Ascorbic Acid - ACS Publications - American Chemical Society

22 Antioxidant Properties of Ascorbic A c i d i n Foods WINIFRED M. CORT

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Ascorbic acid and its esters function as antioxidants with some substrates by protecting double bonds and scavenging oxygen. Activity of ascorbates has been shown on vegetable oils, animal fats, vitamin A, carotenoids, citrus oils, and in fat-containing foods such as fish, margarine, and milk. Ascorbates also scavenge oxygen out of aqueous solutions and out of certain oxygen-containing compounds. This oxygen scavenging ability has resulted in ascorbic acid addition to beer, wine, meat, and bread. Ascorbic acid also lowers the oxidation state of many metals and valence may thus affect oxidation catalysis. The efficiency of ascorbates as antioxidants is dependent upon the substrate and the compounds to be protected. Because the 2- and 3-positions of ascorbic acid must be unsubstituted, the two free radicals formed at these positions may be intermediates in scavenging oxygen and inhibiting radical formation at double bonds.

A

ntioxidants a r e l i s t e d i n the C o d e

of F e d e r a l R e g u l a t i o n s ( C F R )

• u n d e r f o o d a n d c h e m i c a l preservatives (1,2).

T h e listing includes

p h e n o l i c s s u c h as b u t y l a t e d h y d r o x y a n i s o l e ( B H A ) i n d e x e d i n C h e m i c a l Abstracts

as

[phenol,

(l,l-dimethylethyl)-4-methoxy-25013-16-5]

butylated hydroxytoluene ( B H T )

m e t h y l 128-37-0] as w e l l as t e t r a b u t y l a t e d h y d r o x y q u i n o n e [(1,4-benzenediol,

and

[phenol, 2 , 6 - b i s ( l , l - d i m e t h y l e t h y l ) - 4 -

2 - ( l , l - d i m e t h y l e t h y l 1948-33-0)],

(TBHQ)

the thiodipropio-

nates, tocopherols, bisulfites, a n d ascorbates. O n l y the l a t t e r t w o scavenge o x y g e n o u t of s o l u t i o n .

T h e rest of t h e a n t i o x i d a n t s h a v e b e e n

used

p r e d o m i n a n t l y i n fats a n d oils, a n d i n most cases, w i t h t h e e x c e p t i o n of tocopherols

a n d ascorbyl palmitate ( A P ) ( I )

[l-ascorbic

ester

6-hexa-

decanoate 137-66-6], a t t h e l e g a l l i m i t of 0 . 0 2 % of t h e f a t . P r o p y l gallate 0065-2393/82/0200-0533$06.00/0 © 1982 American Chemical Society

In Ascorbic Acid: Chemistry, Metabolism, and Uses; Seib, P., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

534

ASCORBIC

(PG)

[benzoic

a c i d , 3 , 4 , 5 - t r i h y d r o x y p r o p y l ester

121-79-9],

ACID

although

l i s t e d w i t h t h e other a n t i o x i d a n t s i n 1973, is n o w l i s t e d i n 184.1660 as a n a n t i o x i d a n t w i t h a m a x i m u m l e v e l of 0 . 0 1 5 % i n f o o d . E t h o x y q u i n ( E M Q ) [ q u i n o l i n e , 6 - e t h o x y - l , 2 - d i h y d r o - 2 , 2 , 4 - t r i m e t h y l 91-53-2] has b e e n

used

i n a n i m a l feeds a n d the c a r r y - o v e r i n a n i m a l s is l i m i t e d to 5 p p m . E M Q is a l l o w e d i n p a p r i k a a n d c h i l i p o w d e r at 100 p p m . N o r d i h y d r o g u a i a r e t i c a c i d ( N D G A ) , a l l o w e d p r i o r to 1968, is n o w i l l e g a l a n d foods c o n t a i n i n g it are d e e m e d a d u l t e r a t e d . T h e use of ascorbic a c i d ( I I ) , as w i t h other a n t i o x i d a n t s , i n v o l v e s


the

specific

substrate i n t o w h i c h t h e y

are i n c o r p o r a t e d .

I n certain

c i r c u m s t a n c e s , a d d i t i v e s that are present i n foods a n d beverages i n a c t i v a t e d a g i v e n a n t i o x i d a n t w h e n u s e d i n the same f o r m u l a t i o n . I n other cases, Certain a n t i o x i d a n t s r e a c t e d c h e m i c a l l y w i t h the m a t e r i a l s t h e y w e r e i n t e n d e d to s t a b i l i z e .

L-ascorbyl palmitate HO

L-ascorbic acid

OH

HO

OH

HO-CH CH OC(CH ), CH 2

2

4

3

II

A s c o r b i c a c i d w a s s t u d i e d i n fats m a n y years ago a n d r e v i e w e d b y Chipault (3)

w h o r e p o r t e d s o m e a c t i v i t y , alone a n d w i t h other a n t i

oxidants, i n l a r d , cottonseed o i l , meats, fish, m a y o n n a i s e , v e g e t a b l e fats, b a k e d a n d f r i e d foods, m i l k p o w d e r s ,

a n d i r r a d i a t e d foods.

In

these

systems h e reports that ascorbic a c i d d o u b l e s t h e s t a b i l i t y of l a r d i n t h e presence of either t o c o p h e r o l or N D G A . A s c o r b i c a c i d w a s u s e d to p r e v e n t the o x i d a t i o n of o l i v e o i l , m i l k , arachis n u t o i l , l a r d , e t h y l ester of l a r d , cottonseed o i l , p o r k , a n d b e e f fat ( 4 ) ;

d a t a s h o w i n g a c t i v i t y alone a n d as a synergist are r e v i e w e d .

A s c o r b y l l a u r a t e , m y r i s t a t e , p a l m i t a t e , a n d stearate w e r e s i m i l a r l y a c t i v e , a l t h o u g h o n l y A P is l i s t e d as a p r e s e r v a t i v e i n C F R .

Ascorbic

acid

s y n e r g y w i t h t o c o p h e r o l w a s also r e v i e w e d . T a p p e l ( 5 ) s h o w e d that ascorbic a c i d acts s y n e r g i s t i c a l l y w i t h f o o d a n t i o x i d a n t s a n d because of t h e great increase i n effectiveness r e s u l t i n g f r o m a s m a l l a m o u n t of ascorbic a c i d , he suggested m i x t u r e s w o u l d b e effective i n p r e v e n t i n g o x i d a t i v e r a n c i d i t y i n meats, p o u l t r y , a n d fish.


22.

CORT

535

Antioxidant Properties of Ascorbic Acid

Ingold (6)

d i d not r e v i e w ascorbic a c i d p e r se, b u t d i d present a

c h a p t e r o n m e t a l catalysis i n c l u d i n g the m e t a l content o f v e g e t a b l e oils a n d t h e effect of v a l e n c e state of the metals o n o x i d a t i o n o f fats a n d oils. H e reports cobalt, manganese, c o p p e r , i r o n , a n d z i n c at the h i g h e r valences a c t e d c a t a l y t i c a l l y t o o x i d i z e m a n y substrates. T h e a u t h o r d i s c u s s e d t h e a n t i o x i d a n t a c t i v i t y of ascorbic a c i d i n r a d i a t i o n - i n d u c e d free r a d i c a l s , fats i n emulsions,

fluid

milk, and frozen

fish.

