Analyses of phenolic and flavonoid compounds by high-pressure liquid chromatography

ANALYTICAL

102,171-175

BIOCHEMISTRY

(1980)

Analyses of Phenolic High-Pressure

and Flavonoid Compounds Liquid Chromatography

J. M. HARDIN AND C. A. Department

of Agronomy.

Altheiner

Laboratory,

University

by

STUTTE

of Arkansas,

Fayetteville,

Arkansas

72701

Received July 16, 1979 A technique to separate complex phenol& extracted from plant tissue has been developed using high-pressure liquid chromatography. The compounds, in glycosidic and ester linkages, can be collected as separation occurs. After hydrolysis, flavonoid components as well as the benzoic and cinnamic acid derivatives can be separated and identified with one chromatographic analysis of tissue samples. These techniques may be helpful in toxicity studies and in determining the role of phenolics in plants.

High-pressure liquid chromatography (hplc)’ offers high speed and sensitivity often difficult to attain with other chromatographic techniques. Recently, hplc methods have been reported for the analysis of phenolic compounds (43, particularly the benzoic and cinnamic acid derivatives. With these techniques 80 min replaced the laborious 48 to 72 h required for development of paper chromatograms. The purposes of this study were to develop methods for extending techniques previously applied to the benzoic and cinnamic acid derivatives to include more complex flavonoid compounds and for separating the complex compounds (phenolic esters and glycosides) extracted directly from plant tissue. MATERIALS

AND METHODS

Chemicals. Standard solutions of lo-” M included gallic, gentisic, protocatechuic, p-hydroxybenzoic , ,salicylic , vanillic, caffeic , syringic, benzoic, umbelliferone, p-coumaric, coumarin, ferulic, sinapic, and cinnamic acids and the flavonoids-naringenin, hesperetin, quercetin, and kaempferol. An additional standard solution contained all the ’ Abbreviations used: hplc, high-pressure liquid chromatography; BAMW, butanol:acetic acid:methanol: water.

compounds. Butanol and methanol were high grade distilled in glass solvents obtained from Burdick and Jackson Laboratories, Inc. Plant material. ‘Forrest’ soybean [ Glycine ma (L.) Merrill] plants, used for this experiment, were grown hydroponically in a controlled environmental chamber to the VI0 stage (1). Leaf discs were taken from the fifth trifoliolate and tissue was extracted according to methods previously described by Murphy and Stutte (4). The 2% acetic acid extract, labelled “crude preparation,” was flash frozen and stored until chromatographed. Iwtrumentation. A Waters Associates ALC/GC high-pressure liquid chromatograph with a p Bondapak C,, column was used for analysis of the phenolic compounds. A Model 660 solvent programmer sequenced two Model 600A pumps. Compounds were detected by a Model 440 absorbance detector at 254 nm. Separation procedure. Each sample was thawed, and 5 ~1 was injected into the hplc. The initial solvent, 2% acetic acid (in water), was pumped isocratically for 10 min; it was followed by a 20-min linear gradient to BAMW,,, (Butanol:acetic acid: methanol:water; 5:2:25:6). The final solvent 171

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172

HARDIN

AND

continued isocratically until all compounds had been eluted from the column. The concentration of ammonium acetate in all solvents usedforanalysis was 1.8 x lo-* M. At a flow rate of 1 mV60 s, the approximate run time was 40 min. The remainder of the crude preparation was hydrolyzed within 4 h and analyzed according to procedures previously reported with some modifications (4). The initial solvent was a 60:40 mixture of 2% acetic acid and BAMW,, (2.5:2: 12.5:83) accomplished by the solvent programmer isocratically pumped for 10 min. This step was sequenced by a 20-min linear gradient to BAMW,, and a 30-min linear gradient to BAMW,,. The BAMW,, was pumped until all compounds were eluted from the column. Flow rate was 1 ml/min. RESULTS

AND DISCUSSION

A chromatogram representing the separation of the crude preparation is presented in Fig. 1. Standard solutions, analyzed

STUTTE

through the crude preparation solvent regime, indicated that none of the phenolics represented acids in the free form. These schemes were developed for separation of the phenolic compounds in their natural glycosidic and ester linkages as extracted from the plant. It has been determined that the same general pattern of phenolic constituents exists for several soybean cultivars, but that relative concentrations of the fractions differ among the cultivars (2). With this technique not only can an overall pattern be obtained for soybean or other plants, but with the collection valve, the various compounds represented by the peaks can be collected and hydrolyzed separately to determine the phenolic constituents present in each fraction collected. The retention and resolution data are calculated for the phenolic components from the standard mixture (Table 1). The values for the benzoic and cinnamic acids are slightly different from those previously reported (4) because of the use of the solvent programmer to accomplish the initial

,020

016

L

I

I IO

I 20

I 30

I 40

t 50

MINUTES

FIG. 1. Separation of complex phenolics from crude preparation of Forrest soybean leaf tissue on P Bondapak C,,.

ANALYSES TABLE RETENTION

TIMES

AND

OF

PHENOLIC

AND

I RESOLUTION

DATA

FOR

a

R.?

PHENOLICCOMPOUNDSSEPARATED ON /.&ONDAPAK

C18"

t,.”

