InVivo Release of 14C-Labelled Phenolic Groups from Intact Dietary Spinach Cell Walls During Passage Through the Rat Intestine

J Sci Food Agric 1996,71,459-469

In Vivo Release of 14C-Labelled Phenolic Groups from Intact Dietary Spinach Cell Walls During Passage Through the Rat Intestine Callum J Buchanan,* Graham Wallace, Stephen C Fry Institute of Cell and Molecular Biology, Daniel Rutherford Building, Division of Biological Sciences, The University of Edinburgh, King’s Buildings, Mayfield Road, Edinburgh, EH9 3JH, UK

and Martin A Eastwood Gastro-Intestinal Unit, Department of Medicine, The University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh, EH4 2XU, UK (Received 25 July 1995; revised version received 4 December 1995; accepted 26 February 1996)

Abstract: Feruloyl and p-coumaroyl groups in spinach cell walls (CW) were labelled using [14C]cinnamic acid and fed to rats. In the caecum and colon, ferulic acid (FA) and p-coumaric acid (PCA) were released from the CW. Few feruloyl or coumaroyl groups remained in the CW to be excreted in faeces, and thus the presence of simple phenol-sugar esters provided little protection of the polysaccharides to enzymic attack. Some oxidatively coupled phenols were also released but a portion remained in the CW. The oxidatively coupled phenols accumulated in the gut whereas the FA and PCA were absorbed by the rat. Thus enzyme-resistant fragments, containing oxidatively coupled phenols (and possibly sugar residues), may survive microbial attack by rat intestinal bacteria.

Key words : plant cell wall, phenolics, degradation.

INTRODUCTION Much of the FA and PCA in dicot primary CW is ester-linked to pectins. Sixty percent of the saponifiable

Herbivorous and omnivorous animals may acquire FA in spinach primary CW is esterified to galactose or much of their dietary carbon from the breakdown of arabinose residues in rhamnogalacturonans (Fry 1982, plant cell walls (CW). Aspects of CW architecture which 1983). Most of the remainder is present in an enzyme- confer resistance to phytopathogenic microorganisms in resistant fraction (Fry 1984). In addition, some aromatic the living plant may also limit the microbial degrada- material is released upon saponification as soluble but tion of CW in animal intestines. One limiting factor is chromatographically immobile compounds (in those the presence of phenolic material such as lignin and solvent systems tested) which may include oxidatively phenolic acids which can cross-link CW polymers and coupled phenols. Fry (1984) proposed a role for thus enhance the integrity of the CW. Growing primary peroxidase-mediated phenolic coupling in strengthening CW can be a major part of the dietary intake of plant CW by cross-linking polymers via phenol groups. A material and contain little lignin. However, the presence highly cross-linked CW would also impede the pen- of esterified phenolic acids such as p-coumaric acid etration of hydrolytic enzymes into the CW matrix and (PCA) and ferulic acid (FA) (Fry 1982) may be an therefore limit degradation. important factor in restricting the maceration of tissue We have prepared 14C-labelled spinach CW for use (Kato and ‘Nevins 1985) and subsequent digestion of as dietary tracers to study the microbial breakdown of primary CW in animal intestines. CW in the rat intestine (Gray et al 1993a; Miller er a1

1994). The primary CW from cultured spinach (Spinacia * To whom correspondence should be addressed. oleracea L) cells are similar in composition to CW from

J Sci Food A p i c 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain 459

460 C J Buchanan et a1

spinach parenchyma (Gray et a1 1993a) and are exten- sively degraded in the rat caecum and colon (Gray et a1 1993b; Buchanan et a1 1994a). Radioactivity derived from uronate, methyl and acetyl residues of pectin in spinach CW is rapidly solubilised in oioo by rat intestinal bacteria (Buchanan et a1 1994b, 1995a,b). Although most of the pectin of an intact CW is easily degraded, certain sugar residues may be protected by the presence of phenolic groups. To investigate the metabolism of CW-derived phenolic residues, and the role which phenolic monomers may play in limiting the microbial degradation of CW in oivo, spinach CW containing ' 4C-labelled phenolic residues were prepared and used as a dietary 'spike' in rats.

MATERIALS AND METHODS

Preparation of trans- [U-'"C] cinnamic acid

~-[U-'~C]Phenylalanine (25 pCi; 450 mCi mmol-'; Amersham) was dried and redissolved in 0.625 ml tris- HCl buffer (40 mM pH 8.5) and phenylalanine ammonia lyase (37.5 PI, 50 mu) solution was added. The reaction mixture was incubated at 32°C for 16 h, then acidified with 6 M HCl (0.1 ml). [U-'"C]Cinnamic acid was extracted with 5 x 0-5 ml ethyl acetate. Purity was con- firmed by TLC (toluene/acetic acid 9 : 1) on silica-gel. The yield was 90%.

