Curcuminoid Demethylation as an Alternative Metabolism by Human Intestinal Microbiota

Article abstract of DOI:10.1021/acs.jafc.7b00943

Curcumin and other curcuminoids from Curcuma longa are important bioactive compounds exhibiting various pharmacological activities. In addition to the known reductive metabolism of curcuminoids, an alternative biotransformation of curcuminoids by human gut microbiota is reported herein. A curcuminoid mixture, composed of curcumin (1), demethoxycurcumin (2), and bisdemethoxycurcumin (3), was metabolized by the human intestinal bacterium Blautia sp. MRG-PMF1. 1 and 2 were converted to new metabolites by the methyl aryl ether cleavage reaction. Two metabolites, demethylcurcumin (4) and bisdemethylcurcumin (5), were sequentially produced from 1, and demethyldemethoxycurcumin (6) was produced from 2. Until now, sequential reduction of the heptadienone backbone of curcuminoids was the only known metabolism to occur in the human intestine. In this study, a new intestinal metabolism of curcuminoids was discovered. Demethylation of curcuminoids produced three new colonic metabolites that were already known as promising synthetic curcumin analogues. The results could explain the observed beneficial effects of turmeric.

Full text of DOI:10.1021/acs.jafc.7b00943

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Article
Curcuminoid Demethylation as an Alternative
Metabolism by Human Intestinal Microbiota
Supawadee Burapan, Mihyang Kim, and Jaehong Han
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00943 • Publication Date (Web): 12 Apr 2017
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Journal of Agricultural and Food Chemistry
Curcuminoid Demethylation as an Alternative Metabolism by Human
Intestinal Microbiota
Supawadee Burapan, Mihyang Kim and Jaehong Han*
Metalloenzyme Research Group and Department of Integrative Plant Science, ChungꢀAng
University, Anseong 17546, Korea
Corresponding author
*
Phone: +82 31 670 4830. Fax: +82 31 675 1381. Eꢀmail: jaehongh@cau.ac.kr
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Abstract
Curcumin and other curcuminoids from Curcuma longa are important bioactive compounds
exhibiting various pharmacological activities. In addition to the known reductive metabolism of
curcuminoids, an alternative biotransformation of curcuminoids by human gut microbiota is
reported herein. A curcuminoid mixture, composed of curcumin (1), demethoxycurcumin (2),
and bisdemethoxycurcumin (3) was metabolized by the human intestinal bacterium Blautia sp.
MRGꢀPMF1. 1 and 2 were converted to new metabolites by the methyl aryl ether cleavage
reaction. Two metabolites, demethylcurcumin (4) and bisdemethylcurcumin (5), were
sequentially produced from 1, and demethyldemethoxycurcumin (6) was produced from 2. Until
now, sequential reduction of the heptadienone backbone of curcuminoids was the only known
metabolism to occur in the human intestine. In this report, a new intestinal metabolism of
curcuminoids was discovered. Demethylation of curcuminoids produced three new colonic
metabolites that were already known as promising synthetic curcumin analogues. The results
could explain the observed beneficial effects of turmeric.
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KEY WORDS: curcumin, curcuminoids, demethoxycurcumin, demethylation, human intestinal
metabolism,
turmeric
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Introduction
Curcuminoids are major bioactive compounds derived from turmeric (Curcuma longa L.),
which have pharmacological activities, such as antiꢀinflammatory, antibacterial, antifungal,
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antioxidant, antimutagenic, and anticancer effects.
Because of potential pharmaceutical
applications, the metabolism of curcuminoids, including colonic metabolism, has been studied
extensively. Similar to most dietary polyphenols, turmeric curcuminoids, composed of curcumin
(1), demethoxycurcumin (2), and bisdemethoxycurcumin (3) (Figure 1), are poorly absorbed and
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rapidly excreted from the body. It is estimated that more than 75% of ingested curcuminoids
are excreted from the body without absorption, and less than 1% is found as metabolites in
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circulating blood. The major metabolites of curcumin in the plasma are curcumin glucuronide,
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curcumin sulfate, and hexahydrocurcumin. A series of reduced curcumin metabolites are also
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produced by intestinal bacteria, such as Escherichia coli and E. fergusonii.
The enzyme
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responsible for the reduction was purified and characterized as a NADPHꢀdependent reductase.
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One the other hand, many curcumin derivatives have been synthesized to improve the
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bioavailability and pharmacological properties of curcuminoids.
Some of these exhibited
even better pharmacological activities than the natural turmeric curcuminoids. For example,
polyhydroxycurcuminoids have been described as promising Alzheimer’s disease preventatives.
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The epigenetic activity of bisdemethylcurcumin, a demethylated curcumin analogue, was
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particularly highlighted for its neuroprotective effects.
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The discrepancy in curcuminoid fluxes between the ratio of intestinal metabolites from gut
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microbiota and the distribution of phase II metabolites in the plasma attracted our attention. In
particular, less than 1% of reduced curcuminoids in plasma, resulting from microbial
