Human Nutrition and Metabolism

Chlorogenic Acid and Caffeic Acid Are Absorbed in Humans1

Margreet R. Olthof,2 Peter C.H. Hollman* and Martijn B. Katan
Division of Human Nutrition and Epidemiology, Wageningen University, 6700 EV Wageningen,
The Netherlands and *State Institute for Quality Control of Agricultural Products (RIKILT), 6700 AE,
Wageningen, The Netherlands

ABSTRACT Chlorogenic acid, an ester of caffeic acid and quinic acid, is a major phenolic compound in coffee;
daily intake in coffee drinkers is 0.5–1 g. Chlorogenic acid and caffeic acid are antioxidants in vitro and might
therefore contribute to the prevention of cardiovascular disease. However, data on the absorption of chlorogenic
acid and caffeic acid in humans are lacking. We determined the absorption of chlorogenic acid and caffeic acid in
a cross-over study with 4 female and 3 male healthy ileostomy subjects. In such subjects, degradation by the
colonic microflora is minimal and absorption can be calculated as the amount ingested minus the amount excreted
in ileostomy effluent. The ileostomy subjects ingested 2.8 mmol chlorogenic acid and 2.8 mmol caffeic acid on
separate days in random order and subsequently collected ileostomy fluid and urine for 24 h. Absorption of

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chlorogenic acid was 33 ⫾ 17% (mean ⫾ SD) and of caffeic acid 95 ⫾ 4%. Traces of the ingested chlorogenic acid
and 11% of the ingested caffeic acid were excreted in urine. Thus, one third of chlorogenic acid and almost all of
the caffeic acid were absorbed in the small intestine of humans. This implies that part of chlorogenic acid from
foods will enter into the blood circulation, but most will reach the colon. J. Nutr. 131: 66 –71, 2001.

KEY WORDS: ● phenolic compounds ● chlorogenic acid ● caffeic acid ● absorption ● ileostomy
● humans

Phenolic compounds form a substantial part of plant foods. tween coffee intake and colon cancer in some epidemiologic
Most of these phenolic compounds are antioxidants in vitro studies (9 –13) might be explained in part by the chlorogenic
(1) and antioxidants might protect against cardiovascular dis- acid present in coffee. However, there are no data on absorp-
ease. tion of chlorogenic acid or caffeic acid in humans. The major
A major class of phenolic compounds are hydroxycinnamic problem in measuring the absorption of chlorogenic acid and
acids, which are found in almost every plant (2,3). The major caffeic acid in humans is their bacterial degradation in the
representative of hydroxycinnamic acids is caffeic acid, which colon (14). Thus, measurement of fecal excretion of chloro-
occurs in foods mainly as an ester with quinic acid called genic acid and caffeic acid would lead to an overestimation of
chlorogenic acid (5-caffeoylquinic acid) (Fig. 1). Coffee is a the amount absorbed. Therefore, we determined the absorp-
major source of chlorogenic acid in the human diet; daily tion of chlorogenic acid and caffeic acid in healthy ileostomy
intake in coffee drinkers is 0.5–1 g; coffee abstainers will subjects, who lack a colon. Ileostomy subjects were successfully
usually ingest ⬍ 100 mg/d. Other dietary sources of chloro- employed previously to determine the absorption of flavonoids
genic acid include apples, pears, berries, artichoke and au- (15), coffee diterpenes (16) and dietary polysaccharides (17).
bergines (4). Knowledge concerning the absorption of chloro-
genic acid in humans is essential to evaluate possible health
effects in vivo because the absorbed fraction of chlorogenic SUBJECTS AND METHODS
acid will enter into the blood circulation and thus can induce
biological effects in the blood circulation. Furthermore, the Subjects. The study was approved by the Ethical committee of
the Division of Human Nutrition and Epidemiology. We recruited
fraction that is not absorbed will enter into the colon where it subjects by approaching volunteers who had participated in previous
might have biological effects. Chlorogenic acid and caffeic studies at our division (15,16). Exclusion criteria were as follows:
acid are antioxidants in vitro (1,5), and they might inhibit the signs of diseases related to the gastrointestinal tract; resection of ⬎ 50
formation of mutagenic and carcinogenic N-nitroso com- cm of the terminal ileum; an ileostomy that did not function properly;
pounds because they are inhibitors of the N-nitrosation reac- use of drugs that influenced gastrointestinal transit; present illness;
tion in vitro (6). Further, chlorogenic acid can inhibit DNA pregnancy or lactation. Four women and three men, with a mean age
damage in vitro (7,8). Therefore, the inverse association be- of 63 y (range: 46 –74 y), and a mean body mass index of 27.1 kg/m2
(range 23.3–34.9 kg/m2) were admitted to participate and signed an
informed consent form. All subject had had a total colectomy be-
1 tween 7 and 27 y ago for ulcerative colitis or polyposis coli. The
Supported by the Foundation for Nutrition and Health Research, The Neth-
erlands. subjects were healthy, based on a medical questionnaire, normal
2
To whom correspondence and reprint requests should be addressed. blood values for hemoglobin, hematocrit and white blood cell counts,
E-mail: [email protected]. and absence of glucose and protein in urine.

