Antioxidative activity of green tea catechins in canola oil

CPL ELSEVIER

Chemistry and Physics of Lipids 82 (1996) 163 172

CHEMISTRY

AND

PHYSICS OF LIPIDS

Antioxidative activity of green tea catechins in canola oil Z.Y. Chen*, P.T. Chan Department of Bioehemistrv, Tile Chinese Unicersity ~/' Hong Kong, Shatin, New Territories Hong Kong Hol ~ Kong

Received 19 February 1996: revised 24 April 1996: accepted 24 May 1996

Abstract

Jasmine tea is one of the most popular beverages consumed in China. In the present study, green tea catechins (GTCs) were extracted from jasmine tea and their antioxidative activity was examined in canola oil heated at 95°C for various time. The yield of crude GTC extracts was 7.4% of dry tea leaves and it mainly consisted of 51.2% ( - ) epigallocatechin gallate (EGCG), 18.7% ( - ) epigallocatechin (EGC), 12.3% ( - ) epicatechin (EC) and 11.8% ( - ) epicatechin gallate (ECG). Both the oxygen consumption test and the fatty acid analysis showed that the GTC extracts exhibited strong antioxidative effect. At 200 ppm, four major epicatechin derivatives tested demonstrated varying antioxidative activity in the decreasing rank of EGC > EGCG > EC > ECG. They were even more protective than BHT against lipid oxidation in canola oil under the same conditions. Furthermore, thermal loss of the GTCs was significantly less compared to that of BHT in canola oil heated at 95°C. Faster thermal loss of BHT was attributed to volatilization and steam distillation due to the high temperature. We conclude that the CoTCs as a mixture of EGC, EGCG, EC and ECG may replace BHT as antioxidants in processed foods. Keywords': Epicatechin; Epicatechin gallate; Epigallocatechin: Epigallocatechin gallate; Jasmine tea

1. I n t r o d u c t i o n

( - ) epicatechin gallate ( E C G ) and ( - ) epigallocatechin gallate ( E G C G ) . Increased c o n s u m p t i o n

T h e r e is an increasing interest in green tea catechins ( G T C s ) as p r o t e c t i v e agents a g a i n s t free radicals a n d c a r d i o v a s c u l a r disease. These G T C s are m a i n l y epicatechin derivatives including ( - ) epicatechin (EC), ( - ) epigallocatechins (EGC),

o f green tea has been shown to be a s s o c i a t e d with d e c r e a s e d serum t o t a l cholesterol a n d triacylglycerol, a n d therefore be inversely related to risk o f c o r o n a r y heart disease [1,2]. Effect o f d r i n k i n g green tea on p l a s m a l i p o p r o t e i n s is c h a r a c t e r i z e d b y decreasing l o w - d e n s i t y l i p o p r o t e i n cholesterol while increasing h i g h - d e n s i t y l i p o p r o t e i n choles-

* Corresponding author, Tel.: + 852 2635 4781: fax: + 852 2603 5123: e-mail: B583743(~hpgS0a.csc.cuhk.hk

terol [1]. In c o n t r a s t , d r i n k i n g black tea seems to have no effect on p l a s m a t o t a l cholesterol [3,4].

00It9-3084,96/$15.01) ,~'; 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0009-3084196)02587-X

164

Z.Y. Chen, P.T. Chan / Chemistry and Physics o/'Lipid~ 82 (1996) 163 172

OH

OH

Ho

o, H I ~ I

6, " ~ ' ~ 4

0

OH

8

H

5'

, ri

OH

Epicatechin (EC)

o. ~OH

Epicatechin gallate (ECG) OH

Epigallocatechin (EGC)

OH

Epigallocatechingallate (EGCG)

Fig. 1. Structures of ( - ) epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC) and epigallocatechin gallate (EGCG).