Preparation of spinach cell walls labelled with trans- [ U-'"C] cinnamic acid

Cell suspension cultures of spinach (Spinacia oleracea L cv Monstrous Viroflay) were grown in flasks containing 100 ml of sterile medium (Fry and Street 1980) with 5 g litre- ') glucose as sole non-radioactive carbon source plus trans-[U-'"C]cinnamic acid (5 pCi). The inoculum was 1-2 g fresh weight per flask of day-14 cells. After 14 days incubation under standard conditions (Fry 1982), the cells were harvested (packed cell volume 15-25%) by filtration, washed with water and frozen in 10 g por- tions. Each portion was thawed into 100 ml of buffer (2 g litre-' sodium docecyl sulphate, 10 mM ascorbic acid and 20 mM N-[2-hydroxyethyl]piperazine-N'-[2- ethanesulphonic acid] (HEPES, Na', pH 7.4)) and son- icated in an MSE 'Soniprep' fitted with a 19 mm diam- eter titanium probe which vibrated at 23 kHz with an amplitude of 24 pm. Completion of cell disruption was checked microscopically after staining with Evans' blue. Each suspension was filtered through 64 pm nylon mesh, and the CW-enriched residue was washed in the HEPES buffer and stirred in 100 ml of phenol/acetic acid/water (2 : 1 : 1, w/v/v) (PAW) for 16 h at 20°C to extract proteins. The suspension was filtered through sintered glass and the PAW treatment repeated until

the filtrate no longer contained protein as determined by the ammonium formate/acetone precipitation test (Fry 1988). The CW residue was rinsed in water, de- starched in 900 g litre-' DMSO at 20°C for 16 h (Selvendran et a1 1985), filtered on nylon mesh, re- washed in water, dialysed against 5 g litre-' chlorbutol (l,l,l-trichloro-2-methyl-2-propanol) and freeze-dried. The final specific activity of the (phenolic-'"C)-labelled CW was 10 500 DPM mg- '.

Gavage of cell walls to rats and measure of radioactivity in tissues

A sample of the labelled CW was suspended in distilled water (4 mg ml-') and 1 ml of the suspension was fed to male Wistar rats (42000 DPM per 150 g rat) by gavage. Each rat was maintained in a metabolic cage and supplied with food and water as previously described (Buchanan et a1 1994a). C 0 2 was collected by drawing air through the cage and through two 50 ml samples of Carbo-sorb@ (Canberra Packard, Berkshire, UK). Aliquots (1 ml) of Carbo-sorb@ were assayed for I4C using 10 ml of Permafluor V@ (Canberra Packard). Each cage had a metal grill which minimised coproph- agy and enabled faeces and urine to be collected separa- tely. At the end of the experiment the rats were killed by cervical dislocation and immediately dissected. Gut contents and faeces were mixed with cold water (4°C) to minimise enzyme activity. The gut tissues were washed with water (4°C) and the washings and gut contents pooled and a sample was removed for scintillation counting. Gut contents (1 ml) was mixed with Optisolv (1 ml, LKB, UK) and incubated at 60°C overnight in a tightly capped vial. Hionic fluor (12 ml) was added and the '"C assayed using a TRI-CARB@ 4430 liquid scintillation counter. The remaining gut contents were analysed as described below. The organs were identified and tissue samples or the whole organ were removed, weighed and frozen. Samples of tissue (50-100 mg) were solubilised using Optisolv (2 ml) at 60°C for 2-3 h fol- lowed by 12-16 h at 37°C. After tissue digestion was complete, methanol (2 ml) was added to ensure complete solubilisation of lipid and scintillation fluid (12 ml, Hionic-fluorTM, Canberra Packard) then added and the samples assayed for '"C.

Analysis of gut contents and faeces

The gut contents were centrifuged (2500 x g, 30 min, 4°C) and the pellet was separated from the supernatant. The pellet was washed with buffer (Tris-HC1, 10 mM, pH 6.8) and separated by centrifugation. The supernatant and washings (total final volume = 21 ml) were pooled and the pellet and the pooled supernatant fraction analysed immediately or frozen at -20°C.

Phenolic acid metabolism in the rat 461

Analysis of supernatant

Samples of the supernatant (2 ml) were assayed for I4C by scintillation counting. NaOH (400 pl, 0.5 M) or HCI (400 pl, 0.5 M) were added to further samples (2 ml) of the supernatant which were then dried at 60°C. The dried samples were resuspended in water (2 ml) and assayed for 14C as described above. The volatile 14C was calculated as two fractions: 14C which was volatile from acid but not from alkali (volatile acids) and 14C which was volatile from both acid and alkali (alcohols).