0022-3166/01 $3.00 © 2001 American Society for Nutritional Sciences.

Manuscript received 1 June 2000. Initial review completed 24 July 2000. Revision accepted 27 September 2000.

66
 ABSORPTION OF CHLOROGENIC ACID AND CAFFEIC ACID 67

mmol) caffeic acid (Fluka Chemie) or 220 mg (0.3 mmol) quercetine-

3-rutinoside (Rutosidum DAB; BUFA B.V., Uitgeest, The Nether-
lands). The placebo and the quercetin-3-rutinoside supplements were
part of another study; these results will be reported elsewhere. Sub-
jects received the supplements as a powder and were instructed to add
200 mL hot water and to consume the beverage within 5 min after
preparation. Subjects ingested the supplements between 0700 and
0900 h at home, together with a light breakfast that we provided. The
breakfast consisted of wheat bread, cheese, strawberry jam, milk and
the coffee and tea substitutes; subjects had a free choice. After this
breakfast with supplements, subjects were allowed only to drink water
for 3 h.
Collection of ileostomy effluent and urine. On d 5, 6, 10 and 14,
subjects collected one sample of ileostomy effluent and urine just
before ingestion of the supplements. After ingestion of the supple-
ments, they collected ileostomy effluent and urine during 24 h.
During the daytime, they changed the ileostomy bags every 2 h and
immediately stored the bags on dry ice to minimize degradation of the
contents by residual bacterial flora. At night, subjects had to change
the bags as often as possible.
Subjects collected urine in 0.5 mL plastic bottles, with 0.13 g
FIGURE 1 Structure of caffeic acid (I) and chlorogenic acid (II) thymol (# 8167; Merck, Amsterdam, The Netherlands) as a preser-
vative and stored the bottles with urine on dry ice immediately after

