Debate Paper

Frindp,~s (Lakowicz, I.R., nd.), pp. 128-176, Plenum Press 11 Beechem, J.M. and Brand, L (1985) A,,-mu. Rev. B/ochem. 54, 43-71 24 12 Demchen~, A.P. (1986) U/~'av/o/et . ~ of Pro~/ns, 2S

Spdn~er-Veda8 26 13 Burstein, E~,., Vedenldna, N~S. and Ivkova, M.N. (1973) R~a~ac/~m.

/V~ofob[o/. 18, 263-276 27 14 Talbot, J.C., Dufourcq, J., de Bong, J., Fauo0n, J.R. and Lurson, C

(1979) FEBSLet~ 102,191.-193 28 15 Effink, M.R. (1994) B/aphys. 1. 66, 482-501 16 Valeur, B. and Weber, G. (1977) F/~.,~ochem. Pho~ob~o/. 25, 441-444 29 17 Bat~, C.A., Brady, J. and Sawyer, L (1994) Tmnds Food ~ci. Technol.

5, 261-265 3~ 18 Kaptanas, R., Bukolova, T.G. and Burshtein, ~A. (1973) Mo/. B/o/.

(Mo~ow) 7, 753-759 [in Russian] 31 19 Mills, O.E. (1976) Biochim. Biophys. Acta 434, 324-332 32 20 Kaplanas, R., BuEolova-Odova, T.G. and BursMein, E.A. (1975) Mo/.

B/o/. (Moscow) 9, 795-804 [in Russian] 33 21 Wahl, P., Timasheff, S.N. and Auchet, I.C (1%9) BiochemistryS,

2945.-2949 22 Fusate, R.D. and Long. P-S. (1980) B/ochhn. B/op/p/s. Ac~ 625, 28-42 34 23 Brown, E.M., C.arroll, RJ., Ffeff~', P.L and Sampugna, J. (1983) L/p/ds

18,111-118 Mad.eriCh, D.H. and Fnigips, M.~. (1992) $d~ce 256, 789-794 Mebsem, G. (1986) B / o c / ~ 2 5 , 236-244 Yar~, H.C, Rendy, MM., Burke, C and S~d~& GM. (1994) e[oc/~m/say 33, 518--525 Yans, H.C., Pzedy, M.M., Mickeh~, J.R., Louis, CF. and SI ~ G.M. (1993) B/opt. I. 64, A.303 LaPode, D.C., Kc~,r, CH.~Ohvin, B.B. and S~otm, D.B. (19~1) B / o d ~ 20, 3%5-3972 Graham, Z., Le~is, P.C , ~ 1 G e ~ , I. ( 1 ~ ) I. e/o/. O~.,. 261,608-613 l,wor, G.T., Sond, S.M., (3~an~, P. and Sb~y, C.W. (1991) ,q.-ck. e ~ . e/op/~. 289, 39-46 s~n~, v.w. (1991) c ~ xev. ~0od sci. Ted~d. 3O, ~¢;-533

D.). and Menbse~, P ~ (i 994) I. A~r/c. Faod C/~m. 41, 59-63 Taylor, D.L, Wal~oner, A.S., ~ , R.F., Lanm, F. and B~rl~, R.R. (1986) A p p l ~ of F ~ in ~he Biomedical ~iences, Alan R. Liss Be~'ey, r,J.., Thomman, ~K., rdebe, RJ. and Hamwi~, P.~. (1~5) e/a~d~/~s 3, 356-3~6

Review

We have been actively involved in the isolation and charac- terization of endogenous plant anfioxidants that are believed

to inhibit lipid peroxidation and offer protect~z a~ainst

oxidative damage to membrane functions. Antioxidants have been isolated from conventional food sources, such as tea (green and black), sesame and wild rice, and also from other

plant sources, such as rice hulls, and crude plant drugs. Data

on new types of water-soluble and lipid-soluble plant anti-

oxidants are provided, and the biolegical activity and func-

tionality of these antioxidants are discussed.

The contribution of

plant food antioxidants

to human health

Narasimhan Ramarathnam, Toshihiko Osawa, Hirotomo Ochi and Shunm Kawakishi

It is well known that humans, as they grow older, become less active, have an increased probability o f illness, and generally experience a loss o f o p t i m u m function o f all physiological systems. Evolutionary processes have engendered the su,~ival o f individuals o f all living species until the time that they are able to r e p ~ l u c e and pass on their genetic codes to their off- spring. At sexual maturity, an age-dependent process sets in, progressing gradually, and leading eventually to the death o f an individual, if death has not occun'cd from another means before then t.