The remaining supernatant (15 ml) was dried, resuspended in water (10 ml) and was acidified with 2 ml glacial acetic acid and then extracted with butan- 1-01 (3 x 10 ml). Aliquots of the butanol and aqueous phases were assayed for I4C. The butanol phase (containing free phenolic acids) was dried (rotary evaporator) and resuspended in butanol (0.5 ml) and the aqueous fraction was freeze-dried and resuspended in water (0.5 ml). Both fractions were separated by TLC as described below.

TLC of free phenolic acids

Free phenolic acids (in butanol) were applied as a streak to a Whatman silica gel plate (PLKF5 150 &mm thick) and developed in benzene/glacial acetic acid (9 : 1, v/v) twice in the same direction. External markers (caffeic, p-coumaric, sinapic (Aldrich), ferulic and cinnamic acids) were run alongside the streaked sample and visualised at 254 nm. Bands (0.5 cm) were scraped off the plate and 1 ml butanol added. The butanol and silica gel were assayed for 14C using 12 ml of scintillation fluid (Hionic-fluorTM) as described above.

TLC of 14C in aqueous phase

The aqueous fraction (500 p1) was spotted as a streak onto a cellulose TLC plate (Whatman LKZF, 250 pm) and the plate developed using butanol/acetic acid/water (12 : 3 : 5, v/.v/v). External markers 3-0-(3-O-feruloyl-a- L-arabinopyranosy1)-L-arabinose (Fer-Ara,) and 4-0- (6-0-feruloyl-~-~-ga~actopyranosyl)-D-ga~actose (Fer- Gal,) were liberated from spinach CW by the action of Driselase and purified by gel permeation chromatog- raphy using Bio-Gel P-2 (Bio-Rad) as described by Fry (1988). The external markers (FA, Fer-Ara, and Fer- Gal,) were run alongside the streaked sample and visualised at 254 nm (fluorescence after exposure to ammonia vapour).

Analysis of particulate fraction: NaOH digestion

A sample of the particulate material was hydrolysed using 10 ml NaOH (0.1 M) at 25°C for 1 h. The NaOH-

insoluble material was separated by centrifugation (3000 x g, 30 min, 4°C) and samples of the NaOH- soluble and NaOH-insoluble material were assayed for 14C as described above. A portion of the remaining NaOH-soluble material (2 ml) was acidified (1 ml TFA, 2 M) and extracted with butan-1-01 (3 x 5 ml), dried, resuspended in 0.5 ml butan-1-01 and applied to a silica-gel TLC plate and run in benzene/acetic acid (9 : 1, v/v) as described above.

Driselase digestion of particulate fraction

A further sample of the particulate fraction was washed (2 x 5 ml) in methanol (to remove any I4C loosely associated with bacterial cells) and the pellet separated by centrifugation (3000 x g 30 min, 4°C). The methanol fractions were pooled and a sample assayed for 14C. The remaining methanol-soluble material was dried using a rotary evaporator and resuspended in 0.5 nil of methanol and applied to a cellulose TLC plate as described below.

Residual methanol in the pellet was removed by drying at 37°C. The pellet was resuspended in 10 ml pyridine/acetate buffer (pyridine 10 ml litre- ', acetic acid 10 ml litre-', v/v, chlorbutol 5.0 g litre-', pH 4.7) containing 50 g litre-' Driselase (a commercial fungal (Irpex lacteus) extract containing cellulase, xylanase, pectinase, mannanase and other endo- and cxo- polysaccharidases), which was partially purified as described by Fry (1988), and incubated for 40 h at 20°C. The undigested material was separated by centrifugation at 2500 x g for 30 min. Samples of the supernatant and pellet were assayed for 14C, and the remaining soluble material freeze-dried and resuspended in 0.5 ml of pyridine/acetate buffer. The Driselase- soluble material or the methanol-soluble material was applied to a cellulose TLC plate as a streak. The plates were developed in butanol/acetic acid/water (12 : 3 : 5; v/v/v). External markers (FA, Fer-Ara, and Fer-Gal,) were run alongside the streaked sample. 0.5 cm bands were scraped off the plate and assayed for I4C.

Materials

Unless otherwise stated all chemicals were obtained from Sigma and were of the highest purity available.