H e also discussed

the

q u a n d a r y of m e t a l reactions vs. v a l e n c e state. U r i ( 7 ) has p r e v i o u s l y n o t e d that F e


Fe

2 +

+ R O O H -> F e

3 +

2 +

reacts w i t h p e r o x i d e s :

+ OH"+ RO•

thus, the o r g a n i c peroxides s u c h as l i n o l e i c h y d r o p e r o x i d e s are

decom

p o s e d to c h a i n - i n i t i a t i n g a l k o x y r a d i c a l s . W h e n ferrous p h t h a l o c y a n i n e is a d d e d to a s a t u r a t e d s o l u t i o n of q u e r c e t i n i n e t h y l benzoate t r e a t e d w i t h m e t h y l l i n o l e a t e h y d r o p e r o x i d e , a d e e p r e d c o l o r forms t h a t is a t t r i b u t e d to the f o r m a t i o n of a n o - q u i n o n e at t h e 3' -f- 4 ' - p o s i t i o n of t h e flavone.

A l s o , b l e a c h i n g of

/^-carotene

i n e t h y l benzoate

by

methyl

l i n o l e a t e h y d r o p e r o x i d e does not take p l a c e u n t i l ferrous ions are a d d e d a n d t h e n this b l e a c h i n g is c o m p l e t e d w i t h i n 10 m i n . T h e f o r m a t i o n of the a l k o x y r a d i c a l i n this r e a c t i o n is the basis of the o x i d i z i n g p o w e r , w h i c h U r i refers to as " r e d u c t i v e a c t i v a t i o n . " I n his o u t l i n e of a c t i v i t y of different m e t a l catalysts he lists C o , M n , a n d C e 2 +

2 +

3 +

as most a c t i v e .

H o w e v e r , he shows r e a c t i o n of a l l the l o w e r o x i d a t i o n states of t h e metals w i t h p e r o x i d e . O n the other h a n d , a l l the metals i n the l o w e r o x i d a t i o n state, that is, C o , F e , V 2 +

2 +

2 +

, C r * C u , M n , r e a c t e d w i t h o x y g e n to f o r m 2

+

2 +

the h i g h e r v a l e n c e state. I n c e r t a i n systems a s c o r b i c a c i d w a s so effective i n l o w e r i n g t h e v a l e n c e state of metals that it w a s u s e d i n a n a l y t i c a l c h e m i s t r y Ascorbic acid was used w i t h gold, lead, bismuth, tellurium,

(8).

copper,

p h o s p h o r u s , u r a n i u m , halogens, m e r c u r y , a n d cobalt. I n a d d i t i o n to the reports o n the a s c o r b i c a c i d effect o n fats a n d metals, there are n u m e r o u s reports that i n c e r t a i n m e d i a ascorbic a c i d m a y r e m o v e o x y g e n f r o m s o l u t i o n ; p h e n o l i c a n t i o x i d a n t s are not effective i n scavenging.

Experimental Studies reported here use standard assay methods for antioxidants such as thin layer tests (Schaal oven) and the active oxygen method ( A O M ) (9) assayed by measuring peroxide formed, and an emulsion system using hemo globin peroxidation of safflower oil measuring removal of oxygen from solution ( 1 0 ) . Experiments on oxygen scavenging were performed i n all glass equip ment and in glass bottles with metal closures with a measured volume of headspace air. Experiments on additional substances, vitamin A , carotenoids, and citrus oil also were performed in thin layer tests measuring the loss of vitamin A


536

ASCORBIC ACID

at 325 n m and apocarotenal at 460 n m . Experiments w i t h water solutions and sodium nitrite were also included. Ascorbic acid derivatives including ascorbic acid 2-sulfate ( I I I ) , ascorbyl palmitate and laurate, sodium ascorbate, 2,3-di-Omethylascorbic acid ( I V ) , benzoylascorbic acid, isopropylideneascorbic acid ( V ) , and 3-0-[(dimorpholino)phosphinate]-5,6-0-isopropylidene L-ascorbate ( V I ) were also studied i n the test systems. A l l figures reported here were collected i n duplicate and were w i t h i n a 1 0 % variation.

L-ascorbic acid-2-sulfate (potassium salt)

2,3-di-O-methylascorbate


P 3 CH

H co

HQ OS0K

3

3

CHOH 2

IV

III

3 - 0 - [ (dimorpholino) phosphinate]-5,6-0isopropylidene-L-ascorbate 5,6-isopropylidene L-ascorbic acid n

0

C H 3

\ /0—CH

CH /%4V 3

r~\ / \ / 0 N /3 2\ \_/ U 0 3 \ /0 H,C 0—CH

H

C

C

VI

Results A

series of a n t i o x i d a n t e x p e r i m e n t s i l l u s t r a t i n g c o m p a r a t i v e

anti

o x i d a n t a c t i v i t y i n t h e specific systems tested is p r e s e n t e d i n T a b l e s I - I X . Because

m a n y of

t h e f o o d - g r a d e a n t i o x i d a n t s e x c e p t ascorbates

tocopherols are l i m i t e d i n t h e U n i t e d States to 0 . 0 2 %

and

o f t h e fat o r o i l ,

T a b l e I compares t h e a c t i v i t y i n s o y b e a n o i l at t h a t l e v e l .

Although

a s c o r b i c a c i d a n d A P are m o r e a c t i v e t h a n B H T a n d B H A , t h e y are n o t


22.

as a c t i v e as P G a n d T B H Q . ascorbates.

537


CORT

T a b l e I I compares t h e a c t i v i t y o f v a r i o u s

S u b s t i t u t i o n i n t h e 2- or 3-position o f a s c o r b i c a c i d m a k e s

i t n o l o n g e r a c t i v e as a n a n t i o x i d a n t , w h e r e a s c o m p o u n d s

substituted i n

the 6- o r 5,6-positions are a c t i v e a n t i o x i d a n t s . I n T a b l e I I I , a n t i o x i d a n t activities of a s c o r b i c a c i d a n d A P are greater t h a n B H T a n d B H A i n safflower, sunflower, p e a n u t , a n d c o r n o i l . O n p o r k a n d c h i c k e n f a t , w h i c h a r e l o w i n tocopherols, t h e ascorbates a r e less effective t h a n B H T a n d B H A ( T a b l e I V ) ; ascorbates are synergistic w i t h tocopherols ( I I ) . W h e a t g e r m o i l w a s s t u d i e d i n s i m i l a r experiments, a n d i t r a p i d l y


forms peroxides ( T a b l e V ) . A s c o r b i c a c i d a n d A P w e r e m o r e

efficient

a n t i o x i d a n t s here t h a n w a s B H T . T o investigate t h e effect o n w h e a t , w h o l e berries w e r e a d d e d to w a t e r a n d m i x e d w i t h 0.1 g of a n t i o x i d a n t s / 100 g o f berries a n d t h e n d r i e d at 6 0 ° C o v e r n i g h t . T h e d r i e d mass w a s p u l v e r i z e d i n a W a r i n g b l e n d e r a n d 10 g w a s a d d e d t o p e t r i dishes a n d t i t r a t e d d a i l y f o r p e r o x i d e after e x t r a c t i o n i n t o c h l o r o f o r m - a c e t i c

Table I.

acid.