Nm.mim~m Rm~r~he~ and Hbe4temo Od~ are at the ~pan I r ~ u ~ fcr the ConU'ol d Agin~ Nikken Rx~s Co., Lid, 723-I, Hamoka, Fukumi City, Shizuoka - 437-01, ]apan. T e d ~ Osmu ( ~ n g author) and S~we ~ E 4 ~ i are at the Depa~m~,: of AppI~I and Biok~al Sciences, School of A ~ k u m , Nagoya Unive~i(y, Chikusa-Ku, Nagoya - 464-01, Japan (fax: +81-52-789-41201.

! ~ ~ d ~ f m ' m ~ e n in

Oxygen, a vital component for the survival o f the human species, is present in the atmosphere as a stable tdptet biradical (302), in the ground state. Once inside the h,nmn body, it can be ~-~usformed, by a four- electron reduction pve~ess, to water, producing a super- oxide radical (Oz.), a hydxoxyl radical (.OH) and hydxo- gen pezoxide (l~zO z) as the reactive i m m n e d i a ~ (Fig. 1). Singlet oxygen ('0~) is formed from the ©x¢itcd s ~ e o f various seusifizers such as chlorophyll, acridine and other pign~nts. Among the nmjor cellular and extra- cellular components, proteins, enzymes, lipids, DNA and RNA form the primary targets for these w.active oxygen ~pccics. However, o "x~ation o f the unsaturated fatty ~ componenm or" cell membranes is the oxidat- ive event that occuxs most z~equently inside the human body z.

Trends in Food Science & Technology March 1995 IVol. 6] ©i595, E~ia.sci~ce ud ogz4.224,e~s~sog.so 75

l h | [ H " l | a " l | H " v H02. H O f H O .

I t tO, H,O,

H20

Fig.1 Scheme to illu~ate the formation of reactive oxygen species

(shown in bold type) from the stable triplet oxygen, 302 .

L i p i d oxidation has both positive and negative effects. A t l o w levels, the peroxidetion products o f lipids are responsible for the desirable aroma o f fried foods 3, and some o f the characteristic flavor properties o f cooked meats o f d ~ e r a n t spscic.s4, roasted nuts s, and so on. On the othar hand, lipid per6xidation not only poses prob- lems in ~ development o f rancidity in processed foods 6, hot also causes serious damage to the human hodyL ' T h e excess production o f reactive oxygen species, particularly hydroxyl radicals, can easily in- itiate lipid oxidation in the cell membrane, resulting in the f o ~ o n o f lipid peroxides. Many researchers have shown that lipid peroxides and reactive oxygen spec%s are involved in the development o f a vsriety o f diseases, inelnding cancer, and also accelerate aging. Several sec- on&~y woducts o f lipid oxidation, especially malon- dialdehyde and 4-hydroxynouenal, are themselves re- active, they have been shown to react with biological components such as proteins, amino acids and DNA s. Maloudialdehyde, which has been shown to be formed both enzynmtic~ly and non-eazymatically, has been implicated in a~ng, mu~genesis and carcinogenesis 9.

The metabolic processes that involve complex oxi- datiou-redaction reactions are governed collectively by enzymes, hormones, trace elements, and so on. Most living o r g a s m s possess extremely efficient defense and protective systems in those cells that me essential for defending the organism against oxidative stress induced by reactive oxygen species. However, the capa- bility o f such protective systems gradually decreases with age, resulting in disturbances in the normal redox e q ~ b f i u m that is esteblished in healthy systems. Such d i s ~ c e s lead to the malfunctioning o f the vital or- gans, tim gradual loss o f immunity to diseases that tend to be conu'acted with aging, and eventually death. The endogenous antiorddants distributed in and around liv- ing cells, which r e l u m e the various oxidefion--reduction reactions, are therefore seen as a potential class o f deter- minants o f longevity t°. Thus, in order to replenish the age-induced loss in the capability o f endogenous anti- o x i ~ t defense mec~sms, there is a need to identify new phytocbemicals that could be made readily avail- able by the regular intake o f conventional foods.

lnT~cl d | ~ ~/le en ~ m n ~ a l ~ Smoking is one o f the major causes proposed for

cancer around the globe, and has been implicated in

over 30% o f the cases. Improper diet among subjects, especially in industrialized nations, is another major factor in 35% o f cancer and degenerative diseases as- sociated with aging. On the contrary, inflammation due to chronic infection, especially in under.developed nations, has been cited as the cause in over 30% o f the cases, whereas occupational h a z ~ s (2%) and environ- mental factors (1%) are the other minor causes". Thus, if all the external factors could be couu'olled and kept within reasonable limits, cancer and degenerative dis- eases associated with aging could be adequately regu- lated by the intake o f a healthy diet.