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voiding. We checked the completeness of the urine collection by
Study design and supplements. Subjects followed a diet that was assessment of recovery of 270 ␮mol lithium in urine. It was ingested
low in chlorogenic acid and quercetin from d 1 to 14. The diet was daily by the subjects as lithium chloride dissolved in 10 mL of tap
low in quercetin because one of the supplements we tested was water from 7 d before the first urine collection. Lithium chloride is
quercetin-3-rutinoside. To ensure adherence to the dietary guide- completely absorbed and 95% is excreted in urine (20,21). Urinary
lines, we gave the subjects a list of forbidden foods and beverages. recovery of lithium was 94 ⫾ 12% (mean ⫾ SD), indicating good
Foods were prohibited if they contained ⬎ 15 mg/kg of quercetin or compliance in collecting urine.
chlorogenic acid. Beverages were prohibited if they contained ⬎ 4 Sample preparation. The ileostomy bags were kept frozen with
mg/L of quercetin or chlorogenic acid (4,18,19). Because subjects liquid nitrogen during separation of the plastic bags from the con-
were not allowed to drink coffee and tea during the study, we supplied tents. The frozen contents were freeze-dried, ground to pass through
coffee and tea substitutes. The coffee substitute was an extract made an 0.5-mm sieve and stored at ⫺20°C until analysis. We thawed the
of chicory, rye and barley (“Swiss coffee-like,” Tayala AG, Birsfelden, urine bottles in a water bath of ⬃40°C, pooled and mixed urine per
Switzerland) and the tea substitute was an extract of a mixture of subject and per supplement day, froze aliquots of urine in liquid
herbs (“Droommix,” Piramide, Veenendaal, The Netherlands). We nitrogen and stored the urine samples at ⫺80°C until analysis. We
analyzed the coffee substitute and tea substitute for quercetin and prepared the samples collected before breakfast (presupplement sam-
chlorogenic acid, and the amounts were within the range of the ple) and the final collection at the end of the 24-h collection period
dietary guidelines (results not shown). Compliance with the dietary (final sample) separately.
guidelines was good. None of the subjects reported consumption of Incubation of chlorogenic acid and caffeic acid with gastrointes-
any foods or beverages that were on the list of forbidden foods and tinal fluids. To check for degradation of chlorogenic acid and
beverages during the study. Furthermore, the low chlorogenic acid caffeic acid in gastrointestinal fluids, we incubated them in vitro in
and caffeic acid excretion in presupplement ileostomy effluent con- gastric juice and duodenal fluid, and ex vivo in ileostomy fluid. We
firmed that subjects adhered to the dietary guidelines (Table 1). incubated 30 mg of chlorogenic acid and 15 mg of caffeic acid with
On d 5, all subjects consumed a placebo supplement, which was 3 mL human gastric juice and 9 mL of water at 37°C for 0.5 and 2 h
200 mL of water. On d 6, 10 and 14, all subjects consumed one of the (22,23). Similar amounts were incubated with 3 mL human duodenal
following supplements in random order: 1000 mg (2.8 mmol) chlo- fluid and 9 mL water at 37°C for 1 and 4 h, corresponding to the
rogenic acid (Fluka Chemie, Buchs, Switzerland) or 500 mg (2.8 average and maximal transit time in the small intestine (24,25).

TABLE 1
Intake of chlorogenic acid and caffeic acid and subsequent excretion in ileostomy effluent over 24 h in 7 subjects1

Excretion

Presupplement
Supplement Intake sample2 24-h excretion3 Final sample4 Absorption

% of
mg intake

Chlorogenic acid5 1000 2.3 ⫾ 2.5 667 ⫾ 165 2.6 ⫾ 3.8 33 ⫾ 17

Caffeic acid 500 0.3 ⫾ 0.6 27 ⫾ 18 0.1 ⫾ 0.1 95 ⫾ 4

1 Values are means ⫾ SD.

2 Ileostomy effluent sample collected before ingestion of the supplements; amounts represent the mean of 6 subjects because 1 subject did not
collect the presupplement ileostomy effluent.
3 Includes the final but not the presupplement sample.
4 Ileostomy effluent sample collected at the end of the 24-h collection period.
5 Excretion of chlorogenic acid was calculated as the sum of the excretion of 3-caffeoylquinic acid, 4-caffeoylquinic acid and 5-caffeoylquinic acid
in ileostomy effluent.
68 OLTHOF ET AL.