Phenolic compounds

(mins)

k’

Gallic

5:21

Gentisic Protocatechuic

621 7:21

,390 ,675 ,909

p-Hydroxybenzoic

IO:27

1.714

Salicylic Vanillic Caffeic

12:15 13:27 16:42

2.182 2.494

Syringic

18:00

3.338 3.615

Benzoic Umbelliferone

20:36 24:00

4.351 5.234

p-Coumaric Coumarin Ferulic

26:03 30:36 31:39

5.766 6.948

Sinapic Cinnamic

35:54 47:12

Naringenin Hesperetin

63:00 66:12

1 I .260 15.364 16.195

Quercetin Kaempferol

67:42 79:45

16.584 19.714

” Flow rate b Abbreviations

1 ml/min, used:

7.221 8.325

1.73

2.32

1.35 1.89

1.70 4.08

1.27

2.00

1.14 1.34 1.10

1.21 2.66 1.00

1.18

1.69

1.20 1.10

1.93 1.07

1.20 1.04

2.01 0.48

1.15 1.35

1.61 3.27

1.36 1.05 1.02

3.39 0.62 0.26

1.18

2.00

t,, =

3 min 51 s. I, = retention time: s =’ ‘/!(a - l/o)fi(~:/l

k’ = t, + G):

FLAVONOID

173

COMPOUNDS

BAMW, mixture and the variation in method. Not all of the values for the components are optimum, i.e., 1 5 k’ 5 10; however, the more compounds that are separated from one sample, the harder it is to obtain optimum k’ values for all components. Wulf and Nagel reported the separation of flavonoids; however, two analyses and two different solvent systems were required for benzoic and cinnamic acid derivatives and flavonoid components (5). While extracting plant material for benzoic and cinnamic acid derivatives, some flavonoid components are solubilized and will also be freed upon acid hydrolysis. The fact that benzoic and cinnamic acid derivatives and flavonoids can be identified and quantitated from one sample by one chromatographic analysis is a great advantage. Chromatograms of the standards and a hydrolyzate of a soybean tissue extract are presented in Figs. 2 and 3. The following phenolics have been identified from the soybean tissue: gallic, protocatechuic, p-hydroxybenzoic, salicylic, vanillic, caffeic, p-coumaric, and ferulic acids. Although

4

IO

20

30

I 50

40 MINUTES

I 60

I 70

1 80

FIG. 2. Separation of phenolic compounds on PBondapak C,, by hplc sensitivity 0.02. Flow rate 1 mlimin. (1) Gallic acid, (2) gentisic acid. (3) protocatechuic acid. (4) p-hydroxybenzoic acid, (5) salicylic acid, (6) vanillic acid, (7) caffeic acid, (8) syringic acid. (9) benzoic acid. (IO) umbelliferone. (I I) p-coumaric naringenin. (17)

acid, (12) hesperetin.

coumarin. (13) (18) quercetin,

ferulic acid, (19) kaempferol.

(14)

sinapic

acid,

t 15)

cinnamic

acid,

(16)

174

HARDIN ,020

1

.016-

L

AND STUTTE

I

1 IO

I 20

f 30

I 40

I 50

I 60

I 70

I 60

MINUTES

FIG. 3. Separation of phenolic compounds from hydrolyzed soybean leaf extract (cultivar: Forrest) on p Bondapak C,, by hplc. (I) Gallic acid, (3) protocatechuic acid, (4)p-hydroxybenzoic acid, (5) salicylic acid, (6) vanillic acid, (7) caffeic acid, (11) p-coumaric acid, (13) ferulic acid, (14) sinapic acid, (16) naringenin, (18) quercetin.

extraction procedures may vary somewhat for different plant species and tissues, the same procedure for separation and identification is applicable and has been used successfully for a variety of species and tissues (Hardin and Stutte, unpublished data). This technique proves useful for a number of studies on plant phenolics. Toxicity studies, particularly in relation to natural host plant protection from pests, may benefit greatly from this separation procedure. The toxicity of compounds is influenced by side chains such as free hydroxyl groups (3). If the groups are complexed as they occur naturally in the plant tissue, they are of little value for specific studies on plant host protection from invading pests. By using only free standards of compounds, toxicity studies could be misleading. However, the various fractions may be collected as they elute from the column and can be resuspended into a solvent suitable for incorporation into the diets of such pests as insects, fungi, and bacteria. The

technique can also be valuable for studies of genetic differences in relation to phenolics or in investigations of the physiological roles phenolics play in plant growth and reproduction. In being able to extract phenolics and isolate these for use in particular studies, we can observe their presence, the quantities occurring, and the combinations in which they occur throughout the physiological growth and development of the plant. Comparisons can be made in the phenolic distinctions between genetic types within species. However, since free phenolics are not normally found in great quantities in plant tissue, the way the acids combine may be the characteristic that determines their physiological roles or their toxicity to pests. To study the effects of combinations, we must be able to isolate and analyze the particular acids and flavonoids free and in combinations. The procedure outlined for one chromatographic analysis is rapid and efficient in making these needed separations.

ANALYSES

OF PHENOLIC

AND FLAVONOID

REFERENCES 1. Fehr, W. R., Caviness, C. E.. But-mood, D. T.. and Pennington, J. S. (1971) Crop Sci. 11, 929-93 1. 2. Hardin, J. M. (1979) Master’s thesis, Univ. of Arkansas, Fayetteville. Ark,

COMPOUNDS

3. Greathouse. Amer.

175

G. A., and Rigler, N. E. (1940)

J. Bat.

27, 99-108.

4. Murphy, J. B., and Stutte. C. A. (1978) Anul. Biochem.

5. Wulf.

86, 220-228.

L. W.. and Nagel.

Chromotogr.

116, 271-279.

C. W. (1976) J.