This suggests that the beneficial effect of drinking green tea over black tea is attributed to the content of GTCs because in the former, GTCs remain unchanged while they are degraded by fermentation in the latter. It is generally believed that an assortment of compounds including superoxide anions, cholesterol oxide, hydroxyl radicals, and lipid peroxyl radicals may trigger lipid oxidation in vivo and accelerate the atherosclerosis process [5], whereas GTCs delay autoxidation reaction by inhibiting formation of free radicals or interrupting the propagation of the free radical chain reaction and attenuate the progress of atherosclerosis and thrombosis [6,7]. In fact, GTCs have been shown to inhibit lipid oxidation in the lowdensity lipoprotein [8,9]. GTCs may also offer an alternative in protecting fats and oils in foods from oxidative rancidity [10-12]. We have previously examined the antioxidative properties of the ethanol extracts from various Chinese teas. The ethanol extracts from green tea and white teas exhibited a stronger inhibition on lipid oxidation in canola oil than butylated hydroxytoluene (BHT) [13]. In contrast,

the ethanol extracts from black tea and darkgreen tea showed no or little antioxidative activity [13]. The varying protection of tea ethanol extracts against lipid oxidation may due to the distinct manufacturing processes. Green and white teas are non-fermented products in which GTCs are mostly preserved while GTCs in black tea and dark-green tea are extensively oxidized to form browning polymers either by polyphenol oxidase or non-enzymatic browning reaction [14]. GTCs are characterized by sharing a similar backbone with varying number and location of hydroxyl groups (Fig. 1). However, information on relative activity of individual epicatechin derivatives as antioxidants in foods is limited [15]. In the present study, EC, ECG, EGC and E G C G were examined to determine the relationship between their chemical structure and antioxidative activity against lipid oxidation in canola oil. It was also of interest to study loss of GTCs during the heating processes. This was performed by periodically measuring the remaining GTCs in canola oil heated at 95°C.

Z.Y. Chen, P.T. Chan /Chernistry and Physics o/' Lipids 82 (1996) 163 172

2. Materials and methods

2.1. Reagents EG, ECG, EGC and EGC were obtained from Kurita Industrial Co., Ltd (Tokyo, Japan) and the purity was verified by high performance liquid chromatography (HPLC). Canola oil without addition of any synthetic antioxidants was obtained from Lam Soon Marketing Service LTD (Kowloon, Hong Kong). Jasmine tea (Encore Trading Co., Hong Kong) was purchased from a local tea shop.

2.2. GTC extraction The method described by Agarwal et al. [16] was modified and used to extract total GTCs from jasmine tea. In brief, 10 g of jasmine tea leaves were extracted three times with 140 ml of hot distilled water (80°C). The infusion was cooled to room temperature, filtered, and then extracted with equal volume of chloroform to remove caffeine and pigments. The remaining aqueous layer was saved and extracted twice with equal volume of ethyl acetate. The ethyl acetate phase containing GTCs was then removed using a rotary evaporator under vacuum. The resulting crude G T C extracts were dissolved in 10 ml of distilled water and freeze-dried overnight.

2.3. HPLC analysis of GTCs The individual epicatechin derivatives in the GTC extracts were separated using an Alltech Model 525 HPLC (Deerfield, IL, U S A ) e q u i p p e d with a ternary pump delivery system. In brief, 10 #1 of G T C extracts (2 mg/ml) was injected onto the column (Microsorb MV, 250 x 4.6 mm, 5 #m, Rainin, Woburn, MA, USA) via a rheodyne valve (20 #1 capacity, Alltech, Deerfield, IL, USA). A gradient of methanol in water was used at a flow rate of 0.7 ml/min (0 7 min, 28% methanol changing to 40%; 7 - 1 4 min, 40% methanol changing to 52%; 14-20 min, 52% methanol changing to 28%). The separated epicatechin derivatives were monitored using an evaporative light scattering detector (Model M K III,

165

Burtonsville, MD, USA) and a SP 4600 integrator. Individual epicatechin derivatives were identiffed by comparing the retention time of known standards or adding known standards to the sample.