FIGURE 2 Chromatograms of chlorogenic acid isomers and of caffeic acid in standard mixture (A) and ileostomy effluent (B). Peak 1:
3-caffeoylquinic acid or 4- caffeoylquinic acid; Peak 2: 5-caffeoylquinic acid; Peak 3: 3-caffeoylquinic acid or 4- caffeoylquinic acid; Peak 4: caffeic
acid

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Gastric juice and duodenal fluid were obtained from two fasted (Hollman, unpublished results). The detection limit in urine was 0.8
healthy volunteers with a colon and stored at ⫺20°C. mg/L urine for chlorogenic acid and 0.4 mg/L for caffeic acid. Lithium
We also studied the stability of chlorogenic acid and caffeic acid was measured in undiluted, acidified urine by atomic absorption
ex vivo during collection of ileostomy fluid and during sample prep- spectrophotometry (27).
aration in the laboratory. For this purpose, two ileostomy subjects,
who also participated in this study, followed a diet low in chlorogenic
acid and quercetin for 4 d. On d 4, they applied three ileostomy bags
in total, one bag with 300 mg chlorogenic acid mixed with ⬃5 g of RESULTS
strawberry jam, one with 150 mg caffeic acid mixed with 5 g of
strawberry jam and one with 5 g of strawberry jam only. Strawberry The HPLC method used for the determination of chloro-
jam was used as a vehicle for chlorogenic acid and caffeic acid genic acid and caffeic acid in ileostomy fluid showed well-
powder. Strawberry jam itself does not contain chlorogenic acid or resolved isomer peaks. Quantification was not hampered by
caffeic acid. The subjects allowed ileostomy fluid to drain into the bag
for ⬃2 h and kneaded the contents regularly. The ileostomy fluids
potential interferences from the sample matrix (Fig. 2). Of the
were stored and analyzed as described. ingested chlorogenic acid, 67% was excreted in ileostomy
Analysis of chlorogenic acid and caffeic acid in ileostomy effluent fluid, whereas only 5% of the caffeic acid was excreted (Table
and urine. Chlorogenic acid and caffeic acid in ileostomy effluent 1). Traces of the ingested chlorogenic acid and 11% of the
were extracted simultaneously by mixing 0.500 g freeze-dried effluent ingested caffeic acid were excreted in urine (Table 2).
with 25 mL 40% (v/v) aqueous methanol containing 2 g tert-butyl- Chlorogenic acid and caffeic acid were recovered almost
hydroxyquinone/L. The effluent extract was refluxed at 90°C for 1 h completely after in vitro incubation in gastric juice and duo-
with regular swirling, allowed to cool down and subsequently brought denal fluid and after ex vivo incubation in ileostomy fluid
to 50 mL with methanol. The effluent extract was then sonicated for
5 min and filtered through a 0.45-␮m filter for organic solvents
(Table 3). Thus, the amounts not excreted in ileostomy ef-
(Acrodisc CR PTFE; German Sciences, Ann Arbor, MI) before fluent were likely absorbed rather than degraded in the gut or
HPLC injection. For HPLC analysis, we injected 20 ␮L of the in the ileostomy bag. The absorption of chlorogenic acid
effluent extract onto an Inertsil ODS-2 (GL Sciences, Tokyo, Japan) therefore equaled 100% ⫺ 67% ⫽ 33% and that of caffeic acid
column (4.6 ⫻ 150 mm, 5 ␮m particle size) protected by an MPLC 95%.
Newguard RP-18 (Brownlee; Applied Biosystems, San Jose, CA)
column (3.2 ⫻ 15 mm, 7 ␮m particle size) using acetonitrile/0.025
mol/L phosphate buffer, pH 2.4 (8:92) as the mobile phase, at a flow
rate of 1 mL/min. The columns were placed in a column oven set at TABLE 2
40°C. Ultraviolet absorption was measured at 325 nm. Excretion of
chlorogenic acid was calculated as the sum of the excretion of Intake of chlorogenic acid and caffeic acid and subsequent
3-caffeoylquinic acid, 4-caffeoylquinic acid and 5-caffeoylquinic acid excretion of chlorogenic acid and caffeic acid
in ileostomy effluent (Fig. 2). Isomers of chlorogenic acid were in urine over 24 h in 7 subjects1
produced by incubation of a solution of 5-caffeoylquinic acid (pH 8)
at 100°C for 30 min (26). The limit of detection, i.e., the concen- 24-h excretion
tration producing a peak height three times the standard deviation of
the baseline noise, was 3 ␮g/g freeze-dried ileostomy effluent. Supplement Intake Caffeic acid Chlorogenic acid2
For analysis of caffeic acid in urine, urine was treated with ␤-glu-
curonidase/sulfatase before analysis. For analysis of chlorogenic acid mg
in urine, urine was not treated with ␤-glucuronidase/sulfatase. Urine
for measurement of chlorogenic acid and caffeic acid was acidified Chlorogenic acid 1000 1.7 ⫾ 0.8 2.9 ⫾ 1.5
and brought onto an SPE extraction column. The column was eluted Caffeic acid 500 53.4 ⫾ 14.1 ND
with ethyl acetate. Ethyl acetate was evaporated and acids were
derivatized with bis(trimethylsilyl)trifluoroacetamide. Derivates were 1 Values are mean ⫾ SD; ND, not detected.
injected onto a capillary column CP-Sil-5 (Chrompak, Middelburg, 2 Excretion of chlorogenic acid was calculated as the excretion of
The Netherlands) and separated using a temperature gradient. On- 5-caffeoylquinic acid. 3-Caffeoylquinic acid and 4-caffeoylquinic acid
line mass spectrometry was used to quantify and identify the acids were not present in large amounts in urine.
 ABSORPTION OF CHLOROGENIC ACID AND CAFFEIC ACID 69

