Res. Chem. Intermed, Vol. 26, No. 7,8, pp. 817-828 (2000) �9 VSP 2000

THE ESR STUDY ON THE PROTECTIVE EFFECT OF GRAPE SEED EXTRACT ON RAT HEART MITOCHONDRIA FROM THE INJURY OF LIPID PEROXIDATION

JUNTAO GAO 1, HUIRU TANG 2, and BAOLU ZHAO a~

1 Laboratory of Visual Information Processing, Department of Molecular and Cell Biophysics, Institute of Biophysics, Academia Sinica, Beijing 100101, P.R. CHINA 2 Institute ofFood Research, Norwich Research Park, Norwich NR4 7NA, UNITED KINGDOM

Received 5 March 2000; accepted 13 June 2000

AbstractmThe effect ofgrape seed extraet (GSE) on the lipid peroxidation ofrat heart mitochondria was studied using the spin trapping agent a-(4-pyridyi-l-oxide)-N-t-butylnitrone (4-POBN) with electron spin resonanee (ESR) speetrometer. Two fatty aeid spin labels, 5-doxyl-stearic acid (5- DOXYL) and 16-doxyl-stearic aeid (16-DOXYL) were used to study the effeet of GSE on mitochondrial membrane fluidity. The results showed that the GSE eouid scavenge the lipid free radieals generated ffom the mitochondria of rat hearL and protect the mitochondrial membranes damaged by lipid peroxidation. Both the scavenging effect and the protective effeet ate better in water phase than in lipid phase. And the data indicate that perhaps trimers and other polymers have better scavenging effeet on lipid free radicals than monomers.

INTRODUCTION

Mitochondria ofheart muscle cells serve as the place for the generation of energy by oxidative phosphorylation, where substrate is oxidized by molecular oxygen and the released energy is used to synthesize ATE The mitoehondrial membrane is therefore the major target for the damage of lipid peroxidation in vivo. The mitochondrial membrane mainly consists of unsaturated fatty acids and proteins. The production of oxygen free radicals and the related damage are the main source of injury in the process of ischemia-reperfusion and other cardiac diseases [1-3]. The final product of lipid peroxidation - malondialdehyde (MDA), can make the membrane lipid and membrane proteins crosslink with each other, then change the structure of membrane proteins and decrease the mobility of the membrane. The fluidity of membranes is related closely with the function of cells. Therefore, the physical state of membrane lipid can affect the activity of membrane protein and the function of cells, such as

818 B. Zhao et al.

enzyme activity and the ion transmission across the membrane [4]. Grape seeds contain many plant polyphenols generally called procyanidins

(PC). Antioxidants, such as SOD, catalase and Vitamin E can protect heart against ischemia-reperfusion damage, indicating that oxygen free radicals may contribute to this injury. The scavenging effect ofthese polyphenols on free radicals is far better than Vitamin C and Vitamin E [5], especially their activities in vivo are much better than other antioxidants [6]. Procyanidins easily form chemical bonds with collagen, stablize the cell membrane and have good antienzyme activity [7]. Recent work reported that wine-drinking correlated with the lower possibility of degenerative diseases such as heart disease [8]. It has also been speculated that polyphenols were the major components for these protective functions. And there is no report about the protective effect of GSE on mitochondrial membranes in the literature. For these reasons, we selected grape seeds as our source of antioxidants. We got polyphenols from aqueous phase and lipid phase extracts ofGSE and investigated their protective effects on rat heart mitochondria.

MATERIALS AND METHODS

Materials

Wistar white rats (6 weeks, 150-200 g), female, were obtained from the animal center of our institute; the spin trapping agent a-(4-pyridyl-l-oxide)-N-t-butylnitrone (4- POBN) was purchased from Sigma Chemical Co.; 5-doxyl-stearic acid (5-DOXYL) and 16-doxyl-stearic acid (16-DOXYL) were purchased from Aldrich Chemical Co.; Sephadex LH-20 was obtained from Fluka Co. AII other regents made in P.R. China

were ofAR level.