2,4. Measurement of o_\:vgen eonsumption The method previously described by Chen et al. [17] was used to monitor oxygen consumption. In brief, 1 ml of hexane containing 200 mg of canola oil was placed in a glass tube (150 x 16 mm, o.d.). One ml of ethanol containing 0.04 mg G T C extracts or individual epicatechin derivatives was also added to the reaction glass tube. The components were mixed thoroughly. The hexane and ethanol were removed under a gentle stream of nitrogen at 45°C. The final concentration of GTCs or individual epicatechin derivatives was 200 ppm in canola oil. The reason for choosing this concentration of GTCs and individual epicatechins in canola oil is that maximum 200 ppm of a single or a mixture of antioxidants is generally permitted in fats and oils in many countries. The reaction tube was then flushed with air and sealed tightly with a rubber stopper obtained from an evacuated blood collection tube (100 x 16 mm, o.d., Becton-Disckinson, Rutherford, N J, USA), which usually maintains a vacuum for 2 3 years. The sealed tube was leak-free and was verified by filling the tube with nitrogen gas and monitoring by gas chromatography (GC) if headspace oxygen concentration decreased. Oxidation was conducted at 95°C _+ 2°C. The headspace oxygen was sampled periodically with a gas-tight syringe and analyzed in a HP 5890 series II gas-solid chromatograph (Palo Alto, CA, USA) fitted with 1/8 x 6 inch stainless-steel column packed with Molecular Sieve 5A (60/80 mesh) and a thermal conductivity detector. The percent oxygen in the headspace was calculated from the ratio of the oxygen to nitrogen. After headspace oxygen analysis, the canola oil was extracted with 10 ml chloroform and an aliquot containing 20 mg canola oil was taken for fatty acid analysis. Theoretically, the frying temperature (180°C) should be set in the present study. However, the preliminary test showed that the headspace oxygen was de-

166

Z.Y. Chen, P.T. Chan/ Chemistry and Physics o/ Lipids 82 (1996) 163 172

pleted with 2 - 3 h at 180°C. There was a variation between the tube measured at first and the replicate tube measured last. Therefore, oxidation was conduced at 95°C instead, where the headspace oxygen decreased from 21 to 3% within 2 3 days, and the variation among the replicate tubes was minimum.

then transferred into a GC vial after centrifugation at 500 x g for 10 min. The remaining BHT was analyzed using a gas liquid chromatograph as described above in the fatty acid analysis. A typical gas-liquid chromatogram of BHT and H A M E is shown in Fig. 2.

2. 7. Thermal loss of GTCs 2.5. Fatty acid analysis Fatty acids of heated canola oil with or without addition of GTC extracts or individual epicatechin derivatives were converted to the corresponding methyl esters with a mixture of 14% BF~ in methanol (Sigma Chemical Co., St. Louis, MO, USA) and toluene (1:1, v/v) under nitrogen at 90°C for 45 min [17]. Fatty acid methyl esters were analyzed on a flexible silica capillary column (SP 2560, 100 m × 0.25 mm, i.d.; Supelco, Inc., Bellefonte, PA, USA) in a HP 5980 Series II gas-liquid chromatograph equipped with a flameionization detector (Palo Alto, CA, USA). Column temperature was programmed from 180 to 220°C at a rate of l°C/min and then held for 10 rain. Injector and detector temperature were set at 250°C and 300°C, respectively. Hydrogen was used as the carrier gas at a head pressure of 15 psi.