In 1945, the average life span o f Japanese people was 50 years for men and 53 years for women. Recently published figures indicate that women in Japan can expect to live for 81A years and men for 75.6 years t2. Many factors such as improvements in public sanitation, the introduction o f new antibiotics, and awareness o f eating habits and health through proper education have been suggested to be responsible for the increase in the average life span in Japan. However, o f these factors, a combination o f the best o f Western food and o f tra- ditional Japanese food is currently the focus o f attention for improving the health o f old people in Japan.

Recent publications indicate that there is much evi- dence that plant antioxidants play an important role in biological systems in vitro as agents for antioxidafive defense. Antioxidant compounds have already been found in numerous plant materials such as oilseeds, cereal crops, vegetables, fruits, leaves and leaf wax, barks and roots, spices, herbs and crude plant drugs. The isolation and identification o f several plant antioxidents from leaf wax, rice and sesame seeds have been under- taken by us. With this concept o f food and health in mind, this article will emphasize the contribution o f plant food antioxidants to human health, in general. The biological activity and functionality o f some o f the com- ponents will als0 ha discussed.

Green tea consumplion and impliealiom on imnkm I m l ~

The custom o f drinking green tea in Japan originated from China in ~800AD. Green tea has been valued from ancient times as a powerful and miraculous medicine, vital for the maintenance o f health. In recent years, research on green tea has progressed to such an ex- tent that this belief has almost been proved to be true. Epidemiological studies have shown that the death rate from cancer is surprisingly low in the Skizunka Prefecture o f Japan, a major green-tea-producing re- gioulL Animal studies, using mice, have also proved the beneficial effects o f components o f green tea, mainly ca~chin, in both inhibiting the initiation o f can- cer and suppressing the growth o f tumor cells t4.

Green tea also has beneficial effects on the regulation o f blood cholesterol levels. A rapid increase in the levels o f low-density lipoprotein (LDL) was observed in the blood p i ~ m a o f rats fed with a fat-rich diet, wbereas rats fed the same diet supplemented with 1% catechin, the main antioxidant in green tea, showed

76 Trends in Food Science & Technology March 1995 [Vol. 6]

remarkably reduced levels o f LDL cholesteroL However, the catechin supplement did not have any impact on the levels o f high-deusity fipoprotein (HDL), an essential cholesterol component ~s. Green tea ca~chin is also befieved t o regulate blood pressure, and can help t o lower blood sugar levels. Extracts o f green tea or green tea catecbln, when fed to diabetic mice at a dose o f 1%, reduced blood sugar levels by 20-35%. Tolbutamide, a synthetic blood-giuco~-Iowering chug, lowered blood sugar levels by only 15%, when ~ n i n i s t e r e d orally t6.

The membranes o f red blood cells are known to con- rain high levels o f polyunsaturated lipids, and are there- fore very susceptible to oxidation. We have evaluated the antioxidetive activity o f tea polyphenois by a simple and convenient/n vitro entioxidative assay system that uses rabbit erythrucyte (red blood cell) meml~rane 'ghosts'. [The rabbit erytlm3cyte membrane ghost sys- tem was prepared by first washing commercial rabbit blood with isotonic buffer solution several times and then subjecting it to centrifugation. The resulting cells were lysed in phosphate buffer (10raM) and then ultra- centrifuged.] Etytim~yte ghosts prepmed from both human and rabbit red blood cell membranes showed almost the same extent o f lipid peroxidafion, induced by t-butylhydroperoxide tT. Thus, the evaluation o f the in vitro antioxklative activity o f the tea polyphenois was carried out using the rabbit erythrecyte membrane ghost system. O f the four tea catechins evaluated (Fig. 2), (-)-epicatechin gallate end (-)-epigallucatechin gallate produced the strongest l~otection against lipid peroxi- datinn, and were also found to be more active than the standard antioxidants a-tucopherol and pmpyl gallate z.