RESULTS AND DISCUSSION

Labelled phenolic acids in cell walls as dietary tracers

Phenolic residues in spinach CW were l4C-1abelled by supplying cultured spinach cells with tr~ns-[U-'~C]cinnamic acid. Cinnamic acid is rapidly taken up by plant cells and may be converted to several hydroxycinnamic

462

TABLE 1 Distribution of radioactivity after gavage of (phen~lic-'~C)-labelled cell walls

C J Buchanan et a1

Fraction Radioactivity" (YO of gavage) derived from (phenoli~-'~C)-labelled C W after

~ ~

2 h 4 h 6 h 18 h

Gut contents Stomach

Soluble Particulate

Small intestine Soluble Particulate

Soluble Particulate

Soluble Particulate

Soluble Particulate

Caecum

Colon

Faeces

Urine CO, Body tissues' % recoveredd

3.2 f 2.2 25.1 f 0.8

4.0 f 1.6 40.5 f 12.5

0.2 f 0.0 0.5 f 0.1

0.1 f 0.0 0.2 f 0.0

N D ~ ND

0.9 f 0.5 0.0 f 0.0 25.8 f 2.1

1 OQ.4

0.2 f 0.0 9.9 f 0.4

1.9 f 0.3 23.7 f 3.4

15.4 f 5.9 10.3 f 3.3

3.3 f 3.1 2.6 f 0-3

0.0 f 0.0 0.0 f 0.0 1.7 f 0.4 0.0 f 0-0 32.9 f 10.3

102.0

0.0 f 0.0 1.8 f 0.0

2.7 f 0.2 21.7 f 2.3

19.8 f 8.6 8.7 f 2.3

7.7 f 1.6 6.7 f 0.2

0.3 f 0.1 0.2 & 0.0 2.6 f 0.0 0.0 f 0.0 41-5 f 1.8

113.8

0.0 f 0.0 0.4 f 0.1

0.2 f 0.1 0.2 f 0.1

0.3 f 0.3 0.3 f 0.1

1.6 f 0.9 1.7 f 0.3

10.9 f 2.7 8.1 f 3.4 20-1 f 2.2 0.3 f 0.3 34.3 f 3.0

78.4

Mean % of gavage f SEM, n = 3. The percentage was calculated as the percentage of the gavage in each case, and then the average of the percentages was calculated. * No faeces produced.

tivity in each individual organ plus the radioactivity in the remaining carcass.

tissues and excretions.

The % radioactivity in the body tissues represents the sum of the total radioac-

The I4C recovered represents the total recovery of the gavage in the body

acids (eg p-c umaric, caffeic, ferulic or sinapic) via hydroxylation (by 0,) and methylation (by S- adenosylmethionine) reactions. Hydroxycinnamoyl conjugates of glucose or Coenzyme A are the probable precursors for the intraprotoplasmic biosynthesis of hydroxycinnamoyl-polysaccharides (Wallace and Fry 1994; Fry 1987; Myton and Fry 1994). Thus exoge- nously supplied [U-'4C]cinnamic acid may be expected to label a variety of CW bound phenolic residues. CW labelled in this manner have been characterised and found to contain 14C-labelled feruloyl and coumaroyl residues (Fry 1984). Although caffeic acid is an interme- diate in the conversion of coumaric acid to ferulic acid there are few or no caffeoyl residues present in the primary CW of spinach. Sixty percent of the feruloyl residues in spinach CW are esterified to galactose or arabinose residues of polysaccharides, and are released upon Driselase digestion as the feruloyl disaccharides. Fer-Ara, , Fer-Gal, . Most of the coumaroyl groups appear to be linked in a similar manner. A significant proportion of the 14C was incorporated into Driselase- resistant material that upon saponification yielded chro-

matographically immobile material presum oxidatively coupled phenols (Fry 1984).

d to includ

The labelled CW were used as a dietary tracer to investigate the metabolism of phenolic groups in intact CW during passage through the intestine of the rat. The degradation of phenolic acids can be more realistically determined using intact CW than would be possible using purified polymers (or phenolics) whose fate would differ from the analogous compounds in situ in the CW.