One ml of hexane containing 200 mg canola oil and 1 ml of ethanol containing 3 mg GTCs extracted from jasmine tea were similarly placed in a test tube (150 z 16 mm, o.d.). The hexane and ethanol were evaporated under a gentle stream of nitrogen at 45°C. The final concentration of GTCs was 1.5% in canola oil. The sample was then flushed with air and heated in the air at 95°C for various time. The sample was then cooled at room temperature followed by adding 2 ml distilled water containing 0.5 mg/ml ( + ) catechin (C) as an internal standard. Two milliliters of hexane were then added into the mixture and centrifuged at 900 x g for 10 min. The aqueous

'

~

~

2.6. Thermal loss o[" B H T One milliliter of hexane containing 200 mg canola oil and 1 ml of chloroform containing 3 mg BHT were delivered into a test tube (150 x 16 mm, o.d.). The solvents were evaporated under a gentle stream of nitrogen at 45°C. The final concentration of BHT was 1.5% in canola oil. The test tube was then flushed with air and heated in the air at 95°C. After heating, the sample was saponified in order to isolate the remaining BHT. In brief, the sample dissolved in 4 ml of 95% ethanol containing 1% K O H was refluxed at 90°C for 55 min. The mixture was cooled at room temperature followed by adding 2 ml of hexane containing 1 mg/ml heptadecanoic acid methyl ester (HAME) as an internal standard. Three ml of distilled water were then added and the mixture was vortexed thoroughly. The hexane layer was

~i I' '1~' j i if, i,

I !'ijI

I

,!l/J i

1o

20 Minutes

3o

Fig. 2. Gas liquid chromatographic trace of butylated hydrox-

ytoluene(BHT) and heptadecanoic acid methyl ester (HAME). See text for the conditions of separation.

Z.Y. Chen, P.T. Chan ,' Chemistry and Phv.s'ics of Lipi~A' 82 (1996) 163 1 72

~ ~ ~ ~ !t

167

subjected to the analysis of variance, and the means were compared between treatments by using D u n c a n ' s m u l t i p l e r a n g e t e s t [18]. This was done by running data on the PC ANOVA software (PC ANOVA For the IBM Personal Computer, Version 1.1, 1985, IBM, Armonk, New York).

~

3. Results

) i

i

I

i

i

i

0

s

x0

is

z0

2s

Minutes Fig. 3. HPLC separation of jasmine tea catechins. See text for the conditions and Table l for percent composition. Peak identification: ( - ) epigallocatechin (EGC); ( + ) catechin (C, internal standard); ( ) epigallocatechin gallate (EGCG): ( - ) epicatechin (EC) and ( - ) epicatechin gallate (ECG).

layer containing GTCs was subjected to HPLC analysis as described above in the HPLC analysis o f GTCs. A typical HPLC chromatogram of GTCs is shown in Fig. 3. For the accurate cornpar±son, 1.5% GTCs or 1.5% BHT was chosen instead of 0.02% (200 ppm). This was because there was a significant variation in recovery of 200 ppm GTCs or BHT from canola oil. 2.8. Statistics

All the experiments were repeated 2 - 3 times. Data were pooled from each experiment in which 3 - 5 replicates (total 6 - 9 reaction tubes/time point) were conducted. Data for the headspace oxygen consumption and fatty acid analysis were

The yield of GTCs was 7.4_+ 0.3 g/100 g dry jasmine tea from three determination. The composition of GTCs is shown in Table 1. E G C G was the major component and accounted for 51.2% followed by EGC, EC and ECG in the decreasing order. In canola oil heated at 95°C, 80% headspace oxygen was consumed within 36.5 h under conditions examined while, in the presence of 200 ppm GTCs, only 6% headspace oxygen was depleted instead (Fig. 4). This clearly showed that the GTCs exhibited a significant protection to canola oil from lipid oxidation after 23 h of heating (P < 0.01 ).

The results from the fatty acid analysis were in agreement with those from the oxygen consumption test. The more the headspace oxygen was consumed, the more the tinoleic acid and ~-linolenic acid were oxidized. Addition of 200 p p m GTCs significantly prevented loss of linoleic acid and u~-linolenic acid in canola oil heated at 95°C for 36.5 h. In contrast, linoleic acid and ~-linoTable I Composition

of CTCs extracted from jasmine tea

Epicatechins

Absolute (g'100 g

tea)

Relative (%, total

GTCsl

Epicatechin (EC) 0.9 ± I).2 Epicatechin gallate 0.9 ± 0.1

12.3 ± 0.6 11.8 ± 0.3

Epigallocatechin

1~. 7 ± 0.9

(ECG)

1.4 + 0.1

(EGCI 3.8 ±0.2 gallate(EGCG) Unknown 0.4 ± 0.2 Total 7.4_+0.3 . . . . . . .