On the applications front, we have compared the enti- oxidant pmparties o f green tea collected from different tea-preducing regions in Japan. Samples were p r u c m ~ at various times during the tea-producing season, such as during early, mid- and l ~ spring and earl],' summer. Aqueous extraction o f the tea samples was carried out under controlled conditions, so as to give the extract the optimum aroma, taste and color c h a r ~ i s f i c s . The extracts were tested for t h e i r antioxidant activities, as determined by the suppression o f the oxidation o f de- oxyrihose induced by hydroxyl radicals and also o f the autoxidation o f linoleic acid. The superoxide radical and hydroxyl radical scavenging properties o f the tea ex- tracts were also determined by the use o f electron spin resonance (ESR) spectrometry. Although differences in the antioxidem and nulical-scavenging properties o f extracts could not he detected between samples col- lected from different regions o f l~roduction, seasonal differences had a pronounced effect on t h e i r activities.

Ten leaves collected between late spring and early summer were found to be extremely effective in scav- enging superoxide radicals (Table 1). The autoxidetion o f linoleic acid and the oxidation o f dcoxyribuse in- duced by hydroxyl radicals were also found to he sup- g e s s e d more effectively by extracts from leaves g o - cured in late spring. However, the extracts obtained from these samples had a strong astringent taste, and also lacked the desirable fresh green tea aroma. These

0 0 -

5 0 -

1 2 3 4 5 6 7 8

U-q~2 Antioxklative activity of ~een tea polyphenols (final concentra~m: IOOW~), compared with a ccmuol .~mple and a-t~:ophe~, and de.mined by d~e rabbit en~'~oc/te gh~t system. (Z }, C o n t . ; (2), a-tocophe~; (3), (+)-catechin; (4), ( - ) . . e p ~ i n ; (5), ( + } . g a J ~ n ; (6), ( - ~ i n ; ~, ( - ~ o ~ h ~ n ~ e ; ~ (8), (-~,al~.,~echm g~,~. (Data taken from Ref. 2.)

characteristics have been carefully studied at the Japan Institute for the Control o f Aging, the i m ~ e m s re- solved, and an instant type o f 'antioxid~t green tea" has been developed (marketed by Nikkea Foods, Fukarei City,/apan). This Ixeduct, which has good antioxident and organoleptic properties, has been conune~ally available since the beginning o f 1993.

~ aneox~4s in Mack tea Black tea is richer than green tea in thenflavins

(Fig. 3), which are dimers o f c, atechins fornted by enzy- matic oxidation during the manufacture o f black tea. In the rabbit erythr~yte ghost system, thenflavin (TFI) was shown t o have stronger antioxidmive activities than a-tucopharol; however, in a rat liver microsom~ sys- tem, it was found to be less potent than a-toenpharol. [The rat liver micrusomal system was ix~mred using fresh liver removed from secriflced Wistar rats. The liver was first homogenized thoroughly, and then the micrusomes were prepared using a differential centrifu- gation method.] Theaflavins were also shown to inhibit the cleavage o f calf thymus DNA induced by H202

Trends in Food Science & Technology March 1995 NoL 6] 77

I T~ 1. ,~pes~dde ~ scaveng~g a¢Ii~,ty (.~DSA) ~

Eady sl~ng 145 000+20 000 I M~-s~ng 170 000+s000 I~le sp~ng 190 000+5000 Early summer 230 000+30 000

(V'C~,min C control) (170 000+5000)

"N. Rarnarathnam et al., unpublished b3500 SOSA Un~,5 Coffe~oo~ go lhe scavenging activity of 1 mg of pure supem~ clismmase e n z ~

in the presence o f horse heart cytnchromec. Hence, theaflavins are expected to be effective in protecting celiu~r DNA against oxidative dema&e. O f the theaflavins tesP.d, theaflavin digallate (TF3), which has two gallic acid moieties, exhibited the strongest antioxidative

(a) (b) O H

o. o. o _LJ'~ °" 0 ~ 0 0 ~ 0 ~ ~ o s C - ~ " O H

HO HO " I ~ 0 " ~ 0 ( ~

.o.cioT.L<>-o. " C v " . o . o . "y"'-""OH o .

OH OH

(c) O H (d)

O f ~ y O H OH ~ ~ #,~ OH

/ C- OH~ ~OH ~ ~ ~OH O O O H

HO HO

o.° . o .... o .

" C ~ " ' o o. " ~ o~ o . ~ , . "C_ .~_ ~OH

~" "OH OH O H O H

f'ifr 3 ~uctures of black tea pofyphenols: (a), theaflavin (TF1); (b), theaflavin monogallate A (TF2A);

(c), theaflavin digallate (TF3); and (d), theaflavin monogallate B (TF2B) (bold type indicates the gallic acid moieties).

activity in the rabbit erythrocyte ghost system. Based on a detailed investigation o f structure-activity relationships, we concluded that the gallic acid moiety is probably important for the antioxidative and antimutagenic activi- ties o f tbeaflavins ts. The protective roles o f other water- soluble antioxidents on mutagenicity and DNA damaging activity induced by reactive oxygen species a,-zl 'dpid per- oxidation products are currently being investigated. In ad- dition, attempts are being made to develop anti-oxidant- rich health drinks that contain extracts from green and black tea in combination with other natural extracts.