Comparative Antioxidant A c t i v i t y in Soybean O i l

Antioxidant

(0.02%)

Days to Reach PV* 70

None BHT BHA PG TBHQ Ascorbic acid AP

5 8 7 15 25 10 14

Note: a-Tocopherol content 10 mg % , total tocopherols 95%, thin layers, 45°C. ° P V is peroxide value (milliequivalents per kilogram). Table II.

Comparative Antioxidant A c t i v i t y of Ascorbic A c i d Derivatives in Soybean O i l Antioxidant

(0.05%)

Days to Reach PV 70

None L-Ascorbic acid D-Isoascorbic a c i d ( e r y t h o r b i c ) L-Ascorbyl palmitate L-Ascorbyl laurate D-Isoascorbyl l a u r a t e L-Ascorbic acid 2-sulfate 2,3-Di-O-methyl-l-ascorbic acid 3-Benzoyl-L-ascorbic acid 5,6-Isopropylidene-L-ascorbic acid 2,3:4,6-Di-0-isopropylidene-2-keto-L-gulonate 3 - 0 - [ (Dimorpholino) phosphinate] - 5 , 6 - O - i s o p r o p y l i dene-L-ascorbate

5 17 17 21 21 21 5 5 5 23 5 5

Note: Thin layers, 45°C.


538

ASCORBIC

Table III.

ACID

Comparative Antioxidant A c t i v i t y in Vegetable O i l Substrate Oil

Antioxidant

Safflower

Sunflower

Peanut

Corn

6 10 8 11 10

6 9 8 10 10

15 15 15 26 NR"

12 13 15 21 NR"

None B H T (0.02%) B H A (0.02%) A P (0.01%) Ascorbic acid (0.02%)


Note: Thin layers, 45°C. Data given as days to reach P V 70. ° N R = not run. Table IV.

Comparative Antioxidant A c t i v i t y in Animal Fats

Antioxidant

(0.02%)

Pork Lard

None BHT BHA Ascorbic acid AP a-Tocopherol a-Tocopherol and A P

Chicken

3 18 26 8 9 15 28

Fat

5 15 20 10 10 13 28

Note: Thin layers, 45°C. Data given as days to reach P V 20. Table V .

Comparative Antioxidant A c t i v i t y in Wheat G e r m O i l Day

Antioxidant None B H T (0.02%) A P (0.2%) A P (0.06%) A s c o r b i c a c i d (0.06%) B H T (0.06%)

1

2

3

4

11

10 8 0 0 0 0

35 29 18 0 0 0

86 76 50 NR' NR' NR"

102 110 98 0 0 70

NR* NR* NR" 70 12 NR"

Note: Thin layers, 45°C, data given as peroxide values. ° N R = not run. U n t r e a t e d controls r e a c h e d p e r o x i d e v a l u e ( P V ) 70 i n 11 d ; those w i t h B H T t r e a t m e n t l a s t e d 20 d , w i t h T B H Q 24 d , a n d w i t h A P 31 d . Results o n c o m p a r a t i v e a n t i o x i d a n t efficiency

on

crude p a l m o i l

( T a b l e V I ) a g a i n s h o w a s c o r b i c a c i d to be v e r y a c t i v e . Studies m a d e o n curiosity ( T a b l e V I I ) .

h u m a n fat were performed

s i m p l y because

of

O x i d a t i o n w a s not v e r y r a p i d , b u t t h e fat c o n

t a i n e d 14 m g % t o c o p h e r o l s (8.5 m g %

g a m m a a n d 5.5 m g %

alpha).

A P h a d a c t i v i t y o n this fat t h a t m a y or m a y not b e c a u s e d b y s y n e r g i s m


22.

539


CORT

Table V I .

Comparative Antioxidant A c t i v i t y in Crude Palm O i l Antioxidants

Days to Reach 70 PV 33 44 45 60 45 60 100

None B H T (0.02%) B H A (0.02%) P G (0.02%) T B H Q (0.02%) A s c o r b i c a c i d (0.02%) A s c o r b i c a c i d (0.1%)


Note: Thin layers, 45°C.

Table VII.

Compartive Antioxidant A c t i v i t y in H u m a n Fat PV on Days Shown

Antioxidant None B H T (0.02%) B H A (0.02%) P G (0.02%) T B H Q (0.02%) a-Tocopherol (0.02%) A P (0.02%) A P (0.1%) A P (1.0%)

^

7

16

21

89

64

5 0 2 0 0 3 0 0 0

19 0 18 0 0 19 0 0 0

24 0 23 0 0 26 13 0 0

52 12 55 4 0 60 43 0 0

425 42 270 18 0 426 317 63 0

,

Reach 20 21 45 21 78 ND* 21 28 64 ND"

Note: Thin layers, 45°C, human fat obtained from new cadaver rendered in auto clave. Fat filtered through cheesecloth and Whatman N o . 2 paper. ° N D = not detected in 64 d.

of the tocopherols, a p h e n o m e n o n

to b e discussed later.

The unusual

result is the d r a m a t i c effects of T B H Q . T h e A O M test, i n w h i c h a i r is b u b b l e d at a constant rate at 9 8 ° C , shows a s c o r b i c a c i d a n d A P to be a c t i v e ( T a b l e V I I I ) . T h e

unexpected

result here is t h e v e r y large a c t i v i t y of a s c o r b i c a c i d . H o w e v e r , B e r n e r et a l . (12)

r e p o r t e d a n A O M v a l u e of 8.3 for B H A , w h i c h o n the a d d i t i o n

of a s c o r b i c a c i d at 0 . 0 3 % b e c a m e 44.2; this v a l u e w a s t h e i r h i g h e s t A O M v a l u e . I n a recent r e v i e w , P o r t e r ( 1 3 ) n o t i c e d this a n o m a l y a n d i n c l u d e d i t i n his t h e o r y of l i p o p h i l i c , a m p h i p h i l i c classification of a n t i o x i d a n t s . T o d e t e r m i n e q u i c k l y i f a c o m p o u n d h a d a n y a c t i v i t y as a n a n t i o x i d a n t , a h e m o g l o b i n - c a t a l y z e d e m u l s i o n test (10)

was developed

at t h e same t i m e a s i m i l a r test w a s d e v e l o p e d b y B e r n e r et a l . ( 1 2 ) . t h e system (10)

and In

the a n t i o x i d a n t s w e r e a d d e d at 100 /xg a n d c o m p a r e d to

B H T at the same c o n c e n t r a t i o n . A s c o r b i c a c i d a n d A P are w e a k i n this s y s t e m b u t t h e y e x h i b i t some a c t i v i t y , w h e r e a s

ascorbates

substituted


540

ASCORBIC

Table VIII.

Comparative Antioxidant

Antioxidant

Activity

Soybean Oil

Crude Palm Oil

5 11 9 14 13 26 43 NR" 150

11 31 30 50 NR" 17 NR' 51 95

None B H T (0.02%) B H A (0.02%) P G (0.02%) A P (0.02%) T B H Q (0.02%) Ascorbic acid (0.02%) C i t r i c acid (0.1%) Ascorbic acid (0.1%)


ACID

Note: A O M , 98°C, data given as hours to reach P V 70. N R = not run. a

Table IX.