51.2 ± 1.5

Epigallocatechin

6.0 ± 1.9

.

100 . .

.

Z.Y. Chen, P.T. Chan /Chemistry and Physics of Lipids 82 (1996) 163 172

168

25

~.~ 20

m--~li~-°~°~°~o • ~~-o'~ ~

=

,

*

+ GTCs

1_5

,,

c~ o

10

~-

"o 09 -r

"~l

Can01a

5 0

0

'

'

'

'

lO

20

30

40

HouPs

Fig. 4. Effect of green tea catechin (GTC) extracts on oxidation of canola oil at 95 ± 2°C. Data are expressed as mean +_ SD/n = 8 reaction tubes. @, canola oil; C,, canola oil + 200 ppm GTCs. * represents a significant difference between the canola oil control and the sample with addition of 200 ppm GTCs at the same time point (P < 0.01).

lenic acid in the heated canola oil were significantly decreased compared with these in the unheated canola oil. Oleic acid and total saturated fatty acids were significantly increased in the heated canola oil compared to these in the unheated canola oil (P < 0.05, Table 2) but these fatty acids remained unchanged in the canola oil with addition of 200 p p m GTCs. F o u r epicatechin derivatives examined demonstrated varying inhibitory effects on lipid oxidation in canola oil (Fig. 5). E G C is most effective against lipid oxidation followed by E G C G , EC and E C G in the decreasing order. The oxygen consumption test indicated that all four epicatechin derivatives tested showed stronger anti-oxidative activity than B H T at the concentration of 200 ppm. The fatty acid analysis also revealed that addition of the four epicatechin derivatives significantly protected linoleic acid and ~-linolenic acid from thermal degradation ( P < 0 . 0 5 , Table 3). In addition, loss of linoleic acid and ~-linolenic acid in the samples with addition of 200 p p m epicatechin derivatives was significantly less c o m pared with that containing 200 ppm B H T (Table 3). A m o n g the four epicatechins, the fatty acid analysis showed E G C was more protective than E C G against oxidation of linoleic acid and ~-linolenic acid (P < 0.05, Table 3). However, the fatty

acid analysis was not sensitive to detect any significant difference in antioxidative activity among EGC, E G C G and EC. No difference in thermal loss between individual epicatechin derivatives was observed. To simplify the presentation, only data for total G T C s as a sum of EGC, E G C G , EC and E C G are shown. At the concentration of 1.5%, thermal loss of G T C s at 95°C in canola oil was compared to that of BHT. As shown in Fig. 6, loss of G T C s in canola oil was significantly less than that of B H T under the conditions described (P < 0.01). After heated for 44 h, 30°/,, G T C s while 67% B H T added to canola oil was lost. Furthermore, loss of G T C s reached to 65% whereas that of BHT reached to 90% after 98 h of heating at 95°C. Table 2 Effects of total green tea catechins (GTCs) on change in unsaturated fatty acids of canola oil (wt% of total fatty acids)

heated at 95°C for 36.5 h

Unheated Canola

Heated Canola

Canola + GTCs

Palmitic acid

5.2_+0.P

Stearic acid

4.8_+0.1 h 2.0 _+0.1

2.1 ± 0.1

1.8 _+0.1

Arachidic

0.6 _+0.1

0.7 ± 0.1

0.6 _+0.1

Oleic acid Vaccenic acid Linoleic acid

54.4 _+ 0.1 h 2.9 + 0.1 21.5 _+0.1 ~'

60.9 _+ 0.2 ~ 3.3 + 0.1 17.8_+0.2 b

55.3 _+ 0.3 b 3.0 + 0.1 21.6 + 0.2 ~

18:2 isomers

0.6+0.1 b

1.0+0.P

0.7+0.1 u

~-Linolenic

7.9 _+_0.1 ~'