Metabolism of (phenofi~-'~C)-CW in the upper intestine

After rats were dosed with labelled CW the presence of I4C in the body tissues and excreta of the rat was determined as an assessment of the degree to which the phenolic residues of (phenolic-14C)-CW were metabolised and taken up by the host. Two hours after gavage most label was present in the stomach and small intestine with only a minor proportion reaching the lower gut. As the labelled CW passed through the upper intestine, no significant 14C was excreted as 14C0,. However a


TABLE 2 Analysis of soluble fraction from rat intestinal contents

Source Volatile from ?A of total radioactivity" derived from soluble fraction of gut contents of rats fed (phenolic-14C)-CW after

4 h 6 h 18 h

Caecum Acid-onlyb Alkali' Non-volatiled

Partitioning into butanol Partitioning into water

Colon Acid-only Alkali Non-volatile


Faeces Acid-only Alkali Non-volatile


1 .o 0.7

13.7 8.8 5.0 0.0 0.3 3.0 2.2 0.8

N D N D ND ND ND

2.2 0.1

17.5 10.9 6.6 0.2 0.1 7.4 4.3 3.1

ND N D ND ND ND

ND' ND N D N D ND ND ND N D ND N D

0.1 0.0

10.8 4.9 5.9

The % of the total radioactivity gavaged to each animal. Acid-only volatile material is calculated as the 14C volatile from acid corrected to

Alkali-volatile material is calculated as the total activity in the gut contents minus

Non-volatile material is calculated as the I4C remaining after drying in acid condi-

ND; not determined since radioactivity too low for accurate measurements.

account for the loss of alkali-volatile material.

the activity remaining after drying in alkali.

tions.

significant proportion of the label was present in the body tissues or had been excreted in urine (Table 1) indicating that some label had been released from the CW and absorbed by the host animal. In both the stomach and small intestine 14C was associated mainly with the insoluble fraction (typically 89% of the stomach contents and 91% of the small intestinal contents), although a small amount was soluble. Phe- nolic acids which had been released from CW may have been rapidly absorbed by the gastric or intestinal mucosa and therefore were not evident in the gut contents. Phenolic-sugar esters are hydrolysed by porcine pancreatic esterases (Kato and Nevins 1985) although the removal of phenolic ester groups in the small intestine may be limited by the inability of enzymes to pen- etrate the intact CW. Ester bonds are alkali-labile and alkaline conditions such as those found in the mamma- lian small intestine result in the slow hydrolysis of methyl and acetyl ester groups from intact spinach CW (Miller et al 1995). However, feruloyl esters are con- siderably more alkali-stable (Fry 1982) than methyl and acetyl esters and thus would be essentially resistant to non-enzymic hydrolysis in the gut. Westendorf and Czok (1978, 1983) demonstrated that FA and PCA are rapidly excreted in urine and bile when given to rats

intraduodenally, the rate of excretion of FA being greater than that of PCA (20% and 5% of dose excreted in bile in 2 h, respectively). Thus some of the minor fraction of 14C which was present in the soluble fraction in the small intestine may be due to biliary excretion of phenolic acids absorbed from the caecum and colon, especially at the later time points (4, 6 and 18 h post gavage). The removal of phenolic groups from CW in the small intestine may also be due to bacterial activity. The rat small intestine (especially the terminal ileum) is known to contain fermentative bacteria (Macy et a1 1982) and thus the microbial digestion of CW (and the release of phenolic acids from the CW by the action of bacterial esterases) may be initiated in the small intestine prior to entry into the caecum. This may account for the observed incorporation of 14C into the body tissues prior (2 h post-gavage) to the bulk of the radioactivity reaching the caecum. Although 14C accumulated in the rat body tissues it is not known if the 1 4 C was incorporated into the structural tissue components or if the presence of 14C in the body tissues may be attrib- uted to the lipophilic nature (at physiological pH) of the free acids. Free phenolic acids can act as anti-oxidants (Graf 1992) and a putative site for this action is lipid- rich tissues. 14C was excreted in the urine of the rats


although the low specific activity of the label in the urine precluded analysis of the nature of the I4C. It has been shown in rats that FA is dehydroxylated and excreted as 3-hydroxypropanoic acid and to a lesser extent vanillic acid (Booth et a1 1957; Teuchy and Van Sumere 1971).

Solubilisation of (Phenoli~-'~C)-CW in the caecum and colon

Most of the phenolic groups in the CW survived passage through the upper gut and reached the caecum. Upon reaching the caecum a significant proportion of the radioactivity derived from (phen~lic-'~C)-CW was solubilised and free phenolic acids (coumaric and ferulic) and soluble, chromatographically-immobile phenolic material were released. Little of the soluble I4C in the caecum and colon was volatile under either acidic or alkaline conditions (Table 2). Most of the soluble label on acidification partitioned into butanol (Table 2). The butanol fraction, which would contain any free phenolic acids, was analysed by TLC. The major unlabelled phenolic acids present were coumaric, caffeic and ferulic acids (data not shown). In the butanol extract from caecal contents (4 h post-gavage) the 14C- labelled material co-chromatographed with coumaric and ferulic acids (Fig la) and there was a minor peak remaining at the origin which was not identified but may contain oxidatively cross-linked phenolics (Fry 1984). 5,S-Diferulic acid, which chromatographs between caffeic and coumaric acids in this solvent system, was not observed. Soluble I4C which had passed into the colon (Fig 1 b, d) or had been longer in the caecum (Fig lc, 6 h post-gavage) contained lesser