A n f i o x i ~ t i v e components found in rice seeds Rice (Orym s a t i v a ) is the principal cereal food in

Asia, and the staple food for nearly half o f the world's population. We have recently undertaken a comparative investigation o f the germination potential o f rice seeds from the subspecies japonica and indica during long-term storage. We have observed that the indica rice seeds, on average, retained their ability to germinate when stored at 30°C, even after one year, whereas cultivars o f the japon/ca subspecies showed a gradual decline in their germination potential after storage for six months at 30°C 19. We hy-

pothesized that the differences in the storage stability ofjapon/ca and indica rice seeds could have been due to the differences in the effectiveness o f the defense systems in the rice hull, which until now was believed to offer only physical protection to the rice grain. Model experiments were undertaken to induce accelerated aging by initiat- ing lipid peroxidetion at 30°C, using hydroxyl radicals generated during the T-irradiation o f the seeds. As shown in Table 2, the seeds showed wide differences in their germination potential and thioberbitu~ acid CI'BA) values depending on whether or not their hulls were intact during irradia- tion z°. It was concluded that the hull fraction o f long-life (indica) rice seeds must contain some unique antioxida- five oanstituents 21. S'nnilar results were obtained when accelerated aging was induced in rice seeds stored with and without intact hulls at a temperature o f 6 0 0 C ' .

A large-scale extraction, isolation and purification process was used to identify the active components as flavonoid substances. One o f the ac- tive components, when tested by the /n ~ o t l ~ T ~ ~ wes frond to he as active as a-tncopherol, and was identified as isovitexin, a C-glycosyl- flavonoid2L A study to determine the mechanism by which these hull antioxidants offer protection is cor- rentiy being undertaken u. Also, food

78 Trends in Food Science & Technology March

applications o f ric~ hull antioxidants are being explored. In the cosmetic in- dustry, attempts are being made to use rice huh antioxidants in the formu- lation o f body lotions end creams used mainly for the protection o f the skin against the adverse effects o f aging.

Recently, we have also isolated and identified antloxidafive pigments from St~p.in~c~ black rice. Black rice seeds have the g,~em~a ability to maintain their viability even Ce~my p~na after long-term storage, retaining their S u b s p . ~ o ~ viability longer than the regular white Koshihilcad rice seeds from the same species. Kmeb~ Large-scale isolation and purification o f the antioxidative pigments led to the identification o f cyanldine-3-O-~v- ghicopyraneside u. which was found to possess strong antioxidadve activity when tested using the in vitro tlfio- cyanate method. Cyanidine-3-O-~-D-glucopyraneside was also isolated and identified as the antioxidative pigment in black and red beansZ~; thus, studies o f the relationship between the variety and levels o f tbe~e antioxidative pigments in cereal crops and beans are now in progress.

Table ?.. [~k, ct e~ ~ ~ the ~ p ~ b l and ~ , ~ ' , ~ ackl ( ' n ~ qbes ef ~ e (O~mLs~ra) stub, i n ' ~ h ~ at diff~mt chnn ~ h md ~ h a ~ ~ h d ~

at k l i a ~ dine (kGy): at ~ d m t t ~ ) :

0 10 Is o 18 Is

IOO (ioo) 54(28) 0(0) o.l (0.i) 0.11 (0.11) 0.'12 (0.13) 100 (99) 52(26) 0(0) 0,1 (0.1) 0.12(0.13) 0/13(0.15)

'lOO(lOO) 42(22) 0(0) 0.I (0.1) 0,13(0.14) 0.14(0.18) 99 (99) 35(20) 0(0) 0.I co;I) 0.14(0.16) 0.15(0.17)

'The fiSmes in pam~heses refer m values ~ dce seeds irr'~q~ widmut imact bulb bD~a t ~ n from I~f. 20 OD~,Ol~d d e ~ y ~ S35nm

Natural antioxidants in other cereal c n ~ We have also succeeded in isolating the antioxidat-

ive components present in young barley leaves; 2"-0- glucosyfisovitexin was identified as a unique antioxi- dative compound 2e, the activity o f which was deter- mined by the in vitro TBA assay method. Pemxidetion o f methyl linoleate was induced by the hydroxyl radical, generated in a Fenton reaction system (Fig. 4), When 2'-O-glucosyllsovitexin was added to this system. methyl linoleate peroxidetion was drastically suppressed, proving that 2'-O-glucosylisovitexin possesses strong antioxidant activity ~. Also, it has been demonstrated that 2'-O.glucosyfisovitexin protects squalene against peroxidation induced by inadiation with UVB ~. The potential applications o f this antioxidant in vMue-added processed foods are being investigated.