4.7 + 0.2 b

7.6 + 0.2 ~'

18:3 isomers

4.2 _+ 0. P

3.3 _+0. P

4.0_+ 0. P

Polyunsatu-

29.4 ± 0.1 ''

22.5 ± 0.4 b

29.2 ± 0.3"

rated' Monounsaturated2

57.3±0.1 b

64.2±0.3"

58.3±0.3 b

4.6±0.1 b

acid

acid

Others

Saturated 3

1.1 _+0.1

7.5_+0.1 b

1.0±0.1

0.8 _+0.1

8.1_+0.1"

6.9_+0.1 ¢

'Polyunsaturated = Linoleic acid + :~-Linolenic acid.

2Monounsaturated= Oleic acid+ Vaccenic acid. 3Saturated=Myristic acid+Palmitic acid+Stearic acid+ Arachidicacid.

~,.b.~ Means in the same row with different superscript letters differ significantly (p<0.05).

Data are expressed as mean for n = 8 samples.

Z.Y. ('hen, P.T. Chan ,; Chemistry and Phys'ics 0/" Lipi&" 82 (1996) 163 172

25 20 ~ i c ~ o o 15 ~ c# ® ]0 ~m

~

a

m

a

a

--~-m-n-rn,~--~ ÷ EGO b ~kY,.,.'~b +EGCG + EC "~°--~2.~.~o< +ECG ~ + BHT a' Canola

5

70

7///

'

'

'

'

'

16

20

24

28

32

Hours

Fig. 5. Effects of individual epicatechin derivatives on oxidation of canola oil at 95 + 2°C. Data are expressed as mean _+ SD:n = 6 9 reaction tubes. O, canola oil; D, canola oil + 200 ppm epigallocatechin (EGC); ~ , canola oil + 200 ppm epigallocatechin gallate (EGCG); ~ , canola oil + 200 ppm epicatechin (EC): I , canola oil + 200 ppm epicatechin gallate ( E C G ) : ) , canola oil + 2 0 0 ppm butylated hydroxytoluene (BHT). a.b,c.d,,- Means at the same time point with different superscript letters differ significantly (P < 0.05).

4. Discussion Green tea is excellent resources of natural antioxidants, which are mainly epicatechin derivatives including EGCG, EGC, EC and ECG. Jasmine tea, a green tea with addition of jasmine flower, is one of the most popular beverages consumed in China. The extraction method used in the present study yielded 7.4% GTCs of dry tea leaves, which was in agreement with 5.8 9.6% reported in the literature [14,15,19-21]. Although the total GTCs extracted vary with variety of teas, methods used, and different laboratories, the composition of epicatechin derivatives is relatively consistent with E G C G being most abundant ( > 50%) followed by EGC ( > 18%), EC ( > 12%) and ECG ( > 11%) [14,15,19-21]. The total G T C content is also highly dependent on leaf age. It has been reported that the leaf bud and the first leaf are rich in the total GTCs [14,22]. The epicatechin derivatives decrease with leaf age [22]. The crude GTCs exhibited strong antioxidative activity against lipid oxidation in canola oil (Fig. 4). This inhibitory effect of green tea extracts on lipid oxidation is attributed to the content of

