Glycosidically Bound Phenolic and Other Compounds in an

Chapter 6

Glycosidically Bound Phenolic and Other Compounds in an Umbelliferous Vegetable Beverage 1

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Tarik H. Roshdy , Robert T. Rosen , Thomas G. Hartman , Joseph Lech , Linda B. Clark , Elaine Fukuda , and Chi-Tang Ho 2

Downloaded by CORNELL UNIV on August 21, 2016 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0506.ch006

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Center for Advanced Food Technology and Department of Food Science, Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ 08903 An Umbelliferous vegetable beverage to be used by the National Cancer Institute (NCI) for chemical and biological studies directed towards the possible prevention of cancer was analyzed. This beverage consisted of carrot, celery, and tomato juices, with pepper, basil, paprika, garlic and rosemary added as spices and natural antioxidants. This paper reports on the extraction and direct mass spectrometric evidence of bound phenolic and other compounds from an Umbelliferous vegetable beverage. Phenolics observed included ferulic acid, hydroxycinnamic acid, hydroxymethoxycoumarin, homovanillic acid, acetosyringone, coniferol, vanillin and analogs.

It is recognized that compounds in fruits and vegetables may exist at a greater concentration as conjugates than as aglycones. The hydrolysis, for example, during digestion or ripening, of glycosides in vegetables and fruits, is a major pathway leading to the enrichment of flavors. Hydroxy containing species such as phenolics are directly bound to sugars. Compounds such as coumarins, which are lactones, exist as hydroxy acids when bound to sugars. Dihydroxy benzene derivatives yield the corresponding phenolics. Recent work on the bound fractions of pineapple, peach, celery and hog plum has been published (1-5). The NCI under the auspices of Dr. Herbert Pierson, and in cooperation with Dr. Phillip Crandall of the University of Arkansas Food Science Department, has formulated an Umbelliferous vegetable beverage to be used for the chemical and biological studies directed towards the possible prevention of cancer. This beverage consisted of carrot, celery, and tomato juices, with pepper, basil, paprika, garlic and rosemary added as spices and natural antioxidants. Phenolics observed included ferulic acid, hydroxycinnamic acid, hydroxymethoxycoumarin, homovanillic acid, acetosyringone, coniferol, and vanillin. These results were obtained by isolation and extraction techniques using Amberlite XAD-2 resin followed by enzyme hydrolysis with subsesequent determination by gas chromatography (CJC) and GC-mass spectrometry (GC-MS). 0097HS156/92/0506-0085$06.00/0 © 1992 American Chemical Society

Ho et al.; Phenolic Compounds in Food and Their Effects on Health I ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

86

PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH I

Experimental Materials /}-Glucosidase 50,000 units containing approx. 100% protein from Sigma Chemical Co. Supelpak adsorbent, purified form of Amberlite XAD-2 resin from Supelco Inc. Glass column 1 cm I.D. with a 50 cm length from Fisher Scientific Company (for column chromatography). HPLC grade Dichloromethane, acetone, methanol, n-pentane, and water (Fischer Scientific).


Gas Chromatographic & Mass Spectrometry Operating Conditions: a. G C Conditions Tsplitless mode^: GC Type: Varian 3400 Column Type: 60 meter DB-1 Fused Silica Capillary (J&W Scientific). 0.25 micron Column Film Thickness: 0.32 mm Column ID: Carrier Gas: 40 psi High Purity Grade Helium for lml/min. Make-up Gas for FID: 40 psi High Purity Grade Helium 30ml/min Air: 60 psi High Purity Grade 300ml/min Initial Column Temp.: 50°C Initial Hold: 5 min Rate: 27min~ Attenuation Range: lxl0" Final Temp.: 290°C Final Hold: 90 min Total Time: 215.00 min I Z