Antioxidant A c t i v i t y Activity (%BHT)°

Antioxidant" None BHT BHA PG TBHQ AP Ascorbic acid 2,3-Di-O-methylascorbic acid Ascorbic acid 2-sulfate

PV Formed in 10 min

0 100 200 108 100 64 5 0 0

30 28 10 22 25 14 14 30 30

Note: Emulsion test, hemoglobin peroxidation. 100 Atg added to 20 m L 10% stripped sarBower oil emulsion. a

i n the 2- a n d 3-positions h a v e n o a c t i v i t y . T h e d a t a p r e s e n t e d h e r e corroborate

Porter's

theory

because

the

oil-soluble

antioxidants

m o r e a c t i v e i n the e m u l s i o n system a n d the m o r e w a t e r - s o l u b l e

are com

p o u n d s , s u c h as a s c o r b i c a c i d a n d c i t r i c a c i d , are m o r e a c t i v e i n the a l l o i l systems. T o c o p h e r o l w a s effective a n d a s c o r b i c a c i d ineffective i n the p r o t e c t i o n of c i t r u s oils e v a l u a t e d b y a r o m a ( 1 3 ) .

I n a t y p i c a l s t u d y , 5 g of

orange o i l w a s o x i d i z e d i n 7 5 - m L o p e n b r o w n bottles at 45 ° C a n d w a s e v a l u a t e d b y a p a n e l after 6 d , at w h i c h t i m e i t w a s r a n k e d as off-odor, "terpeney."

T h e p e r o x i d e v a l u e of the i n i t i a l o i l w a s z e r o ; the o x i d i z e d

m a t e r i a l h a d a P V of 100. A s a result, days to r e a c h 100 P V w a s u s e d as an endpoint.

C o m p a r a t i v e a n t i o x i d a n t effects o n a n u m b e r

of

citrus

oils a n d o n D - l i m o n e n e [ c y c l o h e x e n e , l - m e t h y l - 4 - ( l - m e t h y l e t h e n y l ) - ( R ) 5989-27-5] are p r e s e n t e d i n T a b l e X . B H A is the m o s t a c t i v e w h i l e A P has n o a c t i v i t y alone b u t does s y n e r g i z e w i t h t o c o p h e r o l .


22.

CORT

541


V i t a m i n A w i t h its five c o n j u g a t e d d o u b l e b o n d s o x i d i z e s to

5,8-

epoxides ( 1 5 ) w i t h s u b s e q u e n t loss of U V a b s o r p t i o n at 325 n m . T h u s , v i t a m i n A w a s s t u d i e d w i t h t h e a d d i t i o n of v a r i o u s a n t i o x i d a n t s ( T a b l e X I ) i n o p e n bottles i n t h i n layers. A l t h o u g h E M Q w a s t h e best, i t is l i m i t e d to use i n v i t a m i n A for a n i m a l feeds. A P a c t i v i t y w a s not great, b u t w h e n a d d e d to a m i x t u r e of t o c o p h e r o l , B H T , a n d d i e t h a n o l a m i d e , A P gave excellent p r o t e c t i o n . K l a u i (16)

has s h o w n s t a b i l i t y of v i t a m i n A

p a l m i t a t e w i t h t o c o p h e r o l , A P , a n d a n a m i n e e q u a l to 1300 h , c o m p a r e d to a c o n t r o l e q u a l to 100 h . H o w e v e r , the a n t i o x i d a n t s i n the d r y m a r k e t


f o r m ( b e a d l e t s ) p r o t e c t the v i t a m i n A i n the beadlets as w e l l as t h e e n d use of the p r o d u c t .

F o r example, spray-dried vitamin A can be

Table X . Antioxidants*

pro-

Citrus Oils Antioxidant A c t i v i t y Orange Orange (Fla.) (Calif.)

None BHT BHA PG eH-a-Tocopherol AP Tocopherol and A P

6 35 44 18 15 6 21

4 32 49 36 10 4 16

Lemon

Grapefruit

Lime

9 24 91 17 9 9 19

3 27 34 20 7 3 7

9 9 15 9 15 9 15

v-Limonene 5 > 56 > 56 > 56 15 5 30

Note: Data given as days to reach 100 P V , 45°C. Antioxidants at 0.02% ; sample size, 5 g in 75-mL open bottles. a

Table X I .

Comparative A c t i v i t y on Antioxidants on Crystalline Vitamin A Acetate Days 2

5

8

12

15

62 None BHT 89 BHA 57 57 PG TBHQ 88 di-a-Tocopherol 89 AP 70 EMQ 98 B H T (at 1.2 g) 87 B H T , Tocopherol, A P , diethanolamine 98 Tocol mix" 95 B H T a n d either e t h a n o l a m i n e d i e t h a n o l a m i n e T w e e n 60 or 80 100

27 79 30 28 82 70 40 91 81 93 89

15 73 19 15 80 57 23 89 74 88 81

10 59 12 10 71 42 18 84 59 85 81

52 9 7 70 33 10 75 56 75 79

89

85

67

60

Antioxidant'

7

Note: Sample size, 5 g in 100-mL open brown bottles, 45°C. Data given as percent retention. A t 100 mg/bottle unless noted. M i x = a-tocopherol 230, diethanolamine 460, Tween 80,1151 mg. a

6


542

ASCORBIC

ACID

d u c e d w i t h o n l y t o c o p h e r o l f o r p r o t e c t i o n , a n d t h e v i t a m i n is stable f o r s e v e r a l years.

However,

if the vitamin

is m i x e d i n flour w i t h t h e

moisture encountered at the mills ( 1 2 - 1 3 % ), i t must have B H T to sur vive (17). Carotenoids

s u c h as /?-apo-8'-carotenal

with

its n i n e

conjugated

d o u b l e b o n d s also f o r m epoxides a n d lose a l l t h e i r c o l o r o n o x i d a t i o n . T h u s , e x p e r i m e n t s u s i n g c o l o r loss at 460 n m w e r e p e r f o r m e d t o s t u d y a n t i o x i d a n t a c t i v i t y . T a b l e X I I shows

that y-tocopherol

followed by

B H A a n d a - t o c o p h e r o l a r e t h e best a n t i o x i d a n t s f o r a p o c a r o t e n a l , a n d i f


A P is a d d e d , b o t h t o c o p h e r o l s a r e t h e v e r y best.

Ascorbic acid a n d A P

b o t h h a v e some a n t i o x i d a n t a c t i v i t y f o r a p o c a r o t e n a l w i t h o u t t o c o p h e r o l . Oxygen

scavenging

experiments

were

performed

on

compounds

d i s s o l v e d i n o x y g e n a t e d d i s t i l l e d w a t e r i n b r o w n bottles w i t h 2 c m of 3

h e a d s p a c e , t e s t i n g a l l t h e c o m p o u n d s at 20 m g / b o t t l e .

T h e bottles w e r e

s h a k e n i n a r o t a r y shaker at 150 r p m f o r 19 h at r o o m t e m p e r a t u r e . I m m e d i a t e l y after o p e n i n g , o x y g e n i n s o l u t i o n w a s r e a d o n t h e O r i o n Electrode

(Orion

Research)

(Table

XIII).