169

epicatechin derivatives. To prove this, we have been able to determine the relative antioxidative activity of each epicatechin derivative. The results clearly demonstrated: ( I ) t h e four major epicatechins present in jasmine tea showed varying antioxidative activity in a decreasing order of EGC, EGCG, EC and ECG; (II) most importantly, they were even more effective than BHT; and (III) EGC and EC were more effective than their corresponding gallate derivatives, E G C G and ECG (Fig. 5). The present result was in agreement with that of Hard [15], who showed that 20 ppm EGC was more protective than 20 ppm E G C G while 50 ppm EC was more effective than 50 ppm ECG against lipid oxidation in lard. However, Xi et al. [12] a l s o e x a m i n e d r e l a t i v e a n t i o x i d a t i v e activity of green epicatechin derivatives using the rancimat test and found that E G C G w a s m o s t effective followed by EGC, ECG and EC in a decreasing rank against lipid oxidation in lard. Lunder [10] also reported that the antioxidative activity of green tea extract was positively related to the content of EGCG. The varying effect of individual epicatechins on oxidation was probably related to the number and position of their hydroxyl groups. As shown in Figs. 1 and 5, EGC was more effective than EC against lipid oxidation in canola oil implying that the hydroxyl group at position 5' may play an important role, at least partially, in contribution to the antioxidative activity observed for EGC. Similarly, the role of hydroxyl group at position 5' can be best illustrated when the anti-oxidative activity of E G C G is compared to that of ECG (Figs. 1 and 5). We have also previously cornpared antioxidative activity of various flavonoids [23]. The results also demonstrated clearly protective role of hydroxyl group at position 5' against lipid oxidation when quercetin was compared with myricetin [23]. It is possible that the continuous three hydroxyl groups at position 3', 4' and 5' in EGC are more vulnerable to loss of a proton and their resulting free radical form is more stable than the two hydroxyl groups at position 3' and 4' in EC. By the similar deduction, stronger protective effect of E G C G than ECG was therefore due to the combination of an additional hydroxyl group at 5' and the resultant three continuous hydroxyl groups at position Y, 4' and 5'.

Z.Y. Chen, P.T. Chan / Chemistry and Physics q[ Lip±&' 82 (1996) 163 - 172

170

Table 3 Effects o f i n d i v i d u a l tea catechins on c h a n g e in u n s a t u r a t e d fatty acids of c a n o l a oil (Wt.% total fatty acids) heated at 95°C tbr 31 h F a t t y Acids

Heated Canola

P a l m i t i c acid Stearic acid A r a c h i d i c acid Oleic acid Vaccenic acid Linoleic acid 18:2 isomers ~ - L i n o l e n i c acid 18:3 isomers Others Polyunsaturated 1 Monounsaturated 2 Saturated 3

+ EGC

5.1 + O.P 2.1 -+_0.1 0.7 + 0.1 61.5_+0.6 ~ 3.3 + 0.2 17.3 + 0.3 ~ 0.8 + 0.1 4.6 +_ 0.2 ~ 3.4 -+- 0.l b 1.2+0.1 21.9_+0.5 ~ 64.8 _+ 0.6 ~' 8.2 _+ 0.2"

+ EGCG

4.4 -I- 0.1 b 1.8 + 0.1 0.6 + 0.1 55.3-+0.68 2.9 _+ 0.2 21.5 _+ 0.3 'l 0.6 -t- 0.1 7.6 _+ 0.3 ~' 4.2 + 0.P' 1.1 + 0 . 1 29.1 + 0 . 6 ~ 58.2 + 0.6 b 6.8 + 0.2 ¢

+ EC

4.3 + 0.1 b 1.9_+ 0.1 0.7 + 0.1 56.5__+l.1 b 3.0 _+ 0.3 20.9 ,+ 0.7 ab 0.6 _+ 0.1 7.1 + 0.6 ab 4.1 _+ 0.2 ~' 0.9_+0.1 28.0_+ 1.2 ~b 59.5 _+ 1.2 h 6.9 _+ 0.2 ~

4.5 -+ 0. ]b 1.9 + 0.1 0.7 + I).1 57.3+2.0 b 3.(1 _+ 0.3 20.2 _+ 1.2 ~'b 0.7 ,+ 0. I 6.6 ,+ 0.9"" 3.9 _+ 0.2 "b 1.2+0.1 26.8_+2.1 "b 60.3 _+ 2.0 u 7.3 ,+ 0.3 b~