TABLE 3 collected all ileostomy effluent. Therefore, we conclude that

collection of ileostomy effluent was complete.
Stability of chlorogenic acid and caffeic acid expressed as the The subjects in this study collected ileostomy effluent for
percentage recovered after incubation with human gastric 24 h, which should be long enough to detect all nonabsorbed
juice or duodenal fluid in vitro, or with ileostomy supplement in ileostomy effluent because the mean transit
effluent ex vivo time through the stomach and small intestine is ⬃8 –11h
(31,32). This was also supported by the fact that the amount
Duodenal Ileostomy of chlorogenic acid and caffeic acid excreted in the final
Gastric juice1 fluid1 effluent2 collection of ileostomy effluent at the end of the 24-h period
was similar to that in the presupplement collection (Table 1).
Incubation period Furthermore, the recovery of 84 ⫾ 19% (mean ⫾ SD) of
Supplement 0.5 h 2h 1h 4h 2h quercetin in ileostomy effluent during 24 h after ingestion of
quercetin-3-rutinoside in this study was similar to the recovery
% during the first 13 h in a previous study (15).
Chlorogenic acid 101 99 95 99 98 (97–98) Comparison with previous studies. We found that the
Caffeic acid 98 101 97 93 97 (96–98) absorption of caffeic acid esterified with quinic acid (chloro-
genic acid) is three times lower than that of caffeic acid itself.
1 Mean of duplicate analyses. To our knowledge, there are no previous quantitative data on
2 Mean (range) of recoveries in ileostomy bags on the bodies of 2 absorption of chlorogenic acid and caffeic acid in humans. The
subjects. studies that were done on absorption of chlorogenic acid and
caffeic acid measured the recoveries of these compounds and