Wild rice (Zizania aquatica) is another cereal crop whose applications in the food industry have shown a rapid increase; for example, it is being used in the development o f ready-to-eat soups, breakfast cereal mixes, baking mixes and gourmet salad pt~paratiom. Unlike regular rice, w i l d rice belongs to the genus Z/~n/o, comprising a grass-like family o f plants. Though traditionally grown by native Indians in shallow lakes and streams in northern Minnesota aud mmhem Wisconsin in the USA, and in southern Ontario and Manitoba in Caneda, wild rice is now cultivated as a regular crop, chiefly in Minnesota. Its composition resembles that o f oats, low in fat (less than 1%) and high in protein (12.5-15%).

The application o f wild rice as an extender in the preparation o f processed meat ptvducts has been well investigated ~. 5ensm'y evaluations indicated that beef patties supplemented with wild rice had higher sensory

scores than control pauies. Also, the F a a m s e d la~luct made with wikl r i ~ possessed Zower ¢ h o ~ s t e ~ ~ g and a low "rBARS value (level o f thioharbita~ acid- reactive substances; a meastwe o f the extent o f lipid oxidation). These observations indicated that wild rice might contain inherent antioxidants. As an extension o f their work, Wu e t a / . investigated the chemical natm~ o f the antioxidant system in wild rice, and attributed the antioxidant w~ivity o f wild rice to phytlc acid ~.

Recently, we have begun investigating the aatioxident componen~ o f wild riee. Ethanolic eatracts o f wild d e e were fra~oneted by column chromatography, followed by purification on high-peffommnce liquid chroma- tography (HPLC). Insmunental analy~as have rer-~aled the g e s e a c e o f ¢aher novel antioxidams with stmnge~ antioxidant activities than phytic acid, as determined by t h e / n vitro TBA assay method. The stngtmes o f three o f the active components have been determined. These three active components were 3,4,5-lfingth- oxycinnsmlc acid, a phenolic ~lycoside with 3,4,.5- trintethoxycinnamate and p-hydroxy acetopbeno~ as the aglycone moieties, and a flavonoid ~ycoside with 3 , 4 , 5 - t r i m e t h o x y c i n ~ t e and luteolin as the aglycone moieties ~. Preliminary investigations have also shown the presence o f other biofactors in wild rice that may have promising applications in the development o f an anti-diabetic diet (i.e. a diet rich in functional ingredi- ents that have been shown, in a n / n vitro assay, to re- duea the formation o f advanced glyeation end ptedlgts, which are found at very high levels in blood samples front diabetic patients). At present, we are actively involved in the application o f wild rice in various processed health-foed products in combination with traditional Japanese flavonng ingredients.

Antloxldat~ ~ in q ~ ~ , ~ ~md

Herbs and crude drugs 1 ~ from plant materials are traditionally used in many Asian countries, and their pharmacological effects have been extensively studied.

Trends in Food Science & Technology March 1995 [Vol. 61 79

/ Fenton reaction

HzO z + Fe z+ ~ - O H + - O i l + Fe z*

Peroxidation of methyl linoleate

- O H cis ~ " cis

13 12 11 r I 0 9 - - C H : C H - - C H z - - C H = C H -

I - - H trans cis cis cis cis trans

- - C H - - C H = C H - - C H = C H . . . . C H = C H - - C H - - C H = C H . . . . C H = C H - - C H = C H - (~H--

H + H +

- - C H - - C H = C H - - C H = C H - - - C H = C H - - C H = C H - - C H - [ trans cis cis trans ]

OOH OOH

elg.4 Feroxidation of methyl linoleate, induced by a hydroxyl radical generated in a Fenton reaction system.

However, only a few reports are available on the anti- oxi~five components o f herbs and crude chugs. In view o f Otis, we have screened 32 different types o f herbs and crude drugs obtained from Japanese markets and drug sto~s in Taipei, Taiwan. The levels o f tecophero! defiva- fives in these drug extracts have been quantified using HPLC, and it was concluded that Osbeckia chinensis did not possess any tocopherols. However, all o f the other herbs and crude drugs screened were found to contain tncophero]s as the antioxidative components. These results wompted us to isolate and identify the active principles in the extracts o f O. c'hinensis ~2.

