ANTIOXIDANT ACTIVITY OF PHENOLIC EXTRACTS OF EVENING PRIMROSE (OENOTHERA BIENNIS): A

PRELIMINARY STUDY

FEREIDOON SHAHIDI', RYSZARD AMAROWICZ', YUEHUA HE and MAHINDA WETTASINGHE

Department of Biochemistry Memorial University of Newfoundland

St. John's, NF, A I B 3x9, Canada

Received for Publication November 8, 1996 Accepted for Publication January 10, 1997

ABSTRACT

Phenolic compounds@om ethanolic extracts of evening primrose (Oenothera biennis) were separated into ten @actions using Sephadex LH-20 column chromatography. For individual @actions, UV spectra were recorded and the content of total phenolics determined Antioxidant activity of each isolatedfiaction was examined in a P-carotene-linoleate model system. Fractions were ficrther characterized for the number of phenolic compounds, presence of proanthocyanidins and their antioxidant activity by TLC analysis. Strong antioxidant properties were noted for @actions with high content of total phenolics @actions IV-4. Vanillin-positive compoumh were observed in Pactions VI-X and one compound was tentatively identified as (+)catechid(-)epicatechin. From @action IV, two phenolic compounds were separated by preparative TLC; compound A was most likely an isofavone and the UV spectrum of compound B was similar to that of 2-hydroxychalcone.

INTRODUCTION

Evening primrose (Oenothera biennis) is a major source of y-linolenic acid (GLA, 18:30-6) (Syed Rahmatullah et al. 1994). The potential beneficial effects of evening primrose oil as an anti-inflammatory agent is well recognized (Gustone 1992; Horrobin 1992). However, the meal after oil extraction remains largely unutilized. Therefore, it is prudent to develop methods for producing value-added components from primrose meal following oil extraction. Similar to other plant materials, evening primrose meal might be used as a potential source of phenolic

'Author for correspondence: Tel.: (709) 737-8552, Fax.: (709) 737-4000, e-mail:

2Permanent address: fshahidi@morgan.ucs.mun.ca

Research, Polish Academy of Sciences, Olsztyn, Poland. Division of Food Science, Institute of Animal Reproduction and Food

Journal of Food Lipids 4 (1997) 75-86. All Rights Reserved. Topyright 1997 by Food & Nutrition Press, Inc., Trumbull, CT 0661 I . 75

76 F. SHAHIDI, R. Ah4AROWICZ, Y. HE and M. WETTASMGHE

antioxidants for use in food and nonfood applications. Phenolic compounds present in meals from seed oils are chemically diverse, but generally belong to the phenolic acid, flavanoid and related compounds. The antioxidant activity of seed meals, as such, or their extracts depends heavily on the chemical nature of their active components, the relative concentration of the active ingredients as well as the system under which they are tested. The antioxidant activity of meals from soybean (Hammerschmidt and Pratt 1978), canola (Wanasundara et ul. 1994), cottonseed (Whitten et ul. 1984), mustard (Saleemi et ul. 1993; Shahidi et ul. 1994), sesame (Fukuda et ul. 1985; Shahidi et ul. 1997), flaxseed (Amarowicz et ul. 1993) and other oilseeds (Shukla et ul. 1997) has been reported in the literature. The present study intended to extract and evaluate the activity of the phenolic fractions of primrose meal following oil extraction. The antioxidant activity of phenolics after fractionation in a P-carotene-linoleate model system was also evaluated.

MATERIALS AND METHODS

Samples and Extraction

Samples of evening primrose were obtained from Efamol, Inc. (Kentville, NS). The meal (40 g), after oil extraction, was dried and extracted with 400 mL of ethanol at 80C for 20 min (Amarowicz et ul. 1995a). Solids were separated by centrifbgation and extracted twice more with the same solution. The resultant ethanolic extracts were combined and evaporated to dryness under vacuum at 40C.

Column Chromatography

The ethanol extract (350 mg fkom 4.7 g total) was dissolved in 5 mL of methanol, applied to a chromatographic column (2.5 x 45 cm) packed with Sephadex LH-20 (Pharmacia, Upsala, Sweden) and eluted with methanol (Amarowicz et ul. 1992). The eluates were collected in 20 mL tubes using a fraction collector and their absorbance was read at 280 nm. Based on their absorbance data, eluates were pooled into ten major M o n s , solvents evaporated, and residues weighed.

Analysis of Fractions

The content of total phenolics, as catechin equivalents, in each fraction was determined using the Folin-Denis reagent (Naczk and Shahidi 1989). UV spectra of individual fractions were recorded using a Hewlett-Packard 8452A diode array spectrophotometer.

ANTIOXIDANT ACTIVITY OF EVENMG PRIMROSE 77

Antioxidant Activity

Antioxidative activities of separated fractions were evaluated using a p- carotene-linoleate model system (Miller 197 1). A methanolic solution (0.2 mL) containing 1 mg of each of the dried fractions or 0.3 mg of butylated hydroxyanisole @HA) was added to a series of tubes containing 5 mL of prepared emulsion of linoleate with p-carotene. Systems were kept at 50C in a water bath, aliquots drawn every 15 min and their absorbances read over a 120 min period.