Data Acquisition: The output from the G C was integrated as well as re corded and logged on a V G Multichrom chromatography data acquisition system as well as a strip chart recorder output of Varian 4290 integrator. b. Mass Spectrometry Conditions: Instrument: Finnigan M A T 8230 high resolution mass spectrometer directly interfaced to a Varian 3400 gas chromatograph. Mode: electron ionization. filament emission current. 70 eV. 1 mA 3 kV Accelerating Voltage: Multiplier Voltage: 1800 V corresponding to a gain of 10 Ion Source Temperature: 250°C. G C Conditions: Identical to those used on the off-line GC. Finnigan M A T SS300 data system. Data Acquisition: Methodology One hundred milliliters of the Umbelliferous vegetable beverage was di vided and placed into four 50mL capacity heavy-duty centrifuge glass tubes with screw caps. The tubes were then centrifuged for 20 min at 2500 R P M . Forty grams of dry Amberlite XAD-2 resin was placed into a glass column [50 cm χ 1 cm]. Glass wool was packed tightly at the top and bottom of the



6. ROSHDY ET AL.

Phenolic Compounds in a Vegetable Beverage 87

column to prevent the loss of the stationary phase and to prevent the column bed from rising with the water level and producing air bubbles. The XAD-2 column was sequentially conditioned with water, methylene chloride/n-pentane [1:1, v/v], methanol, and water. The second water elution was used to clear the methanol from the column prior to adding the sample. The supernatant from the centrifuge tubes was then loaded on the top of the glass column. HPLC grade distilled water was used to elute the water soluble (yellow-orange) fraction until the eluted water was clear. HPLC grade n-pentane / methylene chloride [1:1, v/v] was used to elute the second (yellow-red) fraction until the eluted solvent was clear of color. Finally, 100 mL of HPLC grade methanol was used to elute the most polar organic compounds (yellow fraction). The methanol fraction (bound fraction) was concentrated to 1.0 mL using a rotary evaporator with the assistance of a water bath at 50°C. The concentrate was transferred into a 250 mL flask and 100 mL of citratephosphate buffer (pH 5) was added. Nitrogen gas was bubbled into the solution at a slow rate to eliminate traces of solvent which would inhibit the enzyme. The sample was then hydrolyzed by adding 60 mg of /?-glucosidase (5.5 units/mg) and warmed to 37°C for 72 hrs in a shaking incubator. The liberated aglycones were extracted with three 150 mL portions of methylene chloride using a separatory funnel. The organic phase was dried over anhydrous sodium sulfate. This fraction was concentrated to 0.3 mL under a gentle stream of nitrogen gas. Triacontane was added as an internal standard (I.S.). This fraction was flushed with nitrogen and kept in a freezer until analysis. Results and Discussion The methodology for the determination of glycosidically bound compounds in celery has been published (4). This method described the identification and the quantitation of phthalides and coumarins in celery. In our experiments this methodology was applied to the Umbelliferous beverage. The technique involves applying a filtered or centrifuged aliquot of the sample on the top of an XAD-2 resin column. The column is then washed with water to elute very polar interferences (yellow-orange fraction). This eluent represents the sugars, acids and other very water-soluble substances which were adsorbed onto the XAD-2 column. Subsequent elution with methylene chloride/n-pentane eliminated the free organics (not covalently bound to sugars) (yellow fraction). Lastly, methanol was used to elute the glycosidically bound phenolics and alcohols. Twelve liberated phenolic compounds have been identified in the polar fraction (bound fraction) of the Umbelliferous beverage. These compounds are eugenol, vanillin, 4-hydroxyacetophenone, acetovanillone, 3-hydroxyphenyl acetic acid, homovanillic acid, acetosyringone, coniferyl alcohol, homovanillic acid methyl ester, hydroxycinnamic acid, ferulic acid and 7-hydroxy-6-methoxy-l(2H)-benzopyran-2-one. The summary of the twelve phenolic compounds identified in the bound fraction of the Umbelliferous beverage with their molecular weights and their structures are given in Table I. The internal standard used for quantitative purposes, triacontane, was chosen because it was clearly separable from all the components in the methanol extract. Figure 1 shows the GC-MS profile of the liberated compounds from the bound fraction. The phenolic compounds were identified by matching