The whole o f the O. chinensis p,!ant has long been used as a ¢adifional medicine in "falwan, Japan and China. The dried leaf, stalk and stem o f the plant are normally extracted in hot water, and administered as a q u ~ k remedy to a~eviate body pain, fever and cases o f severe influenza. Large-seale purilicati~n has resulted in the isolation and identification o f many different types o f antioxidative components. Most o f the antioxidative activity o f the extracts o f O. chinensis is believed m be due to rennin-type anfioxidams, in particular casuafinin (Fig. 5). These isolated tannins cont~dn an allagic acid moiety in their s~nctures, and have almost the same anti- oxidant activity as efiagic acid itselP 3.

Sesame seeds and oils have been used traditionally in Japan, China, Korea and other Asian c o u n ~ e s for many years. Sesan~ oil, in particular masted sesame oil, is widely used in Chinese and Japanese cooking, and has s~tong anfioxi¢lant activity. Sosanfinol, an active eom- panant o f sesame ol], is unique among anfioxidants because it has a s u p ~ i ~ heat stability ~. Also, sesaminol has been shown to effectively suppress the degradation o f tocopherols in model systems using corn o i P . The antioxid~mt activity o f ser~ninol was evaluated using several/n vitro lipid peroxidatinn systems, in particular

the rabbit etythrncyte membrane ghost and rat fiver micro- somal systems; sesaminol was found to effectively inhibit lipid peroxidation induced by t-butylhydmperoxide in the mierosomes and the erythrocyte ghost membra~les.

The protective role o f sesaminol against oxidative damage has also been reported in studies using cultured human diploid fibroblasts at various in vitro ages 36. Yamashita et al? ? recently reported that sesaminol inhibited in vivo oxidative damage induced by carbon tetrachlodde in senescent-accelerated mice. More re- cently, in an in vivo system, we have found that rats fed a diet supplemented with sasaminol at a dose o f 10mg/100g o f body weight suffered less DNA damage due to oxidative stress induced by carbon tetraciflofide than the control rats. The levels o f TBARS in blood plasma and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine were measured. Although the detailed examin- ation remains incomplete, we have found that sesmninol inhibits both lipid peroxidation and the excretion o f 8-OHdG in urine, as determined by a monoclonal anti- body enzyme-linked immunososhent assay (ELISA) assay method 38. The assay system, which is very accu- rate, fast and convenient, was developed by the Japan Institute for the Con~ol o f Aging as one o f several assay procedures to measure the degree o f oxidative stress and in vivo oxidative damage.

Sesame seeds also contain a large quantity o f water- soluble fignan glucosides ~. These glncosides were found to act as strong water-soluble anfioxidents as well as being precursors o f lipid-soluble anfioxidant lignans. A diet comprising sesame from w~ieh all lipid-soluble antioxidants, including sesaminol and tocopherol, were removed by hexane extraction was fed to rats for two months. This diet, which therefore contained only water-soluble anfioxidants, inhibited both lipid pemxi- dafion in blood plasma and the excretion o f 8-OHdG in

80 Trends in Food Science & Technolosy March 1995 IVol. 6]

urine, as determined by the monoclonel antibody ELISA assay method ~. The application o f the antioxidants found in sesame and sesame oll for the development o f novel health foods is being actively pursued.

~ ~ p t ~ J c mnpene~ tm red wine U W

Polyphenol oligomers, namely procyanidines, are known to be widely distributed in the plant kingdom. They have been successfully applied in the treatment o f several vascular disorders in human~¢ because o f their positive biochemical effects on blood vessels 4e. Pm- cyanidines are also effective free-radical scavengers and inhibitors o f oxidative enzymes such as xanthine oxidas¢ ¢t, as determined by measuring the extent o f con- version o f hypoxanthine to uric acid. The vasorelaxing activity o f wine and other grape p-odncts including grape juices and grape skin exlracts has been well documented 42. The e f f ~ t is believ'~l to be induced by quereitin and tannic acid, compounds lmown to be Was- ent in grape skin.

Phytnehemicals, especially those in wine products, may also play a critical role in reducing mortality from coronary heart diseases in certain soctors o f the French population. A comparative study initiated by the World Health Organization has shown marked differences in mortality and morbidity from coronary heart disease between countries, in patlicular between French and US populations 4~,~4. Despite having a similar dietary intake o f saturated fatty acids, and comparable plasma cholesterol levels, the French subjects were shown to be less sus- ceptible to coronary heart disease than the US subjects.