TLC Analysis of Fractions

Fractions were examined on silica gel 60 TLC plates with a layer thickness of 0.2 mm (Merck, Darmstad, Germany) using petroleum ether-diethyl ether-acetic acid (80:20: 1, v/v/v) (Amarowicz et af. 1995a) and chloroform-methanol-water (65:35:10, v/v/v, lower phase) (Amarowicz et af. 1994). To visualize phenolic compounds, plates were sprayed with a solution of ferric chloride (Krebs et af. 1989). Compounds with antioxidant activity were visualized after spraying a second developed plate with a solution of p-carotene and linoleic acid (Philip 1974). Each fraction and standards of (-)epicatechin, (-)epigallocatechin, (-) epicatechin-3-gallate and (-)epigallocatechin-3-gallate, isolated from green tea (Amarowicz and Shahidi 1996), were also developed on silica gel 60 TLC plates with toluene-acetone-formic acid (30:30: 10, v/v/v) (Lea 1978) and sprayed with a vanillin-hydrochloric acid reagent (Karchesy et af. 1989).

Preparative TLC

Two pure compounds were separated from fraction IV using preparative silica gel 60 TLC plates with a layer thickness of 0.5 mm (Merck, Darmstadt, Germany) and a chloroform-methanol-water (65:35:10, v/v/v, lower phase) as the mobile phase.

RESULTS AND DISCUSSION

Ten fractions (I-X) were separated from the ethanolic extract of defatted primrose meal using Sephadex LH-20 column chromatography according to their absorbance readings at 280 nm (Fig. 1). The chromatogram was characterized by one sharp intense peak (IV), one broad peak (X), one medium size peak (VII) and four smaller peaks (I, V, VI, VIII). Compounds from fractions I1 and IX did not give typical peaks.

The relative contribution of fraction IV in the extract was the highest (26.8%). The relative content of fractions I, I1 and X varied between 10.2 and 16.9% and remaining fractions were present at less than 10% (Table 1). The content of total phenolics in fractions IV-X was very similar and ranged from 132 to 165 mdg.

18 F. SHAHIDI, R. AMAROWICZ, Y. HE and M. WETTASINGHE

35

30 - -

Li 25

a -

- 20 m -

d d 15

10

5

X 'VIII

10 20 30 40 50 60 T u k numbor ( 20 ml Ituk)

FIG. 1. SEPARATION OF PRIMROSE PHENOLIC FRACTIONS BY SEPHADEX LH-20 COLUMN CHROMATOGRAPHY

Fractions 1-111 contained lower amounts of total phenolics (Table 1). W spectra of separated fi?lctions (Fig. 2) indicated absorption bands generally

at 280 nm (I, If, V, VI, M, X). For itactions III, IV and WI, W maxima occurred at 286,274 and 278 nm, respectively. The W spectrum of fraction VII showed two maxima at 256 and 364 nm. With the exception of fraction I, all other tiactions possessed shoulders at 266 to 348 nm.

Figure 3 shows the antioxidant activity of each fraction as compared with that of BHA. The antioxidant efficacy of fractions I, 11,111 was low as they contained small quantities of total phenolics (Table 1). Antioxidant property of fraction V decreased during the second hour of incubation. Meanwhile, antioxidant activity of tiactions IV and VI-X was comparatively strong as approximately 50% of the p-carotene remained unoxidized after 2 h of incubation in the presence of these fractions. Antioxidant activity of fkactions IV and VI-X was comparable to those of extracts tiom rapeseed (Amarowicz et ul. 1995a), crude catechins fkom green tea (Amarowicz and Shahidi 1995), and the most active fractions from extracts of flaxseed (Amarowicz ef ul. 1993) and mustard (Shahidi et ul. 1994; Amarowicz et ul. 1996).

ANTIOXIDANT ACTIVITY OF EVENING PRIMROSE 79

TABLE 1. DISTRIBUTION (%) OF SEPARATED PHENOLIC FRACTIONS AND THEIR CONTENT OF

TOTAL PHENOLICS (MGIG)" AND THEiR UV SPECTRAL DATA

Fractions Relative Total Phenolics I Max, nm Shoulder, nm Content

I

I1

111

IV

V

VI

VII

VIII

IX

10.2

16.9

8.1

26.8

3.9

3.5

9.5

1.4

3.9

59

38

36

165

161

143

140

132

143

280

280

286

274

280

280

256,364

278

280

272

322

328

304,3 14

306

266-282

304,348

3 04

x 15.8 147 280 304 "As catechin equivalents.