88

PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH I TABLE I PHENOLIC COMPOUNDS IDENTIFIED IN THE BOUND FRACTION

COMPOUNDS IDENTIFIED

MW

ppm

CH-CH=CH

Eugenol o r 2-methoxy-4-(2-propenyl)-phenol (C H 0 ) 164

0.51

Eugenol isomer

0.12

1Q

C

12

2

164

STRUCTURE

2

2

OCH

3

H


( 10 12°2>

V a n i l l i n or 3-hydroxy-4-methoxy-benzaldehyde (C H 0 ) 152 8

8

3

4-hydroxy-acetophenone (C H 0 ) 8

8

136

0.24

OCH,

0.35

2

COCH

3

Acetovanillone or 1-(4-hydroxy-3-methoxyphenyl)-ethanone (CçH-oO,) 166 0.89 ^OCH OH ,CH -C00H 2

3-hydroxyphenyl a c e t i c (C H 0 ) 8

8

3

acid 152

0.62

Homovanillic a c i d o r (4-hydroxy-3-methoxyphenyl) a c e t i c a c i d (CçH^OJ 182 0.74


3

6. ROSHDY ET AL.

89

Phenolic Compounds in a Vegetable Beverage

TABLE I Cont. COMPOUNDS IDENTIFIED

mw

ppm

STRUCTURE

Acetosyringone o r 1-(4-hydroxy-3,5-dimethoxy-phenyl)-ethanone (C H O ) 196 0.61 l0

12

COCH

3

A

Hccr y e

*OCH

3


OH Coniferyl alcohol or CH=CH-CHOH γ-hydroxyisoeugenol o r 3-(4-hydroxy-3-methoxyphenyl)-2-propen-l-ol Λ (C H O ) 180 0.61 ^ 2

10

12

3

OCH

3

OH H o m o v a n i l l i c a c i d methyl-ester (C H 0 ) 196 1Q

l2

4

CH -COOCH 2

3

0.68

OCH, OH hydroxycinnamic a c i d o r 3-phenyl-2-propenoic a c i d

164

0.09

( CH=CH-COOH OH Ferulic acid or 3-(4-hydroxy-3-methoxyphenyl)2-propenoic a c i d 194 ( 1 H 0 )

CH=CH-COOH 0.49

C

0

10

4

0CH

3

OH 7-hydroxy-6-methoxy-2 H-1benzopyran-2-one C

192

0.61

H

( 10 8°4)

HO. ^ H CO 3


η


90


t.Bf

0. 5l

0.Θ ι I » » I I [ ι ι ι Rt (min) 20:00 1 ' Scan No. 3 0 0

ι I ι ι ι ι I

I

t

I

ι I ι ι ι ι I ι ι τ ι; ι τ ι ι I ι ι ι ι I ι ι ι ι I ι ι ι ι I ι ι ι t I ι ι ι ι I ι ι ι τ I ·

4Θ:0Θ

Γ

1000

"I

1300

6Θ:0Θ r

. .

20ΘΘ

80:00 M

. . . .

r

100:00

120:00

» . .

2500

140:00 ,

3000

3380

4000

4300

Figure 1. GC-MS profile of aglycones liberated from bound fraction of the Umbelliferous vegetable beverage.



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ROSHDYETAL.