amounts of free ferulic or coumaric acids but a larger proportion of chromatographically immobile material. The increase in the proportion of the chromatographically immobile material compared to the free acids could be due to the retarded release of soluble but chromatographically immobile material from the CW and/or to the favoured uptake of the free acids from the caecum and colon. Some 14C moved from the origin (on pre-absorbent layer) but remained immobile on the separating area of the TLC plate. This material was unidentified but co-migrates with gallic acid.

The 14C which remained in the aqueous phase after butanol extraction (Table 2) was analysed by cellulose TLC. Most of the radioactivity remained at the origin or moved onto the separating layer but was chromatographically immobile (Fig 2). A minor peak co- chromatographed with Fer-Ara, . The immobile radioactivity is likely to be larger but soluble feruloylated (or coumaroylated) oligosaccharides or water-soluble oxidatively coupled phenols attached to sugar residues. The water-partitioning, chromatographically-immobile material which was released from the CW in the caecum may include oxidatively coupled phenols or feruloylated polysaccharides. Some of the immobile material was hydrophilic (remained in the aqueous fraction upon partitioning into butanol) and if this is oxidatively coupled phenols, its water-soluble nature may suggest that there were more sugar residues associated with the phenolic groups (14C-labelled) than was the case with the radioactivity which partitioned into butanol. It is possible that this material consists of highly cross- linked polysaccharides which initially partition into water but partition into butanol as sugar residues are removed from the polysaccharjde. Some putative feruloyl (or coumaroyl) oligosaccharides were detected but

a a a

i o a

a 2 4 6 8 1012 14 16 18 20 c

3 3 0 0 ..i

0 U 1

2 4 6 8 1012 14 16 18 20

per Cin :- I'ei

2 4 6 8 1012 14 16 18 20

2 4 6 8 1012 14 16 18 20

Distance (a)

Fig 1. TLC of butanol-extractable fraction from supernatant of rat caecal (A and C) or colonic (B and D) contents 4 h (A and B) or 6 h (C and D) after gavage of @henolic-14C)-labelled CW. 0, Origin; sp, start of separating area of plate; sf, solvent front; Caf, caffeic acid; Cou, coumaric acid; Sin, sinapic acid; Fer, ferulic acid; cin, cinnamic acid. Distance is measured from the edge of the

plate.


Per -AraZ

Fer-Gaz 100

2 4 6 8 10 12 14 16 18 20

100

100

0

2 4 6 8 10 12 14 16 18 20

Distance (cm)

Fig 2. TLC of aqueous fraction from supernatant of rat caecal (A and C) or colonic (B and D) contents 4 h (A and B) or 6 h ((2 and D) after gavage of Cphen~lic-'~C]-labelled CW. Fer-Ara, = 3-(3-0-feruloyl-a-~-arabinopyranosyl)-~-arabinose; Fer-Gal, = 4-(6-0-

feruloyl-B-D-galactopyranosy1)-D-galactose. Other details as in legend to Fig 1.

did not accumulate and may be transitory. Several soluble lignin-carbohydrate complexes derived from graminaceous CW have been detected in the bovine and ovine rumen (Richards and Beveridge 1975; Conchie et a1 1988; Wallace et a1 1991) and it is possible that soluble phenol-carbohydrate conjugates (derived from spinach CW) may be present in the mono-gastric intestine.

Insoluble radioactivity from (pLenoli~-'~C)-CW in the lower gut

The nature of the I4C in the insoluble fraction from the caecum, colon or faeces was determined by hydrolysis using either NaOH or Driselase. Treatment with NaOH

released 80-90% of the I4C associated with the particulate fraction (Table 3). NaOH effectively hydrolyses ester bonds and thus the released 14C would contain material derived from CW ester-linked phenolics. Phe- nolic material associated with bacterial cells may also have been released by this treatment; 70-90% of the NaOH-solubilised material partitioned into butanol although the proportion of 14C remaining in the aqueous fraction was higher in the colon or faeces than in the caecum. The butanol phase contained predominantly, chromatographically-immobile material although some ferulic and coumaric acid were present (Fig 3).