Preparation o f GSE

Approximately 200g of homogenized grape seeds was extracted three times with Me2CO-H20 (1:1,600ml). The mixture was concentrated under vacuum until all of the acetone had been removed. The extract obtained was washed three times with petroleum ether to eliminate liposoluble substances. The aqueous extract of grape seeds was extracted three times with EtOAc:H20 (2:3). The upper layer (EtOAc phase) and the lower iayer (H20 phase) were concentrated separately under vacuum until all of the EtOAc and I-I20 were removed. So the lipid phase extract and the water

phase extract were obtained [9].

The Protective Effect of Grape Seed Extract on Rat Heart Mitochondria 819

Gel chromatography fractionation Fractionation of the extract (lipid phase substance or water phase substance

separately) of grape seeds was performed in a column (60• filled with Sephadex LH-20 suspended in 95% ethanol. The gel was previously conditioned by successive washing with water and 95% ethanol, in which it was allowed to swell for 24 hours. Elution was carried out with 95% ethanol, as described by Bailon [10], maintaining a flow rate of 2.5 ml/min with the aid o fa peristaltic pump. The absorbance of the eluate was measured at 280 nm. The fractions collected were added to a small volume of water and concentrated under vacuum to eliminate the alcoholic solvent, and 31

fractions were obtained (17 fractions from the lipid phase and 14 fractions from the

water phase). Because there are more polymers and longer oligomers in the end fractions, which have better antiulcer activity [11], so we choose the end fraction of lipid phase substance and water phase substance as our source of research. The end fraction of lipid phase substance was called Lipid Phase Substance (LPS); and the end fraction of water phase substance was called Water Phase Substance (WPS). The LPS and WPS fractions were analyzed by Analytical HPLC.

A nalytical HPLC conditions A BioRAD Model 2700 Solvent Delivery System equipped with a BioRAD 7215

manual injector was used. The column (25x0.46 cm) was a 4-1am Ullracarb C18 ODS 20, thermostated at room temperature. The solvents were: A: acetic acid/bidistilled

water (10:90); B: bidistilled water. A linear gradient was run from 10 volumes of A + 90 volumes of B to 70 volumes ofA + 30 volumes ofB during the first 50 min, followed by another linear gradient from 70 volumes of A + 30 volumes of B to 90 volumes of A + I 0 volumes of B during another 40 min, and then to pure A during the last 20 min. The flow rate was 0.9 ml/min. Between two injections the column was washed isocratically, using methanol/water (50:50,v/v), for 30 min. The fractions were detected at 280 nm with a BioRAD Model 1706 UV/VIS Monitor. Before

injection into the chromatographic system, all extracts were filtered through a 0.45-

~tm membrane filter (Millipore). AII analyses were done in duplicate at least.

Preparation of Mitochondria Mitochondria were isolated from rat hearts according to the literature [12] with slight modification. Rat hearts were minced, washed and then homogenized at 4~ in 0.9% NaCI. The homogenate was centrifuged at 600 g for 7 min. The supematant was then centrifuged at 10,000 g for 10 min. The mitochondrial pallet was washed twice with

a TMS buffer containing 3 mM MgCI, 50 mM Tris-HCL (pH 7.5) and 0.2 mM

sucrose. Then the pallet was suspended in TMS buffer and stored at -20~

820 B. Zhao et al.

Spin trapping of lipid free radicals 15 q mitochondria suspension (Protein Concentration 7.74 mg/mi), 10 ~tl 4-POBN (0.2M), 15 ~tl PBS (pH 7.4), 5 ~ti Fe 2+ (5 mM), 5 ~tl Diethylenetriaminepentaacetic Acid (DETAPAC, 5 mM) were mixed and incubated at 37~ for 30 min, then sucked into a quartz capillary for ESR measurement.

