,her different lots of wheat germ, table oils particularly tTvo or more components act synergistically 1 those corn germ. and soybean oils were also examined, as 1 ~ 1 :is to afford antioxidant protection. from numerous other varieties of vegetable oils, the variations Mattill (9) was t,he first to associate the stability of a fut n.ith were a matter of degree rathrr thnn a dernonstr:itiori of some clifthe presence of natural antioxidants in it. Since that date (19271 fereiit effect. A partial analysis of the crudc oils iiscd is inc~ludcd numerous data have been reported to the e in Table I. of crude antioxidant-containing oils is derr SOLVENTS treatments (1, If). Mattill and co-workers (13' showed that the antioxidant substances in rertairi vegc-tublv oils could be C O L I tigation on the use of so1vt:nt extriic't a i i ~ i i i ~ i ( h i i t 'om crude n-heat germ, C I I I n gc'i i n , anti M ) ~ ~ I C : L I J ceiitrated in the unsaponifiablc, fractions of such oils antl that their activity depcnded on free hytirosyl g w u p ~ . Stcrolz prtwiit in the unsapunitia1)lc frwtioiis n-ei'c' iound t o be del-oitl of antioxid:mt activit?.. 1I:ittill :fO) TABLEI. COlIPAR.4TIvE X S A L Y S I S O F C R U D E VEGETABLE 011,s i S D 91 5 gossypol l,l.esc.nt iri c,,uciecOtrol,firsr rrcorcicti ISOPROP.4XOL E X T R ~ C CT o S C E S T R A T E s PRO11 THE O I L S ed marlicti antioxidant activity. Ex:. Sesamol, a constituent of sesanie oil, is an effective K h ea t \' hea. i t Gerin Germ Boyhe:in S inhitit or for preventing oxidation ( 1 2 ) . Fan-cctt Oil Oil Oil Oil Oil 011 (6) demonstrated that, during thc molecular dis% iree i a t t y acids 3 6 23.6 1 1 8.4 1.8 3,j.I tillation of crude cottonseed oil, the antioxidslits % unsaponifiable 5.3 10.5 1 3 8.4 1 x 9 7 118 5 108.3 119 1 113.2 121.1 115 0 Iodine va1u.e are concentrated in both the first distillate and Saponification value 182 5 184 6 193 0 183.3 189 0 169.0 i n the residue. Golumbic i;) concludes that 1171
INDUSTRIAL AND E N G I N E E R I N G CHEMISTRY
1112
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example, solvent extract c o n c e n h t e s prepared from crude soybean, corn. \vliwt
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Vol. 39, No. 9
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aq u c o u s i so 11 r o p a n i j I about +20" c'. xri' slightly less effectivr a.3 mitimidant:, in fiali 1ivc.r oils tlmn extracts prepared \vith 01 c; aqueous is o 1) I'o 1) ii I I o 1 a t -18' C'. The IJercY'lltagc yield ohtaiiictl nit11 i l w former type solvi'iitb
is.
same type uils ivitli aqueous i J o p r o pan o 1 a I at,ou1 - 2 5 " ( 2 , or l l J \ \ C t are still more p u t i ' i i t as antioxidants. Thc solvmt c>stractconccrltlatl's ('all I>? ~ v a y st o improvc~thc. C I J ~ O I. remove frcv Fatty :icids, part or all of tiic p h phatides, anti t h o i1iartiv-c) sterols, alld I l l u s rut t h concciitratc the activc m t i oxidant principles. 1 Lo\vt ~ c r , in this stutiy ilii. i-I,ude solvent extrac.1 concentratcs ere used \vit,hout such further treatiiiciita. The general estr:ic.tioii method used for 1ir(,paritig the a i i t i o y i d a i i t uxti'act concentrates dtudicd in tliih i e p o r t is r e c o r d e d wlicre (31. This gciieral ostractioii nicthotl i> d S i J applicable fur c o n w i l t rating thu naturally ocrwriiig mitiosidarits in i i h h 1ivc.r oils ( ~ 7 ) . Uricdy. i I i c ~ tlctails of the riicmt Iiciil nri' as t'iI1ll)n.s:
O n t ~ 1lullctr.ed glallls ui' the crude vcgetahle oil t o be extracted ivcre \vc*ighcd into a 1000-id. Crleiinic~yer (:urvc 1.