Using multivariate analyses, it was nostulated that the consumption o f wine was the only die~ry factor respon- sible for this discrepancy, commonly referred to as the 'French paradox '~. An extended investigation demon- strated that the non-alcoholic portion o f red wine, par- ticularly the phenolic substances, inhibited the oxidation o f human LDL in vitro ~.46. The assay was carried out by measuring the levels o f bexanal and conjugaAed dienes formed by a Cuz÷.-catalysed oxidation o f freshly pre- pared human LDL.

In view o f all these observations, it appears that the consumption o f red wine, in moderate amounts, may have a long-term health benefit. Quite often, healthy centenarians in Japan, when interviewed about the secrets o f their longevity, strongly advocate drinking moderate amounts o f sake, the traditional Japanese rice wine, believing it to be one o f the pmbeble reasons for their long life. Work ~ currently being carded out to characterize the non-alcoholic phenolic antioxkhnts in rice wine. Steps are also being taken to investigate other non-alcoholic fermentation pmdncts like soy sauce, and fernzeazed soybean pmdncts such as natto and miso, which have long been used traditionally in Japan.

An~xklatlve defense system in seaweeds Seaweeds aze another foodstuff that has been tra-

difionaliy used in the Japanese diet for centuries. The lipid content o f seaweeds is low, and is rich in eicosa-

[30-

5 0 .

0 ~ 1 2 3 4 5 6 7 8 9

~ . S Antioxidative activities of tannins (6hal concentration: 25 I ~ l isolaL-cl from Os~ck/a c/~nens/s, compared wkh a comrd samite and a ~ , and demm,.ined I~ ~e r a l ~ e ~ m ~ ~ m t sr~'~'n. (I), Coneol; (2), ~-tocophe~; (3), ella~ add; (4), ca~adnin; (~, casumm; (6), pm~cacon~ A;(7), d e ~ ~ A; (8), 2,3-[(5)-404',5,5',6,6'~.~%~x~!g41~'o~ -o-glucopl~nosi~; and (9), 4,6.[($)- 4,4',5,5',6,6'.h~e~EeflO~-D.gh¢opyrmmd~. (Data taken from gel. 33.)

pentaenoic acid, which has been shown to be effective in lowering blood cholesterol levels in rats 47. Despite their high content o f p o l ~ fatty acids, it is w e d known that seaweeds are stable against oxidation during storage. Recently, we undertook an investigation to charactedze the chemical nature o f the defense sys- tem Wesent in seaweeds. Extracts o f brown algae were found to contain strong hydroxyl-radical scavengers, as determined by the ESR s p e c t r o m ~ y m e r l i n . . S o m e o f the e x t m ~ also displayed a strong potential for scav- enging supemxide radicals. Studies are in progress to identify the key antioxidant components in seaweeds. In addition, attempts are being made to utilize seaweed extracts in the lnepezation o f unconventional soups, seasonings and salad dressings, both for oriental food applications and to meet the nceds o f the Western palate.

Conclusions L i p i d pemxidatlon/n vivo ~ s been indicated to be th=

primary cause o f many o f t i ~ cardiovascular diseases such as ~ ~ aud also in cancer and aging. Consneaen, especially in the Western world, were

Trends in Food Science & Technology March 1995 [Vol. 6] 81

adv~sad in the 1980s to reduce their fat intake and to t8 t ~ i r intake of food sources that would provide

more crude fiber. The current emphasis is on drastically 19 ~ g the intake o f red meat, and increasing the intake o f fresh vegetables and fruits. Without douht, 2o c o n s ~ all over tee globe are becoming more health 21 c o n s c i o u s , and are prepared to educate themselves about nu~Ecn. Z~

Scientists in the fields of aging, cancer, diabetes, c a r - diovasctfiar diseases and many of the diseases related to 23 aging are becondng aware of the value o f preventive therapies. Terms like functional foods, designer foods, therapeutic foods, n u ~ e u t i c a l s and the like are already being used by the media. However, the information 24 that is currently available is not sufficient to address 2s all o f the unsolved health problems. There is certainly a strong need to investigate plant products in a more 26 systematic way. Effects that have been observed at the test-tube level need to be interfered c a r e f u l l y , and docu- 27 mented with more in vivo evidence. Traditional foods that have been used for several generations are likely to 28 be the subjects o f in-depth research in the near future. New types o f plant antioxidants and novel biofactors 29 will be developed, and it is very likely that the increased consumption o f plant foods will become the trend o f the 30 next decade. 31

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82 Trends in Food Science & Technology March 1995 Wol. 6]