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their metabolites in urine of rats (33,34).
DISCUSSION We recovered 0.3% of chlorogenic acid in urine after in-
gestion. After ingestion of chlorogenic acid by rats, no chlo-
A maximum of 33% of the ingested chlorogenic acid and rogenic acid was found in urine (35). We recovered 11% of
95% of the ingested caffeic acid was absorbed from the small caffeic acid in urine after ingestion, which was comparable to
intestine in humans. Traces of chlorogenic acid in urine were the recovery of 13% found by (34) after ingestion of caffeic
recovered after ingestion of chlorogenic acid, whereas 11% of acid in rats. After intravenous injection of chlorogenic acid
caffeic acid in urine was recovered after ingestion of caffeic and caffeic acid in rats, only 9% of chlorogenic acid and 26%
acid. This indicates that at least some of the chlorogenic acid of caffeic acid were recovered in urine (35). This indicates that
was absorbed intact. Indications that caffeic acid and chloro- the fraction of chlorogenic acid and of caffeic acid that is
genic acid are absorbed in the small intestine were also found absorbed is metabolized extensively in the body and therefore
in a rat intestine perfusion model (28). However, because the only small amounts are recovered in urine.
recovery in urine was not nearly complete, chlorogenic acid Mechanisms of absorption. The absorption of caffeic acid
and caffeic acid are probably metabolized extensively into esterified with quinic acid (chlorogenic acid) was less than
other compounds after absorption. Unfortunately, data on that of caffeic acid itself. It is possible that chlorogenic acid
metabolism of chlorogenic acid and caffeic acid in the human and caffeic acid are absorbed through different absorption
body are scarce. mechanisms. We can envisage two mechanisms for the absorp-
Validity of ileostomy model. We measured absorption as tion of chlorogenic acid in humans. The first mechanism
the difference between the amount of supplement ingested and might involve absorption of chlorogenic acid as an intact
the amount excreted in ileostomy fluid in subjects without a molecule as indicated by the presence of traces of chlorogenic
colon. Absorption of nutrients in the small intestine of ileos- acid in urine after ingestion of chlorogenic acid in our study.
tomy subjects is probably not affected by the lack of the colon We probably found only traces of the absorbed chlorogenic
(29), as indicated by their normal serum cholesterol concen- acid in urine because chlorogenic acid is metabolized inten-
trations (30), normal absorption of para-aminobenzoic acid sively after absorption (35). The second mechanism might
(15) and of lithium in this study (20,21). involve hydrolysis of chlorogenic acid in the stomach and/or
It is unlikely that appreciable amounts of chlorogenic acid small intestine into caffeic acid and quinic acid before absorp-
or caffeic acid disappeared through degradation in the gastric tion. The caffeic acid moiety and the quinic acid moiety are
juice, duodenal fluid, in the ileostomy bag or during analysis on subsequently absorbed (36,37). If this mechanism plays a role
the laboratory because chlorogenic acid and caffeic acid were in the absorption of chlorogenic acid, we would expect to find
recovered completely after in vitro and ex vivo incubations in caffeic acid in urine as we found after intake of the caffeic acid
gastrointestinal fluids (Table 3). However, we cannot exclude supplement. If we assume that the absorption of chlorogenic
that part of the supplements were lost somewhere in the acid is 33% and the amount of caffeic acid in urine after
gastrointestinal tract; therefore, the absorption values we ingestion of caffeic acid is 11%, then we would expect to
found in this study should be regarded as maximum absorption recover ⬃4% of chlorogenic acid in urine as caffeic acid.
values rather than as fixed absorption values. In subjects with However, we found only 0.3% of chlorogenic acid as caffeic
a colon, absorption of dietary phenolic compounds and their acid in urine after ingestion of chlorogenic acid, which is ⬃10
metabolites in the colon is possible. Therefore, in subjects with times lower than we expected. This indicates that hydrolysis of
a colon, caffeic acid in urine might originate from dietary chlorogenic acid in the stomach or small intestine is not very
chlorogenic acid. important. This is also supported by the fact that we found a
Collection of ileostomy effluent. The amounts of caffeic large amount of the ingested chlorogenic acid unchanged in
acid and chlorogenic acid that were not recovered in ileostomy ileostomy effluent. Thus, this second mechanism likely does
effluent cannot be explained by loss of ileostomy effluent. not play an important role in the absorption of chlorogenic
None of the subjects reported loss of ileostomy effluent during acid. Therefore, we propose that in ileostomy subjects, most of
the 24-h collection periods, and the 9 –11 ileostomy bags that the absorbed chlorogenic acid is absorbed intact and is metab-
subjects collected during 24 h also indicated that they had olized extensively in the liver.
70 OLTHOF ET AL.

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