Crude soup-tin shurh li\t.r oil Carbon-trrated soup-tin sliark liver oil (:ur\c 5. Carbon-treated soup-lin sharh li+rr oil 370 of sol\ent PIl r i l v t antioxidant concentrat- from crude s o ) l w a n oil
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2.
Carlion-treated ~ o u p - t i n s h a r h l i v e r oil t .57c of aolvrnt PXtract n n t i o x i d a u t concentrate from crude eoyhean oil C u r v e .5, Crude soup-fin shurl. liter oil L .ig of sol\cnt *\tract anti\idant concentrate from rrudr i o ) h i i n oil C; .\ d e s t r
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pre,wnce of nitrogen gas t o remove' all trave> of t l i t iolvcnt. The residual oil fractions insoluble in the solvent at thr low teniperature were also freed of solvent traces in a similar manner. Both the boluble cstract fractions and the solrent-insoluble oil fractions were stored in closed vials at +4' C. until they could be of the crude vegetable tested. Table I gives comparative a n oils used, a s well as of the solvent ex eoncentratcb prepared from these oils.
Samples of the crude soup-fin shark liver :ind halibut liver oils ~ ' e r carbon-trcatcd e in cyclohesanc using 2 0 5 of activated carbon ~ S u c l i a rSSS)esactly as reported previously ( 1 ) . T h e carbontreated samples were either used immcdiatt~lyor stored in completely filled closed vials at - 18" C. until the studiei \\.('re conducted. Portions of the crude soup-fin shark liver anti haliliut liver oils, thci same oils ear,lmn-treatecl, and the oils containing thc various vegetable oil materials to be tcsted for antioxidant properties. \\.ere studied for pcrcentage vitamin dtstructioii and pvrosidation a t 34.5" C. by the method outlined in a previous report ( 1 ) . T h e results on vitamin -1and perosidation for the various oils studied are summarized in Figures 2 and 3 and in Table 11. -in csperimeiit is sho\\-n in Table I1 in which 0.50c; a-tocopherol was addc,d to the carbon-treatrd halihut liver oil for comparison. The percentage of vegetable oil material addrd t o the fish liver oils and te6ted for comparative effectivcrirsh, as indicated by rate of peroside formation and percentage vitamin -1 destruction, \vas arrived at somewhat arbitrarily from preliminary studies. One of the aims was to add sufficient of tht. regetahle oil mlvent estract antioxidant concentrates t o the samples of carbon-treated oils to produce oils exhibiting approximately the same rate ah, or a sloa.er rate of vitamin A deqtructioii than that dicpla>-ed by the crude fish liver oils. Comparison of cwrves 1 and 3, Figure 2 , s h o w that \$-hen 355 of the solvc,nt extract of crude soybean oil \vas added to t h e carbon-treated soup-fin shark liver oil. the rcsulting oil exhibited about t h r same vitamin -1stability ab the crude soup-fin shark liver oil. This type of antioxidant concentrates is A \
1173
134 20.6
154 .8 i3
also eqtlally effective for inhibiting osiclaticiii of of oxidizable substancw (.3, . i ) . EFFECT OF PEROXII)4TIOY Oh' \-ITA>II\
iiiaiiy
otliilr tJyt's
..i UESTRIJ(TTIO~