A d d i t i o n a l experiments,

read w i t h i n 5 m i n , showed that the inorganics reacted immediately; h o w ever, after b u b b l i n g i n a i r f o r 5 m i n , o n l y t h e s o d i u m sulfite c o n t i n u e d to k e e p o x y g e n i n s o l u t i o n at zero.

Table X I I .

A s c o r b i c a c i d a n d cysteine

Comparative A c t i v i t y of Antioxidants on Apo-8'-carotenal Day (room temperature)

Day (Jt5°C) Antioxidant* None BHT BHA PG Ascorbic acid AP di-a-Tocopherol dZ-a-Tocopherol dZ-a-Tocopherol ascorbic a c i d dl-y-Tocopherol dJ-y-Tocopherol dJ-y-Tocopherol ascorbic a c i d

took

and A P and

and A P and

4

6

8

12

16

20

28

28

72 100 100 95 80 96 100 100

59 92 100 95 78 88 100 100

40 88 98 92 52 78 95 98

16 70 88 85 29 46 92 92

6 40 72 65 7 16 72 75

2 >1 7 25 59 40 52 36 1 3 1 5 48 28 65 42

80 91 95 93 87 91 95 100

100 100 100

100 100 100

100 100 100

95 100 92

78 82 78

62 72 72

43 48 56

96 96 96

100

100

100

92

75

70

55

96

Note: 200 jig/g in coconut oil. Data given as percent ret ention. 3.3 g Petri plate 150 X 15 mm. ° Antioxidants at 200 fig/g.


22.

CORT

543


Table XIII. Oxygen Scavenging Experiment Effect of Compounds on Residual Oxygen in Solution Compounds Added" None Na S 0 Na S20 Na S0 L-Ascorbic acid Sodium-L-ascorbate 2,3-Di-O-methylascorbic acid 3 - B e n z o y l ascorbate 5,6-Isopropylidene-L-ascorbic acid 3-0-[ (Dimorpholino) phosphinate]-5,6-O-isopropylidene-L-ascorbate Potassium-L-ascorbate 2-sulfate Cysteine H C l C y s t e i n e free base D-Isoascorbic a c i d Dehydro-L-ascorbic acid 2

2

4

2

5


2

3

Oxygen (ppm)

Odor

8 0-1 0-1 0 0 0 8 8 0 8

OK S0 -like S0 -like OK OK OK OK OK OK OK

8 5 0 0 8

OK + odor - j - odor OK OK

2

2

Note: 50-mL brown bottles, closed metal lids, 2 cm of headspace air, shaken 150 rpm for 19 h, room temperature. 20 mg/bottle. 3

a

1 7 - 2 4 h of s h a k i n g i n c l o s e d bottles to c o m e t o c o m p l e t i o n .

As previ

o u s l y s h o w n , i n fat o x i d a t i o n t h e 2- a n d 3-positions of a s c o r b i c a c i d h a v e to b e u n s u b s t i t u t e d to a l l o w o x y g e n s c a v e n g i n g .

M e a s u r e m e n t of

UV

a b s o r p t i o n s h o w e d a n o p t i c a l d e n s i t y at 290 n m of 1.0 a n d at 260 n m of 1.4 f o r t h e s o d i u m ascorbate; these r e a d i n g s m i g h t i n d i c a t e f o r m a t i o n

of

t h e free r a d i c a l i n t h e 2 - p o s i t i o n , w h i c h r e p o r t e d l y absorbs at 290 n m (18). A s c o r b y l p a l m i t a t e , l a u r a t e , a n d stearate w e r e tested i n this sys t e m . H o w e v e r , t h e i r w a t e r s o l u b i l i t i e s w e r e so l o w t h a t t h e y d i d not f u n c t i o n as o x y g e n scavengers u n t i l the p H w a s a d j u s t e d t o 9 to s o l u b i l i z e a l l three esters. S u b s e q u e n t l y t h e y r e m o v e d a l l of the o x y g e n f r o m s o l u t i o n at 20 m g / b o t t l e w i t h 2 c m

3

of h e a d s p a c e a i r .

T h e o r e t i c a l l y 3.3 m g of a s c o r b i c a c i d w i l l c o n s u m e t h e o x y g e n i n 1 cm

3

of h e a d s p a c e

air (19).

A d d i t i o n a l experiments w e r e

performed

w i t h m e a s u r e d a m o u n t of h e a d s p a c e a i r a n d a s c o r b i c a c i d ( T a b l e X I V ) . T h e s e experiments w e r e p e r f o r m e d i n 3 7 - m L bottles w i t h m e t a l l i d s i n a shaker at 150 r p m for 19 h . Six m i l l i g r a m s of a s c o r b i c a c i d r e m o v e d o x y g e n i n the bottles w i t h 1 c m i n the bottles w i t h 2 c m

3

3

of h e a d s p a c e a n d r e d u c e d the o x y g e n

of h e a d s p a c e .

A s c o r b i c a c i d assays m e a s u r e d

b y a b s o r p t i o n at 260 n m i n d i c a t e a l a c k of m a t e r i a l b a l a n c e .

Therefore,

a d d i t i o n a l studies w e r e p e r f o r m e d i n a l l glass bottles ( T a b l e X V ) .


In

544

ASCORBIC

ACID

this e x p e r i m e n t 3.4-3.6 m g of a s c o r b i c a c i d w a s r e q u i r e d for e a c h c u b i c c e n t i m e t e r of h e a d s p a c e , w h i c h is close to t h e t h e o r e t i c a l v a l u e . I n a d d i t i o n a l e x p e r i m e n t s not i n c l u d e d i n this t a b l e , m e t a l caps w e r e i n s e r t e d i n t o the a l l glass e q u i p m e n t ; less t i m e w a s r e q u i r e d to r e m o v e the o x y g e n f r o m t h e solutions a n d m o r e a s c o r b i c a c i d p e r c u b i c c e n t i m e t e r of h e a d space w a s r e q u i r e d . W a t e r solutions

of

ascorbic

acid measured

i n the

U V required

g a s s i n g w i t h n i t r o g e n to s l o w d o w n t h e d e s t r u c t i o n of a s c o r b i c


Nitrogen-gassed

ascorbic

a c i d solutions

spectrophotometrically

at 3 6 0 - 2 0 0

optical density (peak,

260 n m ) .

nm

10 ju,g/mL

were

24 h

no

at for

with

B y comparison,

acid.

scanned

change

10-jug/mL

in

solutions

of a s c o r b i c a c i d , c o n t a i n i n g 8 p p m o x y g e n i n a l l glass e q u i p m e n t w i t h 10 c m

3

of h e a d s p a c e a i r , w e r e s c a n n e d h o u r l y also. W i t h i n 6 h t h e U V

p e a k w a s r e d u c e d , n o n e w peaks w e r e f o r m e d , a n d m o r e t h a n 9 0 % of the a s c o r b i c a c i d w a s gone. A n a e r o b i c d e g r a d a t i o n of a s c o r b i c a c i d i n g r a p e f r u i t j u i c e is a z e r o o r d e r r e a c t i o n ( 2 0 ) , b u t d e g r a d a t i o n of fish is e i t h e r first or zero o r d e r d e p e n d i n g o n the t y p e of fish (21).

Table X I V .