Phenolic Compounds in a Vegetable Beverage91

their electron ionization mass spectra with a NBS (NIST) computer library. Other isomers than those presented are possible. Most of these species were not observed in the free fraction, that is, the fraction which was eluted with dichloromethane/n-pentane. Other bound compounds which were identified in the bound fraction of the Umbelliferous beverage are listed in Table II. Eight liberated nonphenolic compounds have been identified in the polar fraction (bound fraction) of the Umbelliferous beverage. These compounds are benzaldehyde, methionol, benzene methanol, maltol, 2,3-dihydroxy-2-methoxy- 6-methyl-4Hpyran-4-one, 4-(3-hydroxy-2,6,6-trimethyl-l- cyclohexen-l-yl)-3-buten-2-one, lH-indole-3-ethanol, and 7-hydroxy-6-methoxy-2H-l-benzopyran-2-one. This work was initiated as the use of enzymes for the liberation of conjugated species is important in human biochemistry and in flavor chemistry. The human intestine has enzymes generally classified as "fecalase" which liberate aglycones from glycosides, subsequently allowing adsorption of potentially important phytochemicals through the colon. Some of the phenolic and other compounds identified in this study have important antimutagenic activity. Ferulic acid has activity inhibiting formation of nitrosamines in vivo (6) and protecting D N A from electrophilic attack (7). Other phenolics inhibit formation of certain prostaglandins implicated in tumor growth (8). Indole carbinols and some homologs increase the rate of metabolism with subsequent reduction of estradiol, a compound thought important in induction of breast cancer (9). In addition, enricnment of these previously bound conjugates through enzymatic digestion has the potential of increasing yields of naturally occurring flavorants in which the public so greatly demands. Table II Other Compounds Identified in the Bound Fraction of the Umbelliferous Beverage Compounds Identified benzaldehyde methionol benzene methanol maltol or 3-Hydroxy-2-methyl4H-pyran-4-one 2,3-dihydro-3,5-dihydroxy6-methyl-4H-pyran-4-one 4-(5-hydroxy-2,6,6-trimethyl-1 cyclohexen-1 -yl)-3-buten-2-one lH-indole-3-ethanol 7-hydroxy-6-methoxy2H-l-benzopyran-2-one

MW 106 108 108

wm 0.97 0.12 1.76

126

0.94

144

0.07

208 161

0.09 0.83

192

0.61

Acknowledgment: The Center For Advanced Food Technology is a New Jersey Commission on Science and Technology Center. The authors thank the NCI for the funding of the project, and Dr. Herbert Pierson of NCI for his comments and encouragement during the period in which the work was done.


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Literature Cited 1. Adedeji, J.; Hartman, T.G.; Rosen, R.T.; Ho; C.-T. J. Agric. Food Chem., 1991, 39, 1494-1497 . 2. Wu, P.; Kuo, M.-C.; Hartman, T.G.; Rosen, R.T.; Ho, C.-T. J. Agric. Food Chem., 1991, 39, 170-172. 3. Ho, C.-T.; Sheen, L.-Y.; Wu, P.; Kuo, M.-C.; Hartman, T. G.; Rosen, R. T. In Flavour Science and Technology. Thomas, A. F.; Bessier, Y. Eds.; 1990, 77-80. 4. Tang, J.; Zhang, Y.; Hartman, T.G.; Rosen, R.T.; Ho, C.-T. J. Agric. Food Chem., 1990, 38, 1937- 1940. 5. Wu, P.; Kuo, M.-C.; Hartman, T.G.; Rosen, R.T.; Ho, C.-T. Perfumer and Flavorist, 1990, 15, 51-54. 6. Kuenzig, W., Chan, J., Norkus, E. Holowaschenko, H. Newmark, H., Mergens, W. and Conney, A.H., Carcinogenisis 1984, 5, 309-313. 7. Newmark, H.L., Nutr. Cancer, 1984, 6, 58-70. 8. Dehirst, F.E. Prostaglandin, 1980, 20, 209-214 9. Bradlow. L.H.; Michnovicz, J.J., J. National Cancer Institute, 1990, 82, 613-615. RECEIVED

July 16, 1992


Glycosidically Bound Phenolic and Other Compounds in an

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