To determine if the I4C remaining in the residual gut contents was still present as phenol-polysaccharide conjugates, samples of the insoluble fraction from the

lc 100

2 4 6 8 1012 14 16 18 20 U -

2 4 6 8 1012 14 16 18 20

2 4 6 8 10 12 14 16 18 20

2 4 6 8 10 12 14 16 18 20 Distance (a)

Fig 3. TLC of butanol-extractable, NaOH-soluble material derived from the particulate fraction of rat caecal (A and C) or colonic (B and D) contents 4 h (A and B) or 6 h (C and D) after gavage of (phen~lic-'~C)-labelled CW. Other details as in legend to Fig 1.

466

TABLE 3 Saponification products of particulate fractions from gut con-

tents of rats gavaged with (phet~olic-'~C)-CW

C J Buchanan et a1

Source Fraction YO of total radioactivity" derived from gut contents of rats fed

(phen~lic-'~C)-C W after

4 h 6 h 18 h

Caecum Butanolb 9.5 7.2 ND' Aqueouse 0.2 0.8 ND Insolubled 0.6 0.7 ND

Colon Butanol 2.2 5.2 ND Aqueous 0.3 0.8 ND Insoluble 0.2 0.7 ND

Faeces Butanol ND ND 5.6

Insoluble ND ND 1.4 Aqueous ND ND 1.1

The % of the total radioactivity gavaged to each animal. The % of the I4C in particulate material which is soluble after

The YO of the 14C in particulate material soluble after hydro-

The % of the I4C in particulate material which is insoluble

ND; not determined since radioactivity too low for accurate

hydrolysis and extractable into butan-1-01.

lysis but remains in the aqueous fraction.

after hydrolysis.

measurements.

caecum, colon or faeces were washed in methanol (to remove any I4C associated with bacterial cell membranes or contained within bacterial cells) and then digested with Driselase. In the caecum and colon half of the radioactivity was extractable in methanol alone (Table 4) and may include phenolic material within bacterial cells or I4C dissolved in bacterial membranes. In the caecum and colon, but not in the faeces, most of the remaining activity was solubilised by treatment with Driselase (Table 4).

The methanol-soluble I4C from the particulate fraction was analysed by TLC (data not shown). The major peak of radioactivity co-chromatographed with free phenolic acids although there was significant radioactivity which chromatographed in the same region as Fer-Ara, and may include other phenol-sugar conjugates (eg coumaroyl-Ara,). Some of the free phenolic acids (Fig 3) extracted from the particulate material may be present in bacterial cells and therefore no longer associated with the CW. Driselase digestion of the MeOH-insoluble particulate fraction released mainly chromatographically immobile I4C (Fig 4). Some radioactivity co-chromatographed with the Fer-Ara, and Fer-Gal, markers.

The rate of degradation of CW may be influenced by the presence of wall-bound phenolic groups. In rumi- nants CW-derived phenolic acids limit the degradability of forage material (Hartley and Jones 1977) and a similar inhibition may occur in mono-gastric animals.

In uitro the rate of degradation of CW polysaccharides by rumen microorganisms is depressed by free phenolic acids (Jung 1985; Jung and Sahlu 1986) which inhibit the growth and enzyme activity of bacteria such as Pre- votella or Fibrobacter (Chesson et a1 1982; Martin and Akin 1988). For example B-glucosidase, carboxymethylcellulase and xylanase activities of the cellulolytic rumen bacterium Fibrobacter succinogenes S85 were inhibited by low concentrations of PCA and FA (Martin and Akin 1988). CW polysaccharides such as pectins or hemicelluloses may be highly branched and require the co-operation of a variety of different enzymes for complete degradation. The efficiency of this process may be diminished if some sugar residues have phenolic substituents since many polysaccharolytic enzymes are less able to hydrolyse glycosidic bonds involving feruloylated sugars (or other phenolic sugars). Although it is uncertain if hydroxycinnamoyl sugar esters are transported into bacterial cells (and subse- quently hydrolysed) it is likely that some phenolic-sugar esters would be hydrolysed externally by extracellular esterases. The presence of phenolic acids associated with the gut bacteria may be due to non-specific binding (or partitioning of the free acids into bacterial membranes); however it may also be the case that some gut bacteria were actively metabolising phenolic material (Scheline 1968). The ruminal bacterium Bacillus purnilis is capable of degrading FA to 4-vinylguaiacol and PCA to 4- vinylphenol (Degreasi et al 1995) and several other