A s c o r b i c a c i d d e g r a d a t i o n i n peas is

Residual Oxygen in Solution Ascorbic

Headspace

(cc)

0 1 2 3 5 7 10

Acid

Content

(mg)

3

6

9

0.0 1.2 2.2 3.6 4.7 7.0 8.0

0.0 0.0 0.2 2.8 5.1 6.0 7.0

0.0 0.0 0.0 0.3 1.4 5.0 6.0

Note: Closed bottles, metal lids, shaken 19 h at room temperature. Data given as parts per million. Table X V .

Oxygen Scavenging by Ascorbic A c i d with Reduced Metal Contamination Residue Ascorbic Acid

Ascorbic

Acid

none 3 mg 6mg 9 mg

Oxygen

8.0 0.4 0.0 0.0

(ppm)

(mg)

Ascorbic Acid Consumed (mg)

0.0 1.3 4.2 7.2

0.0 1.7 1.8 1.8

Note: 0.5 c m of headspace, glass flasks and stopper; shaken 24 h at room tem perature. 3


22. a

CORT first-order

545

Antioxidant Properties of Ascorbic Acid degradation

(22,23).

F i r s t - o r d e r d e g r a d a t i o n of

ascorbic

a c i d w a s s h o w n i n l i m i t e d o x y g e n , b u t o x y g e n h a d a p r o f o u n d effect o n t h e rate ( 2 4 ) ; these

findings

agree w i t h o u r d a t a . Studies o n

ascorbic

a c i d i n r e l a t i o n to w a t e r a c t i v i t y i n d i c a t e t h a t the l a c k of w a t e r i n the A O M p r o b a b l y prevents a s c o r b i c a c i d f r o m b r e a k i n g d o w n , a l l o w i n g i t to r e m a i n active. F u r t h e r studies o n the a b i l i t y of a s c o r b i c a c i d to scavenge o x y g e n b y r e a c t i n g w i t h n i t r o g e n h a v e b e e n of interest. A s c o r b i c a c i d a d d e d

to

s o d i u m n i t r i t e solutions i m m e d i a t e l y p r o d u c e s b u b b l e s , w i t h the release


of n i t r i c oxide.

Mergens

s t u d i e d the a d d i t i o n of a l l of t h e p r e

(26)

cursors of a s c o r b i c a c i d synthesis as w e l l as the 6- a n d 5,6-derivatives d i s cussed here. H i s system is s i m i l a r to t h a t r e p o r t e d p r e v i o u s l y (27).

The

loss of n i t r i t e w a s m e a s u r e d w i t h i n 1 m i n f r o m a 7 m M s o l u t i o n of so d i u m n i t r i t e . A s c o r b i c d e r i v a t i v e s i n the 6- a n d 5,6-positions w e r e a c t i v e , a n d a l l precursors

were inactive, i n nitrite decomposition.

More

re

c e n t l y M e r g e n s f o u n d t h a t III a n d V I are i n a c t i v e . T h u s , r e a c t i o n w i t h n i t r i t e p a r a l l e l s the r e q u i r e m e n t for o x y g e n s c a v e n g i n g a n d a n t i o x i d a n t i n fats, n a m e l y t h a t the 2- a n d 3-positions

of a s c o r b i c

acid must

be

u n s u b s t i t u t e d a n d r e m a i n as the e n e d i o l or e n e d i o l a n i o n to b e a c t i v e .

Table X V I

om Nitrogen Dioxide by Ascorbates

Oxygen Scavenging Active

Inactive

A s c o r b i c a c i d precursors glucose, sorbose, 2 - k e t o g u l o n i c , etc.

L-Ascorbic acid

AP 5,6-Isopropylidene-L-ascorbic acid

L - A s c o r b i c a c i d 2-sulfate

d-Isoascorbic a c i d

3-0-[ (Dimorpholino) phosphinate]5,6-O-isopropylideneascorbic acid

(erythorbic)

Discussion Metal Effects and Prooxidant Action. i n s o m e situations. K a n n e r et a l . (28)

A s c o r b i c a c i d is p r o o x i d a n t

s h o w e d that C u

2 +

i n c r e a s e d the

o x i d a t i o n of l i n o l e a t e u s i n g loss of ^-carotene as a n i n d i c a t o r .

However,

w h e n sufficient a s c o r b i c a c i d w a s a d d e d to his system, c o p p e r catalysis w a s reversed. F u r t h e r m o r e , w h e n F e

3 +

w a s a d d e d , the a d d i t i o n of ascor

b i c a c i d i n c r e a s e d the p r o o x i d a n t effect.

Previous publications

(29)

h a v e d i s c u s s e d t h e d e a c t i v a t i o n of c o p p e r catalysis b y a s c o r b i c a c i d , b u t i n iron-catalyzed oxidation, F e formed from F e

3 +

2 +

initiates o x i d a t i o n of l i p i d ( 2 ) .

Fe

2 +

is

b y a s c o r b i c a c i d . M a n y foods c o n t a i n metals, a n d t h e


546

ASCORBIC

a d d i t i o n of a s c o r b i c a c i d w i l l l o w e r t h e i r v a l e n c e .

ACID

These lower valence

metals m a y cause p r o b l e m s . T h e r o l e of a s c o r b i c a c i d i n e n z y m e systems has b e e n k n o w n f o r a long time.

T h e ascorbic

a c i d requirement was

a-ketoglutaric a c i d , oxygen, a n d F e

reviewed along

with

i n p r o p y l a n d lysyl hydroxylases

2 +

a n d the p o s s i b i l i t y t h a t a s c o r b i c acid's f u n c t i o n is to k e e p to i r o n as F e (30).

2 +

M o r e r e c e n t l y researchers c l a i m t h a t a s c o r b i c a c i d does n o t p a r

t i c i p a t e i n the h y d r o x y l a t i o n r e a c t i o n b u t is s p e c i f i c a l l y r e q u i r e d to k e e p the enzyme F e

2 +

i n the r e d u c e d f o r m (31).

The peptide-bound proline


is t r a n s f o r m e d to h y d r o x y p r o l i n e a n d a - k e t o g l u t a r a t e to s u c c i n a t e a n d c a r b o n d i o x i d e . A s c o r b i c a c i d as a cofactor f u n c t i o n s to k e e p i r o n as F e i n n o t o n l y p r o p y l h y d r o x y l a s e b u t also l y s y l h y d r o x y l a s e , p h e n y l pyruvate hydroxylase, indoleamine-2,3-dioxygenase,

2 +

p-hydroxyand a-keto-

g l u t a r a t e a n d 5 - h y d r o x y m e t h y l u r a c i l dioxygenases. O n e of the most i n t e r e s t i n g papers o n a s c o r b i c a c i d - C u showed that ascorbic a c i d - C u

2 +

reactions

2 +

catalyzes the f o r m a t i o n of e t h y l e n e f r o m

s e v e r a l precursors. T h e interest i n e t h y l e n e was as a n a b s c i s s i o n agent i n plants.

A l l alcohols,

aldehydes,

ethylene w h e n m i x e d w i t h C u

2 +

acids,

ethers,

and

epoxides

formed

a n d a s c o r b i c a c i d i n 5 - m L c l o s e d bottles

at 30 ° C for 1 h . M e t h i o n a l w a s the most a c t i v e , f o l l o w e d b y p r o p a n a l , p r o p a n o l , p r o p y l ether, e t h y l ether, a n d e t h a n o l . T h i s r e a c t i o n m a y p a r t of the o x y g e n s c a v e n g i n g system b e c a u s e C u acid's

ability

to

scavenge

oxygen.