Figure 1 arid Table I1 show the data obtained on rlie rtxlritiun betnwn peroxidation and percentage vitaniiii -1 tlcstructioti in the samples of crude and carbon-treated soup-fin d i w k liver aiid lialibut livrr oils containing thc various typw of rruclc. v(>getablc: oil+ antl fravtions studic~d. Thcwx rwults iritlicatc t h t , :it a g i w n pc.rcentage of vitamin A dcstruction, the ptrosidiz nunibci,s of the crude fish livor oils containing the solvent extract concentratrs and of the carlion-trcated oils cuntaining the crude vegetat)li, oils, t,he solvtwt-soluble cstract concentrates and the solvent-irisolublr residue fractions from the crude vegetable oils are consitlerably li)n-ei, than thtr pcroside numbcrs of the co saniplt,~. In t he earl)on-treatcd oils, the solvent-iri~iilulilcresidue iraetiuiis ai' types of oils. Thc ptwxntage yield of antioxidant extract cnitrentrate obtained from the n-heat germ oil was considerakily grwt than t h a t obtained from the soybean oil (10.9% as against 5 , 5 ( respectively). This may account in part for the slightly lo\vc.i. antioxidant, potency displaypd by the wheat germ oil extract coticentrate. From a percentage weight yield standpoint, t h(,re is actually more antioxidant in the solvent extract from ivhcat gt'rni oil than from,soyhean oil. Results in Table I1 indicat 3y0 concentration the three crude vegetahlc oils po minor ability t o protect the vitamin A in thr: carbon-treated halib u t liver oil under the conditions employed. The solvt.rit-inxoluble residue fractions exhibit no protective a(-tiori for vitainin .I under t,he same circumstances
conceritrates from tht, thret. crude vegetable oils studied was much sloiver than in the crude halibut liver oil. For example, after a btorage period of 9 days a t 34.5' C. the peroxide numbers of t h e (-rude halibut liver and the carbon-treated halibut liver oil conttiiriing 3 7 of the antioxidant extract concentrates from wheat gcrm arid soybean oils were 80.5, 12.0, and 5.6, respectively. The antioxidant c:xt ract concentrate from crude corn germ oil was slightly less active for inhibiting peroxidation in t,he carbon1 i ~ w t c . t I halibut 1ivc.r oil than in t h e carbon-treated soup-fin shark liver oil. This liehavior is undoubtedly due t o differences iri th(. di'grw and type of unsaturatiori and in tlie glyceride configuixtioil ( i f the, two fish livcr oils. Samples of the carbon-trcasted h:tlihut liver oil mritairiiiig 3 7 of the crude vegetable oils and the solventiiisolu1,lc residiii~i'i,sc.iions from the crude vegetable oils tiisp1;tycd . - l o \ v i ~ ijic:roxiciatioii ~ rates t h m did the lilank carbon-tri:atcd oil. ' I ' h c b rt,siilth indicate that crude wheat germ, corii germ, arid w,ylii,:iii oil.< arc' valuahle sources of antioxidants; from thrsm con. act ivc: for irihibiting vitamin -4 dwtructiori atrri 1 1 fiqli liver oils ea11 tic1 prc,pared by SOIVC iouglr thix crude vc~gc~tahlc oils, as such, po t o inhibit perosiciation aiid vitamin A dr .solvc,nt (1st r w t cwnct'ritratc< from t h(, said oils are highly effccativc. Thtx i ~ i s ~ i l ~ ir,csidutl l i l ~ ~ fr,aCtioiis from the crude vcgctahlc oils l ~ w antioxidant potcticy toivard preventing vitamin clestructioii, hut somewhat greater activity than the crude vc~gct aljle oils tlir~mrelvesfor inhibiting peroxide formation in the C M boil-treated halibut livcr oil. This fact indicates t h a t thc p!!oxide-inhibiting type antioxidants are not removed by the solvent (axtraction tre:rtnient t o the samp extent as are the vitamin A inhibiting tj-pe antiosidants. Certainly the solvent extr'act coilceiitratw contain large amounts