TABLE 4 Driselase digestion products of particulate fractions from gut contents of rats

gavaged with (phenolic-14C)-CW

Source Fraction % of total radioactivity" derived from gut contents ofrats fed

(phenolic-l4C)-C W after

4 h 6 h 18 h

Caecum Methanol-extractableb Driselase-soluble' Driselase-insolubled

Driselase-soluble Driselase-insoluble

Driselase-soluble Driselase-insoluble

Colon Methanol-extractable

Faeces Methanol-extractable

4.9 4.2 1 *2 0.9 1.1 0.6 ND ND ND

4.2 3.5 1 -0 2.1 3.1 0.9 ND ND ND

ND" ND ND ND ND ND 4.1 1.2 2.2

~~~ ~~ ~

The % of the total radioactivity gavaged to each animal. * The Yo of the 14C in particulate material which is extractible by washing in methanol prior to Driselase treatment. ' The % of the 14C in particulate material which is extractible by Driselase treatment.

The % of the 14C in particulate material which is insoluble after Driselase treatment.

ND; not determined since radioactivity too low for accurate measurements.

unidentified rumen bacteria capable of degrading phe- ethylbenzene) and phenols (eg phenol, cresols, 2- nolic acids have also been isolated (Akin 1980; Chen et a1 1988). In sewage sludge FA can be demethoxylated and dehydroxylated under anaerobic conditions and converted to phenylpropionate and phenylacetate by Enterobacter cloacae (Grbic-Galic 1985). In addition other members of the Enterobacteriaceae are able to slowly degrade FA to benzoate and then to an alicyclic acid. A range of aromatic hydrocarbons (eg toluene,

ethylphenol) are also produced (Grbic-Galic 1986).

Radioactivity in faeces

The I4C present in the faeces should be enriched in material which is most resistant to microbial degradation. At 18 h post-gavage, there was little label remaining in the gut contents although 19% of the label

3 0 0 1 0 0

2 0 0

a In 100

0 \ I p d o - -

2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 h Y '; 3 0 0

: 1 0 0

..I

U Y

.rl Q

100

0

2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16

Distance lcm)

Fig 4. TLC of Driselase-soluble material derived from the particulate fraction of rat caecal (A and C) or colonic (B and D) contents 4 h (A and B) or 6 h (C and D) after gavage of (phenoli~-'~C)-labelled C W . Other details as in legends to Figs 1 and 2.


2 4 6 8 1012 14 16 18 20

Fer-Ara2 -

2 4 6 8 10 12 14 16 18 20

coo

500 n 1.D

Fer-Ara2 -

2 4 6 8 10 12 14 16 18 20

7 0 0 100 lf;;.;;i150 Fer-Gai;

0

2 4 6 8 1012 14 16 18 20 2 4 6 8 10 12 14 16 18 2 0

Distance (cm)

Fig 5. Radioactivity recovered from faecal material from rats gavaged with (phen~lic-'~C)-labelled CW. A, TLC of butanol- partitioning fraction from supernatant; B, TLC of water-partitioning fraction from supernatant; C, TLC of butanol-partitioning radioactivity released from the particulate fraction by saponification; D, TLC of soluble material released from the particulate

fraction by Driselase treatment. Other details as in legends to Figs 1 and 2.

present in the initial gavage had been excreted in faeces. Half of the label present in faeces after 18 h was mainly in a soluble form (Table 1). Fifty percent of the soluble label partitioned into butanol (Table 2) and contained chromatographically-immobile 14C, with only minor peaks of coumaric and ferulic acids (Fig 5a). The aqueous material contained predominantly immobile material (Fig 5b). Most of the label associated with the particulate fraction was extractable in methanol alone. Driselase digestion of the particulate fraction released only a small proportion (15%) of the radioactivity (Table 4), most of which was chromatographically immobile (Fig 5d). NaOH digestion of the particulate fraction released most of the label and the majority of the soluble label partitioned into butanol (Table 3). TLC of the butanol-soluble fraction indicated a chromatographically immobile peak, although there was a significant background of free phenolic acids (Fig 5c).

CONCLUSIONS

Although the presence of phenolic groups may slow the breakdown of CW, simple feruloyl and coumaroyl esters do not significantly limit the extent of degradation of CW in oiuo in the rat intestine as evidenced by the almost complete removal of such groups by the caecal and colonic bacteria. However, the presence of oxidatively coupled phenols (chromatographically immobile fraction) is a significant factor in limiting CW digestion since these compounds were the major 14C- labelled phenolic fraction to survive passage through the gut into the faeces.

ACKNOWLEDGEMENT

The authors thank the BBSRC for financial support of this project. Thanks also to Janice Miller (ICMB) and Michael Spencer (Gastro-intestinal labs) for their excel- lent technical assistance. Thanks also to the staff of the Biomedical Research Facility (Western General Hospital) and the Department of Medical Physics (Western General Hospital) for the use of their facilities.

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InVivo Release of 14C-Labelled Phenolic Groups from Intact Dietary Spinach Cell Walls During Passage Through the Rat Intestine