The ESR measurement condition: the ESR spectra were recorded with Brucker ER200 D-SRC ESR spectrometer. Parameters were employed as follows: X- band, 100 kHz modulation with amplitude 1 G, microwave power 10 mW, central magnetic field 3,250 G, sweep width 200 G, temperature 20~

Scavenging effect of GSE on lipid free radicals 15 ~tl mitochondria suspension (Protein Concentration 7.74 mg/mi), 10 btl 4-POBN (0.2 M), 5 ~tl DETAPAC (5 mM), 10 q PBS (pH 7.4) and 5 ~tl water phase or lipid phase of GSE (1 ~tg/ml, 5 p.g/ml, 10 ~tg/ml, 50 ~tg/ml, 100 ~tg/ml, 200 lag/ml, 500 ~tg/ml and 1 mg/mi respectively) were mixed and incubated at 37~ for 10 min, then 5 ~tl Fe 2§ (5 mM) was added and incubated at 37 ~ for 30 min. The sample was sucked into a quartz capillary for ESR measurement [13]. The definition of the scavenging effect of GSE on lipid free radicals is:

Ho - Hx E - • 100%

Ho

where Ho and Hx represent the ESR intensities of the sample without and with GSE respectively.

Spin Labeling mitochondria with 5-DOXYL or 16-DOXYL The lipid peroxidation reaction in mitochondrial system was initiated by Fe2+/Fe 3+ [14]. 80 ~L mitochondria suspension (Protein Concentration 7.74 mg/mi), 50 ~tl WPS or LPS of GSE (0.02 mg/mi) were mixed and incubated at 37~ for 10 min, then 0.05 mM (NI-I4)2Fe(SO4)2 and 0.15 mM FeCI3 were added. Affer incubation with Fe2+/Fe 3+ at 37~ for 10 min, the spin label 5-DOXYL or 16-DOXYL (0.15 mM) was added. Then the mixture was incubated at 37~ for 60 min again before ESR measurement. The order parameter S and rotational correlation time te are defined as follows

[15,16]:

The Protective Effect of Grape Seed Extract on Rat Heart Mitochondria 821

S A,,

A n - A•

- 1/2(Axx + Ayy)

Here AH<0), h(0), h(l), hc_1 ) are shown in Fig. 3, Ax~=6.3, Ayy=5.8, A==33.6, Arnax =

2A,,and Ami, = 2A•

Data Analysis The scavenging effect of GSE on lipid free radicals was shown by the value of IC50. The order pararneter S and rotational correlation time % were calculated to show the membrane fluidity. AII data are expressed as mean + S.D. and analyzed by ANOVA. A difference was considered statistically significant when P < 0.05.

RESULTS

The Scavenging Effect o f GSE on Lipid Free Radicals Generated from Lipid Peroxidation of Mitochondria lnitiated by Fe 2~ /Fe 3+ Figure 1 shows the ESR spectrum of lipid free radical generated from lipid peroxidation of mitochondria initiated by Fe 2+/Fe 3+ and spin trapped by 4-POBN. The hyperfine splitting constants aN=l.56 mT and an=0.297 mT are typical of the

spectrum oflipid free radical-4-POBN adduct (no free radicals could be trapped in the absence of Fe 2§ 3§ or mitochondria) [20,21 ]. The signals were stable for 30 min. So we used the signal amplitude as the parameter to measure the concentration of lipid free radicals generated by the lipid peroxidation of mitochondrial membrane.

Figure 2 shows the scavenging rate of GSE on lipid free radicals generated from the lipid peroxidation of mitochondria. During the incubation at 37~ the lipid free radicals increased significantly with time in rat heart mitochondria. The scavenging efficiency was increased with the concentration of GSE. At the same concentration, the scavenging effect of WPS was greater than that of LPS. The IC50 values ofWPS and LPS are 0.28 mg/mi and 0.59 mg/ml respectively, indicating that WPS is a better scavenger of lipid free radicals in mitoch'ondria than LPS.

The Effect of Lipid Peroxidation on the Mitochondrial Membrane Fluidity, and the Protective Effect o f GSE on the Mitochondrial Membrane ESR spectra ofspin labeled mitochondria with 5-DOXYL and 16-DOXYL ate shown in Figure3. The order parameter (S) calculated from the spectrum is shown in Table 1. From the data labeled by 5-DOXYL, it can be found that S increased evidently at~er lipid peroxidation. When GSE was added, it was found that S decreased, almost

822 B. Zhao el al.

H

Figure 1. ESR spectrum oflipid ffee radical generated from lipid peroxidation of mitochondria initiated by Fe2+/Fe 3+ and spin trapped by 4-POBN. The mitochondria suspension (Protein Concentration 7.74mg/mi) 50 mi was incubated in the mixture of 0.2 M 4-POBN, 5 mM DETAPAC and 5 mM Fe 2§ for 30 min at 37~ in PBS (pH 7.4). The ESR conditions are described in Materials and Methods.