The

authors

be

increases

ascorbic

c l a i m this

reaction

2 +

c a n n o t b e a t t r i b u t e d to c o p p e r i n its l o w e r v a l e n c e state. E i t h e r F e n t o n reagent or a m i x t u r e of a s c o r b i c a c i d , F e , a n d e t h y l 3 +

enediaminetetraacetic

a c i d c a t a l y z e d t h e p r o d u c t i o n of

acetyldehyde

from ethanol, ethylene from methional derivative, a n d methane d i m e t h y l sulfoxide (34).

from

T h e authors c l a i m e d t h a t b o t h h y d r o x y r a d i c a l s

a n d singlet o x y g e n w e r e f o u n d as i n t e r m e d i a t e s , a n d , i n d e e d ,

ascorbic

a c i d s c a v e n g e d b o t h h y d r o x y l r a d i c a l a n d singlet o x y g e n . I n c e r t a i n foods a n d

Ascorbic A c i d and Tocopherol. ascorbic

a c i d a n d A P s y n e r g i z e d other a n t i o x i d a n t s a n d

beverages tocopherols,

a n d s o m e of this d a t a w a s r e v i e w e d ( 5 , 3 5 ) .

B o t h a- and y-tocopherol,

s i m i l a r to a s c o r b i c a c i d , react w i t h C u

3 +

2 +

a n d F e , a n d as a r e s u l t a s c o r b i c

a c i d m a y sacrificially stabilize tocopherols

(36).

H o w e v e r , ascorbic a c i d

t r a n s f o r m s « - t o c o p h e r o x i d e to a - t o c o p h e r o l a n d t h e d i m e r i c k e t o ether to " b i - a - t o c o p h e r y l , " thus r e g e n e r a t i n g t h e t w o a n t i o x i d a n t species oxidized tocopherols

(37).

P a c k e r et a l . (38)

o x y r a d i c a l w i t h ^ - t o c o p h e r o l u n d e r p u l s e r a d i o l y s i s to f o r m r a d i c a l ( a b s o r b s at 425 n m ) .

from

mixed trichloromethylpertocopherol

W h e n ascorbic a c i d was added,

ascorbic

r a d i c a l f o r m e d , a b s o r b i n g at 360 n m ; t h e 4 2 5 - n m a b s o r p t i o n w a s lost. T h e r a p i d i n t e r a c t i o n m a y r e c y c l e t o c o p h e r o l at t h e expense of a s c o r b i c acid.


22.

CORT

547


I n b i o l o g i c a l systems, o x i d a t i o n m a y p r o c e e d

Biological Systems.

t h r o u g h s u p e r o x i d e r a d i c a l 0 ~ , w h i c h forms f r o m m o l e c u l a r o x y g e n b y 2

a d d i t i o n of a single e l e c t r o n ( 3 9 ) . S u p e r o x i d e d i s m u t a s e ( S O D ) acts o n 0 ~ t o y i e l d h y d r o g e n p e r o x i d e a n d o x y g e n , w h i c h reacts w i t h a n o t h e r 2

0 " to p r o d u c e h y d r o x y r a d i c a l . T h i s l a t t e r r e a c t i o n is c a t a l y z e d b y F e ; 3 +

2

t h e h y d r o x y r a d i c a l s are c o n s i d e r e d most toxic.

S u p e r o x i d e r a d i c a l is

p r o d u c e d b y r e s p i r a t i o n a n d a n u m b e r of e n z y m e s , i n c l u d i n g x a n t h i n e oxidase, a l d e h y d e oxidase, d i h y d r o - o r o t i c a c i d d e h y d r o g e n a s e ,

galactose

oxidase, i n d o l e a m i n e a n d 2 - n i t r o p r o p a n e dioxygenases, d i a m i n e oxidase,


and

ribulose-l,5-diphosphate

carboxylase.

O x i d a t i o n of

many

com

p o u n d s , i n c l u d i n g h y d r o q u i n o n e , flavins, t h i o l s , c a t e c h o l a m i n e s , d i a l u r i c acid, herbicides, paraquat, a n d carbon tetrachloride, produce 0 " . 2

Pol

l u t e d a i r f r o m b u r n i n g gasoline, p a p e r , a n d t o b a c c o a n d o z o n e - c o n t a m i n a t e d a i r contains 0 ~ . 2

A s c o r b i c a c i d reacts w i t h 0 " g e n e r a t e d f r o m t h e x a n t h i n e oxidase 2

system a n d m a y p l a y a role against 0 " m e d i a t e d t o x i c i t y . A s c o r b i c a c i d 2

quenches the h y d r o x y l radical ( 4 0 ) . A s c o r b i c a c i d m a y protect against free r a d i c a l s i n t h e l u n g b e c a u s e a s c o r b i c a c i d is f o u n d i n t h e fluid ( 3 9 ) . T h e t o x i c i t y of o z o n e a n d o x y g e n m a y also b e r e d u c e d b y a s c o r b i c a c i d (39).

C a r b o n t e t r a c h l o r i d e m o r t a l i t y i n rats is l o w e r e d b y a s c o r b i c a c i d .

A u t o x i d a t i o n of a s c o r b i c

a c i d d i d n o t generate

0 ". 2

Reduced gluta

t h i o n e reacts w i t h d e h y d r o a s c o r b i c a c i d ( V I I ) a n d recycles a s c o r b i c a c i d .

dehydroascorbic acid

HO-CH

I

C H OH 6 * 0

VII Review of Ascorbic A c i d Mechanisms of Action.

Ascorbic acid a n d

A P h a v e a n t i o x i d a n t a c t i v i t y i n fats, oils, v i t a m i n A , a n d carotenoids. I n these systems A P is a better a n t i o x i d a n t t h a n a r e t h e p h e n o l i c a n t i o x i dants B H T a n d B H A , b o t h f r o m these d a t a a n d others ( 2 9 , 3 5 ) .

Ascorbic

a c i d protects against o x i d a t i o n of flavor c o m p o u n d s i n w i n e , beer, f r u i t s , artichokes, a n d cauliflower (29) presumably b y oxygen scavenging. T h e w e l l - k n o w n f o r m a t i o n of n i t r i c o x i d e f r o m nitrites b y a s c o r b i c a c i d is u s e d n o t o n l y f o r i n h i b i t i o n of n i t r o s a m i n e f o r m a t i o n , b u t also t o p r o m o t e

American CnemicaT Society Library 1155Metabolism, 16th St. and N.Uses; W. Seib, P., et al.; In Ascorbic Acid: Chemistry, Advances in Chemistry; American Chemical Washington, DC, 1982. Washington, D.Society: C. 20036

548

ASCORBIC

the f o r m a t i o n

of

nitrosometmyoglobin

and nitrosomyoglobin

ACID

to

keep

meat red. A s c o r b i c a c i d , a c t i n g as a n a n t i o x i d a n t i n c e r t a i n systems, scavenges o x y g e n out of s o l u t i o n at a r a t i o of 3.4r-3.6 m g / c m

3

of h e a d s p a c e a i r ,

w h i c h is close to the t h e o r e t i c a l v a l u e . I n a l l of the a s c o r b i c a c i d reactions s u b s t i t u t i o n i n the 5- a n d

5,6-

positions does not interfere w i t h its a c t i v i t y . S u b s t i t u t i o n i n e i t h e r t h e 2or 3-position or b o t h makes a s c o r b i c a c i d n o l o n g e r a c t i v e as a n o x y g e n scavenger or as a n a n t i o x i d a n t . O l c o t t a n d L i n (41) Downloaded by UNIV ILLINOIS URBANA on May 16, 2013 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch022

certain antioxidants b y

a t t e m p t e d to l e a r n the m e c h a n i s m of a c t i o n of s t u d y i n g stable free r a d i c a l n i t r o x i d e s .