of the active peroxide-inhibiting type antioxidants: otherwise these concentrates, bvhich arc known to contain the major proportion of the unesterified tocopherols present in the crude vegetable oils, even though they are effective in inhibiting vitamin destruction in the carbon-treated oils, would not have so effectively inhibited peroxide formation. Some of the antioxidant activity of the concentrates is the result of t,ht. synergistic action between the phosphatides, which are present in large aniounts in the extracts, and ot,her antioxidant components also present, such as the tocopherols and chroman-5,6-quinone. Further work is required along these lines before a clear undcrstanding of all the factors involved can be realized. ACKNOWLEDGMENT
The author i~rprc~+~es thanks t o H. J. Konen, now of Schenley Distillers, Inc., for many helpful suggestions and assistanctl in collecting thc data rc,portcd here. LITERATURE
EFFECT O F TIME OY P E R O X I I ~ EF O K I ~ T I O N
oiidary i n iniportancc~ t o The inhibition of pcrosidatioii is the prevention of vitamin A losses in fish liver oil< OII cotilact with air. Antioxidants of the type of a-tocopheid o i ' ~ - t n c n ~ i h i ~ ~ . o I plus lecithin are ineffective for preventing pcroxi(l:i~iiiiii n f i q l i liver oils ( 2 ) . Figure 3 a n d Table I1 shoiv thc results obtained i n this investigation on the peroxidation rate of t h r v:triour w i i p l c i studicd. Curws 3 and 4, as compared with cui'vc' 1, demoiistrate clearly that the soybean antioxidant extract fractions a t a 3 and 5 ~ c l c v ein l the carbon-treated soup-fin shark liver oil arc cxtrenic~ly potent in peroxide-inhibiting principles. .&ftcr 125 hours tlica peroxidation rate of the sample containing 5rc \vas much slo\\-cr than in the samplc~containing 3cc. T h e blank c:trhoii-trc.ated soup-fin sha1.k liver oil (curv(: 2, Figure 3) exhibited a straiglit liiic of oxidation, \\-liich indicates the absence of active ailtioxidants. The rate of peroxide formation in the samples of carbon-trmted halibut livcr oil (Table 11) containing the antioxidant (%\;tract
cum
H u s t o i i . I,. 0 . . I s n . I ~ G ~ .H E J I . 34, , 1486 (1942) Itik!.. 39.2 2 5 fl91Ti. Bustoii,'L. O . ' ( t o S a t l . Oil Products Co.) U. S. Patents 2,345.5 i t i a i i i l 2,345,578 (.1pr. 4, 1944). I h i d . . 2,:345,579,2,345,580, 2,315,960, 2,345,961 (Apr. 4, 1944): 2,389,955 (KO\-.27. 1945) ; other patents pending. I h i i i h r o w . B. .1.(to Natl. Oil Products C o . ) , I h i d . , 2,380,41X
f.JnI>-31, 1045\. I~'a\wett.I.. K. 11...l. SOC.Chem. I n d . , 158,43T (1939). Golutiihii,, C . . .I. A m . Chem. Soc., 64, 2337 (1942). Goluiiil~i~~, C., Oil & S o a p , 20, 105 (1943). Am. .\led. Assoc., 89,1505 (1927). Mattill, H. .I.,J . B i d . Chem., 90,141 (1931). Mattill, H. A,! and Crawford. B., IND. ENG.CHEM.,22, 311 (1930); Hilditch, T . P., arid Sleightholrne, J. J., J . Soc. C'hpm. I n d . , 51,39T (1032); Banks, A , and Hilditch, T. P.. Ihid.. 51, 411T (1932); Elder, L. W., Jr., Oil &- Soup, 18, .4nn. chim. applicata, 30, 141 (1940). :38 (1941): ('iu.a, W., Olrott, H . S . , and Mattill, €I. A , , C'hem. Revs..29,257 (1941). Olcott, H . S.,and Mattill, H. -4.. J . Biol. Chem., 93,65 (1931), 104,423 (1934) ; J . Am. Chem. Soc., 58,1627 (1936); Bradway. E. M.,and Mattill, H. A . , I b i d . , 56, 2405 (1934).