+ ECG 4.4 + 0.11' 1 . 9 - 0.1 0.7 _+ 0.1 56.8 ,+ 0.9 h 3.4 +_ (1.4 20.4 _+ 0.6 h 0.6 + 0.1 6.7 _+ 0.5 b 3.9 + 0 . P 1.2+0.1 27.1 + 1.1 b 60.2 + 1.0 b 7.2 _+ 0.3 b~

+ BHT 4.8 -- O. p,b 2.1 + 0.1 0.7 ,+ 0.1 60.3_+0.8 ~' 3.3 _+ 0.3 18.2 -- 0.7 ~ 0.7 _+ 0. I 5.1 _+ 0.4 ~ 3.6 _+ 0.1 t' 1.2_+0.1 23.3 _+ 1.1 c 63.6 -+ 0.8 ~' 7.9 + 0.4 b

~Polyunsaturated = Linoleic a c i d + :~-Linolenic acid. 2 M o n o u n s a t u r a t e d = Oleic acid + Vaccenic acid. 3Saturated = Myristic acid + Palm±tic acid + Stearic acid + A r a c h i d i c acid. ,,b.~ M e a n s in the s a m e row with different superscript letters differ significantly ( P < 0 . 0 5 ) . D a t a are expressed as m e a n _+ S D for n = 6 - 9 .

The mechanism by which EC was more effective than ECG while EGC was more effective than EGCG as ant±oxidants remained unexplained (Figs. 1 and 5). Perhaps, ECG is larger, less mobile and therefore less effective than EC

1O0 ~

~

80 ~ -~ c

~

40

~

3 GTCs

•

20 BHT 0

I

I

20

40

I

I

I

60

80

100

Hours Fig. 6. T i m e course o f the r e m a i n i n g G T C s and B H T in c a n o l a oil h e a t e d at 95°C. D a t a are expressed as m e a n

_+

SD/n = 6 samples. O, BHT; CL G T C s . * represents a significant difference between G T C s and B H T at the same time

point (P<0.01).

against lipid oxidation. Alternatively, ECG has an additional gallate group, which makes it more hydrophillic, less soluble in oil, and therefore less effective against lipid oxidation compared to EC. By similar deduction, larger molecular size and more hydrophillicity may contribute to less ant±oxidative activity observed for EGCG than that for EGC. Loss of anti-oxidant during the heating processes is one of factors contributing to ineffectiveness of an ant±oxidant. There has been no study regarding thermal degradation of green tea epicatechins. It can be very complex in investigating breakdown pathway of tea epicatechins, and interactions between degradation products derived from lipid oxidation and ant±oxidants themselves during the heating processes. To simplify this, our method aimed at thermal loss of individual tea epicatechins by measuring the remaining content o f GTCs regardless of identification of their degradation products. Our data demonstrated that thermal loss of the GTCs was less than B H T under the conditions examined. Escape of BHT from the frying medium by volatilization and

Z.Y. Chen, P.T. Chan /' ChemLstry and Physics q[ Lipids 82 (1996) 163 172

distillation due to the high temperature was well documented [24 26]. However, the possibility that BHT was thermally decomposed could steam

not be eliminated [27]. Although the loss of GTCs in canola oil by volatilization o r s t e a m distillation was minimum due to larger molecule and higher boiling point, the destruction or polymerization could be initiated by light and heat [22]. Nevertheless, the present results suggest that the GTCs may replace BHT as antioxidants to control lipid oxidation in processed foods. We are currently studying thermal breakdown pathway of GTCs and identifying their possible decomposition products in frying oil. In summary, we examined the antioxidative properties of GTCs isolated from jasmine tea and the individual epicatechins in canola oil heated at 95°C for various time. All the epicatechin derivatires tested were more powerful than BHT as an antioxidant. To generalize the data obtained in the oxygen consumption test, the antioxidative activity of individual epicatechin derivatives tested were in a rank of EGG > E G C G > EC > EGG. The antioxidative characteristic of four common epicatechin derivatives was determined by multiple factors including the balance between hydrophillicity and hydrophobicity, the total number and location of hydroxyl groups on aromatic ring.

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