They

m e a s u r e d the e l e c t r o n p a r a m a g n e t i c resonance ( E P R ) s i g n a l a n d as l o n g as the n i t r o x i d e s i g n a l w a s o b t a i n e d , o x i d a t i o n w a s i n h i b i t e d . W h e n t h a t s i g n a l was n o l o n g e r d e t e c t e d , o x i d a t i o n p r o c e e d e d .

L a t e r (42)

EMQ

n i t r o x i d e p r e v e n t e d the o x i d a t i o n of squalene a n d w h e n t h e E P R n i t r o x i d e s i g n a l was no l o n g e r d e t e c t e d , o x i d a t i o n p r o c e e d e d . T h e y c o n t e n d e d t h a t the free r a d i c a l of the a n t i o x i d a n t keeps the the a l k y l free r a d i c a l i n the l i p i d f r o m f o r m i n g ; therefore, peroxides c a n n o t f o r m .

This explana

t i o n is not u n i v e r s a l l y a c c e p t a b l e , a l t h o u g h the f o r m a t i o n of a r a d i c a l p r i o r to p e r o x i d e f o r m a t i o n of fats a n d oils has b e e n a c c e p t e d f o r m a n y years. O r r (43) effect of C u

2 +

u s e d d i m e t h y l sulfoxide as a free r a d i c a l sink to i n h i b i t the a n d a s c o r b i c a c i d o n catalase a n d ^ - g l u c u r o n i d a s e as w e l l

as the d e g r a d a t i o n of h y a l u r o n i c a c i d . T h e f o r m a t i o n of a r a d i c a l f r o m ascorbic a c i d a n d C u

2 +

i n w a t e r was d e t e c t e d b y E P R (44).

Based on

a n E P R s p e c t r o s c o p i c s t u d y of a s c o r b i c a c i d d u r i n g o x i d a t i o n of m e t h y l arachidonate-enriched

liposomes,

ascorbic

acid may

be

important

p r e v e n t i n g free r a d i c a l d a m a g e i n the c e n t r a l nervous system

F o r m a t i o n of the free r a d i c a l of a s c o r b i c a c i d ( m e a s u r e d at 360 accompanied (18,46)

r e d u c t i o n of t o c o p h e r o l

s h o w e d that the a s c o r b i c

free r a d i c a l s (38).

in

(45). nm)

B i e l s k i et a l .

a c i d free r a d i c a l i n t h e 3-position

absorbs at 360 n m a n d t h a t i n the 2-position absorbs at 290 n m ; also, a s c o r b i c a c i d reacts w i t h superoxide. I n c e r t a i n systems a s c o r b i c a c i d free r a d i c a l o n the 2- a n d 3-positions m a y b e a n i n t e r m e d i a t e i n the a n t i o x i d a n t f u n c t i o n , b u t i t is a s h o r t - l i v e d i n t e r m e d i a t e (18).

A n e l e c t r o n s p i n resonance flow system w a s u s e d to

s t u d y s c a v e n g i n g of a n i t r o s a t i n g agent b y a s c o r b i c a c i d , a n d a t o t a l s p i n free r a d i c a l , w h i c h is the same as t h a t p r o d u c e d b y r a d i o l y t i c o x i d a t i o n (48),

was d e t e r m i n e d

(47).

T h e e v i d e n c e i n this c h a p t e r is that the 2- a n d 3-positions of a s c o r b i c a c i d m u s t be u n s u b s t i t u t e d a n d a v a i l a b l e for a s c o r b i c a c i d to act as a n o x y g e n scavenger or a n a n t i o x i d a n t .


22.

CORT


549

Acknowledgments T h a n k s are g i v e n t o J a n e J e r n o w a n d E . O l i v e t o f o r p r o v i d i n g t h e ascorbic acid derivatives a n d to M . Mergens for experimental data.


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31. "The Role of Ascorbic Acid in the Hydroxylation of Peptide-Bound Pro line," Nutr. Rev. 1979, 37, 26. 32. Hayaishi, O.; Nozaki, N.; Abbott, H. T. In "The Enzymes"; Boyer, P. D., Ed.; Academic: New York, 1974; Vol. 12, p. 119. 33. Lieberman, M.; Kunishi, A. T. Science 1967, 158, 938. 34. Cohen, G.; Cederbaum, A. I. Arch. Biochem. Biophys. 1980, 199, 438. 35. Klaui, H. In "Vitamin C: Recent Aspects of Its Physiological and Techno logical Importance"; Birch, C.; Parker, K. J., Eds.; John Wiley & Sons: New York, 1974; pp. 16-39. 36. Cort, W. M.; Mergens, W.; Green, A. J. Food Sci. 1978, 43, 797. 37. Schuldel, P.; Mayer, H.; Isler, O. In "The Vitamins"; Sebrell, W. H.; Har ris, R. S., Eds.; Academic: New York, 1972; pp. 168-225. 38. Packer, J. E.; Slater, T. F.; Willson, R. L. Nature 1979, 278, 737. 39. Leibovitz, B. E.; Siegel, B. V. J. Gerontol. 1980, 35, 45. 40. Fessenden, R. W.; Verma, N. C. Biophys. J. 1978, 24, 93. 41. Olcott, H. S.; Lin, J. S. "Free Radical Intermediates of Antioxidants," 66th Meet. AOCS, Dallas, TX, 1975. 42. Lin, J. S.; Olcott, H. S. J. Agric. Food Chem. 1975, 23, 798. 43. Orr, C. W. M. Biochemistry 1967, 6, 2995. 44. Moravskii, I. Proc. Acad. Sci. USSR Chem. Sect. 1977, 1, 61; Chem. Abstr. 1977, 86, 111618C. 45. Demopoulu, S. H.; Flamm, E.; Seligman, M.; Power, R.; Pietronigro, D.; Ranschoff, J. Oxygen Physiol. Fund., Proc. Am. Physiol. Soc. Colloq. 1977, 491-502; Chem. Abstr. 1979, 90, 927105. 46. Bielski, B. H. J.; Richter, H. W. J. Am. Chem. Soc. 1977, 99, 3019. 47. Kalus, W. H. Filby, W. G. Experientia 1980, 36, 143. 48. Laroff, G. P.; Fessenden, R. W.; Schuler, R. H. J. Am. Chem. Soc. 1972, 94, 9026. 49. Jernow, J.; Blount, J.; Oliveto, E.; Perrota, A.; Rosen, P.; Toome, V. Tetrahedron 1979, 35, 1483. RECEIVED for review January 22, 1981. ACCEPTED June 22, 1981. ;


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