1.0 I

0.8.

�9 , - . - 0.6.

0.4.

o 1~ 0.2, e-

I--

0 .0 ,

wPS

' ! I ' �9 .....

o'.o o~ o., o'.6 o'.6 11o 1'.2 Concentration( m g/mi)

114

Figure 2. The scavenging effect of GSE on the lipid free radicals generated from iipid pemxidation of mitochondria studied by ESR spin trapping technique. The first peak height of the ESR spectra of lipid free radical-4-POBN adduct represented the relative quantity of the lipid ffee radicals. The spin trapping procedure was described in Materials and Methods.

The Protective Effect of Grape Seed Extract on Rat Heart Mitochondria

to the same as the S value o f control.

823

Am~x

s ~ ]G-DOXY~L

hm~

h(-l)

A//o lmT

Figure 3. ESR spectrum of mitochondrial membrane labeled with 5-DOXYL(top) and 16- DOXYL(bottom). The ESR experimental conditions are described in Materials and Methods.

Table 1 The effect of grape seeds extract (GSE) on the membrane fluidity labeled by 5- DOXYL

S P

Control Peroxidized Peroxidized + GSE (lipid phase substance) Peroxidized + GSE (water phase substance)

0.622i-0.0011 P<0.05 0.669-k0.0062 0.626+0.0064 P<0.05

0.614+0.0038 P<0.05

S is presented as mean :t: S.D. P value is compared with the peroxidized membrane.

824 B. Zhao et al.

Table 2 The effect ofgrape seeds extract (GSE) on the membrane fluidity labeled by 16- DOXYL

~c(• "l~ s) P

Control

Peroxidized

Peroxidized + GSE (lipid phase substance)

Peroxidized + GSE (water phase substance)

1.603•

1.922•

1.499-2-_0.0076

1.622•

P<0.05

P<0.05

P<0.05

S is presented as means + S.D. P value is compared with the peroxidized membrane.

From the data labeled by 16-DOXYL in Table 2, it was found that the rotational correlation time (xc) increased evidently afler lipid peroxidation. When GSE was added, xc decreased, almost to the same as the Tc value of the control.

HPLC Analysis of LPS and WPS In order to study the catechins and procyanidins fractions ofGSE, we compared the location of the peaks on the chromatograms with the commercial standards purchased from Extrasynthese (Genay, France). These standards are: gallic acid, (+)- catechin, (-)-epicatechin, epicatechin 3-O-gallate, procyanidin B3, procyanidin C2 and a procyanidin tetramer. We tried to identify the procyanidins from the spectra corresponding to each peak of the chromatogram of the commercial standards. The results of the analysis on the compounds are presented in Table 3. The data in Table 3 obtained by the HPLC conditions show that there are more complicated compounds in WPS than in LPS. Dimers are the main part ofWPS, but dimers, trimers and other polymers are the main parts of LPS.

DISCUSSION

Lipid peroxidation plays an important role in free radical biology. Unsaturated fatty acids in mitochondria membranes readily suffer lipid peroxidation, which consequently damages the cell membrane, impairs the respiration and oxidative phosphorylation of the mitochondria and disturbs the mechanism of ion transportation. This oxidative process is generally a free radical reaction, producing lipid free radicals [17-19]

The free radicals generated from lipid peroxidation of mitochondria can damage cell membranes, proteins, enzymes and DNA to cause diseases. The end

The Protective Effect of Grape Seed Extract on Rat Heart Mitochondria 825

Table 3 Compounds (water phase substance and lipid phase substance) isolated from grape seeds: distribution by percentage areas under the HPLC conditions employed.