Thin-layer chromatographic results indicated that fractions obtained via column separation contained several phenolic compounds (Fig. 4 and 5). TLC plates developed with a polar chloroform-methanol-water solvent system displayed more compounds than those separated using a nonpolar petroleum ether-diethyl ether- acetic acid as a nonpolar solvent system. Spots from all fractions which were located on the baselines of plates, using both developing solvent systems, possessed antioxidant activity. Furthermore, plates developed with nonpolar systems had one spot with antioxidant activity as detected for fractions I, 11, IV and V, and three spots for fraction 111 (Fig. 4). Application of a more polar solvent system (Fig. 5 ) allowed separation and subsequent detection of more spots (Rf > 0) from different fractions.

The presence of vanillin-positive compounds was noted for fractions VI-X on plates developed with toluene-acetone-formic acid (30:30: 10, v/v/v) (Fig. 6). A compound from fraction VI with an Rf value of 0.63 was isolated and tentatively identified as (-)epicatechin. In fractions VII-X, compounds which were more polar than (-) epigallocatechin-3-gallate were present. Rf values for these compounds, most probably dimers and trirners of proantocyanidines, were 0.27 and 0.25 (fraction VII), 0.38, 0.31, 0.27 and 0.17 (fraction VIII) and 0.33 and 0.28 (fraction IX). In addition, in fractions VIII-X red spots were noted on or close to the baseline; these compounds might be due to polymerized polyphenols.

The UV spectra of two compounds isolated by semi-preparative TLC from fraction IV are shown in Fig. 7. The UV spectrum of compound A exhibited a

80 F. SHAHIDI, R. AMAROWICZ, Y. HE and M. WETTASINGHE

WAVELENGTH nm

250 300 WAVELENGTH nm

FIG. 2. W SPECTRA OF PHENOLIC FRACTIONS ISOLATED FROM PRIMROSE

maximum at 262 nm and was very similar to the spectra of isoflavones such as genistein, which possess an absorption maximum at 261 nm, and sphaerobiside, sophoricoside, prunetin, lanceolarin, orobol or pratensein (all isoflavones) which display an absorption maximum at 262 nm (Mabry et al. 1970). The maximum absorbance of compound B was observed at 276 nm, which is similar to that of 2- hydroxychalcone (Mabry et al. 1970). Further work on the identification of the chemical structure of the antioxidative compounds of evening primrose is in

ANTIOXIDANT ACTIVITY OF EVENING PRIMROSE 81

progress and potential application of extracts of evening primrose meal in different systems is also being examined.

-v- I -v- I 1 -a- 111 -.- IV

+lX --t X +BHA -@-Control -0-v -A-vl evil -f vlll

0.7

0.6

E c 0.5

3 0.4

8 0.3

0 PI *

E 2 0.2 8

0.1

15 30 45 60 75 90 105 120 Time (min)

FIG. 3. ANTIOXIDANT ACTlVITY OF PHENOLIC FRACTIONS OF PRIMROSE IN A p- CAROTENE-LINOLEATE MODEL SYSTEM AS MEASURED BY CHANGES IN

ABSORBANCE VALUES AT 470 NM

ACKNOWLEDGMENTS

We are grateful to Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support in the form of a research grant to Dr. F. Shahidi.

82

Rf

0.6.

F. SHAHIDI, R. AMAROWICZ, Y . HE and M. WETTASINGHE

A 0 0

00 0

0 %

a 0 0 8

Rf Lo-

0.6.

B 0 0

0

0 0

0 0

? W ? w QoQQsge v M w VI

FIG. 4. TLC CHROMATOGRAMS OF PHENOLIC FRACTIONS OF PRIMROSE; chromatograms were developed using petroleum ether-diethyl ether-acetic acid (80:20:1, vMv); plates were sprayed with a solution of (A) ferric chloride to give spots of phenolic compounds and (B) p-carotenelinoleate in order to evaluate antioxidant activity of spots

of fractions I-X.

ANTIOXIDANT ACTIVITY OF EVENlNG PRIMROSE

1.0.

0.6.

83

FIG. 5 . TLC CHROMATOGRAMS OF PHENOLIC FRACTIONS OF PRIMROSE; chromatograms were developed using chloroform-methanol-water (6935: 10, v/v/v) plates were sprayed with a solution of (A) ferric chloride to give spots to phenolic compounds and Q3) pcarotene-linoleate in order to evaluate antioxidant activity of spots of fractions I-X.

0 0

FIG. 6. TLC CHROMATOGRAM OF PHENOLIC FRACTIONS OF PRIMROSE; chromatogram was developed using toluene-acetone-formic acid (30:30: 10, v/v/v) plate was sprayed with a vanillin-hydrochloric acid reagent; I-X fractions from sephadex column; standards; 1, (-) epicatechin; 2, (-) epicatechin-3-gallate; 3, (-) epigallocatechin; and 4, (-)

EpigaIlocatechin-3-gallate.

84

1 0 1

0.8 *

I::: 0.2 *

F. SHAHIDI, R. AMAROWICZ, Y. HE and M. WE'ITASINGHE

1.0

0.8 *

I:::: 0.2.

4

-

4

FIG. 7. W SPECTRA OF COMPOUNDS A AND B SEPARATED FROM FRACTION IV BY A SEMI-PREPARATIVE TLC

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