Compounds Area, %

WPS Gallic acid 6. i Monomers 15. i Dimers 40.6 Trimers 17.3 Tetramers and other polymers 21.5

LPS Gallic acid 6.2 Monomers 20.0 Dimers 40.4 Trimers and other polymers 34.2

product, MDA, was usually measured to detect injury by this process. But lipid peroxidation is a chain reaction, in which free radicals are generated and participated. So it is difficult to study the mechanism by the end product. Here, the lipid free radicals trapped by 4-POBN were measured to detect the injury, which is a more direct method to study lipid peroxidation. With the effect offree radical, the following reactions take place:

LH + R' --~L" + RH

L o" + 02 --+ LOO"

LOO" + LH --+ LOOH + L.

A great number of free radicals (L" and LOO') are produced in this process. This leads to the change of lipid composition in biological membranes and the physiological function of membranes. On the other side, the free radicals will make the protein and DNA damaged, and even iead to cell death.

The above results (Fig. 2, Table I and 2) indicate that GSE reduces the damage of mitochondria caused by lipid peroxidation and has protective effect on the membrane. These polyphenols and their derivatives have good scavenging effect on lipid free radicals.

From the data ofTable 1 and 2, it can be found that both scavenging effect and protective effect ofWPS are better than LPS. Several kinds of monomers, dimers and trimers of the compounds of GSE are shown in Fig.4. The different compounds in WPS and LPS (Table 3) make the different scavenging effect on lipid free radicais.

826 B. Zhao et al.

The percentage of trimers, tetramers and other polymers in WPS is about 38.8%, which is greater than that in LPS, but the percentage of monomers in WPS is less than

LPS. Both scavenging effect and protective effect of WPS ate better. These data show that perhaps trimers, oligomers and other polymers have better scavenging effect on lipid free radicals than monomers.

There is another reason why the scavenging effect and the protective effect of WPS are better than LPS. LPS is easily soluble in organic solutions such as

HO.~~~ OH R2

(+)-Catechin: RI=C~(, R2=H (-)-Epicatechin: RI=H, R2=OH

) . . . - ~

""2" r HO~,,~ _~'~OH OH R2

Procyanidin Dimerl: RI=OH,R2=H Procyanidin Dimer2: RI=H,R2=OH

.o .~) i . .o. _ o. oH~ vo....,.,~oH

Ho~~l ,,,H

~176

Procyanidin trimerl: RI=H,R2=OH Procyanidin trimer2: RI=OH,R2=H

Figure 4 The monomers, dimers and trimers ofpolyphenols extracted from grape seeds

acetone, and not so easily soluble in water. So there are many more nonpolar components that ate not easily soluble in water in LPS than WPS. Because of the many nonpolar components in LPS, the molecules of LPS can not reach the surface

of the membrane easily. Although some of LPS molecules are near the surface because of the pervasion effect, there are many more chances for them to connect with

the nonpolar parts of the protein molecules of membrane because of the lyophobic interaction. Ir is well known that the proteins in mitochondria membrane ate about 75-

The Protective Effect o f Grape Seed Extract on Rat Heart Mitochondria 827

80 percent of the membrane weight. This makes the LPS moleeules more easily

connect with protein, not with lipid. LPS molecules can scavenge the scarce lipid free

radical in the outside space near the membrane. So the protective effect of LPS on

lipid membrane is limited. On the other hand, WPS is easily soluble in water, and it

can reach the surface ofmembrane easily. The WPS molecules readily scavenge the

lipid free radicals that reach the membrane surface. A s a result, WPS forms a

protective layer near the membrane surface and provides protection to membrane.

Thus its protective effect is much better than LPS.

Grape seeds are a rich source ofcatechins and procyanidins, and there are a

number of reports of these compounds showing a potent antioxidant activity [22-24]

and free radical scavenging activity [25-29]. These compounds are good for health of

human beings. Recognition of such health benefits of catechins and procyanidins in

GSE may facilitate the use of GSE asa dietary supplement.

A c k n o w l e d g e m e n t

We thank Professor Qian Wan, Mrs. Meifen Li and Mrs. Jungai Hu for their ESR

technological support, and Professor Shen Wu, Mr. Wei Li, Mrs. Duohua Liu, Prof.

Huwei Liu, Ms.Wei Wei and Ms.Yi Li for their expert advice of HPLC in this study.

This work was supported by a grant from the National Natural Science Foundation

of China anda grant from K. C.Wong Fund.

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