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metabolism, was too small to explain 25% of the orally administered curcuminoids that undergo
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intestinal metabolism.
Therefore, it was hypothesized that other types of curcuminoid
metabolism should be operating in the intestine. To test this, curcuminoids were reacted with
human fecal mixed cells. The result strongly indicated an alternative metabolism of
curcuminoids different from the known reductive metabolism. Curcumin (1) and
demethoxycurcumin (2) were biotransformed by the recently isolated Blautia sp. MRGꢀPMF1
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that catalyzes the demethylation of the methyl aryl ether functional group. This study describes
an alternative metabolism of curcuminoids, and which may encourage the further study of
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curcuminoids as a valuable bioactive ingredient.
Materials and Methods
The experimental protocol was evaluated and approved by the Institutional Review Board of
ChungꢀAng University (Approval Number: 1041078ꢀ201502ꢀBRꢀ029ꢀ01).
Chemicals and Bacterium
Curcuminoids mixture was purchased from Sigma Co. (St. Louis, USA). Curcumin (1),
demethoxycurcumin (2), and bisdemethoxycurcumin (3) were purchased from Phytobean AC.
LTD (Yecheonꢀgun, Korea). HPLCꢀgrade methanol (MeOH) and acetonitrile (MeCN) were
purchased from Burdick & Jackson Laboratories, Inc. (Muskegon, MI, USA). Formic acid for
mass spectrometry (98%) was purchased from Fluka (Buchs, Switzerland). Ethyl acetate (99.5%,
EtOAc), acetic acid (99.5%), N,Nꢀdimethylformamide (99.5%, DMF were purchased from
Samchun Pure Chemicals (Gyeonggiꢀdo. Korea). Gifu anaerobic medium (GAM) was from
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Nissui Pharmaceutical Co. (Tokyo, Japan). The GAM broth was prepared following the
manufacturer’s instructions, and GAM plates were prepared by adding 1.5% (w/v) agar in GAM
broth.
Human intestinal bacterium, Blautia sp. MRGꢀPMF1, isolated from our laboratory (GenBank
accession number: KJ078647), was used in these experiments. Bacterial growth and substrate
conversion experiments were performed under anaerobic conditions, according to published
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methods.
General HPLC Method
A Finnigan Surveyor Plus HPLC with a Thermo PDA Plus detector, equipped with a C18
Hypersil GOLD column (4.6×100 nm, 5 ꢁm, Thermo Scientific, Waltham, MA, USA) was
used for HPLC analysis. The injection volume was 10 ꢁL and the flow rate was 1.0 mL/min. The
mobile phase for the analysis of curcuminoid biotransformation was composed of 0.1% acetic
acid in deionized water (solvent A) and 0.1% acetic acid in MeCN (solvent B). For the eluent
gradient system, solution B was started at 20% and increased to 40% in 8 min, to 50% in 12 min,
to 55% in 15 min, to 70% in 20 min, to 80% in 22 min, and finally to 20% in 30 min.
Biotransformation of Curcuminoids
All the experimental procedures for the biotransformation of curcuminoids were performed
under anaerobic conditions (CO 5%, H 10%, N 85%) at 37 °C. Fresh fecal samples from three
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healthy volunteers were taken in sterilized vials and placed immediately in the anaerobic
chamber. Using a cotton swab, the samples were diluted in saline solution, and the solution was
filtered through sterile cheesecloth. For the preparation of mixed cell cultures, 1 mL of the
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original filtrate was diluted to 10 mL with GAM broth and incubated. After 1 d, 100 ꢁL of the
mixed cell culture was transferred to an Eppendorf tube, and 1 ꢁL of curcuminoids (a mixture of
1 – 3, total 10 mM) was added to begin the biotransformation. After 6 h and 24 h, 1 mL of ethyl
acetate was added to each tube and the reaction was stopped by vortexing. The solution was
centrifuged (10770g) for 10 min, and the organic layer (800 ꢁL) was dried under vacuum. The
dried residue was dissolved in 10 ꢁL of DMF and filtered through a 0.2ꢀꢁm filter (Grace, USA)
for HPLC analysis.
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For the curcuminoid biotransformation with Blautia sp. MRGꢀPMF1, 60 ꢁL of the solution
containing the curcuminoids mixture, 1, or 2 (10 mM in DMF) was added to GAM broth
medium containing the MRGꢀPMF1 strain (6 mL, OD600 of 0.9). Aliquots (100 ꢁL) of this
reaction mixture were transferred to Eppendorf tubes after scheduled reaction times. The
allocated media were extracted with 1 mL of ethyl acetate, and the supernatant (800 ꢁL)
collected after vortexing and centrifugation (10770g for 10 min) was dried under vacuum. The
dried residue was dissolved in DMF (100 ꢁL) and filtered through a 0.2ꢀꢁm filter (Grace, USA)
for HPLC analysis.
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Structural Analysis of Curcuminoid Metabolites by HPLC-MS
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The metabolites derived from curcumin (1) and demethoxycurcumin (2) were analyzed by a
Dionex Ultimate 3000 HPLC system (Thermo Scientific, Waltham, MA) equipped with a C18
reversedꢀphase column (Kinetex, 100 × 2.1 mm, 1.7 ꢁm, Phenomenex, Torrance, CA) and a
diode array detector (DAD). A Thermo Fisher Scientific LCQ fleet instrument (Thermo
Scientific, Waltham, MA) was coupled to the HPLC system for electrospray ionization mass
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spectrometry (ESI−MS) analysis. The mobile phase of the eluent gradient system was composed
of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The initial
composition of the mobile phase was 20% solvent B for 1 min. In a linear gradient, the
composition was changed to 30% solvent B in 5 min, 40% solvent B in 15 min, 60% solvent B in
20 min, 80% solvent B in 22 min and then 20% solvent B remained for 2 min at a flow rate of
0.2 mL/min. ESI−MS analyses were performed in the positiveꢀion mode within the m/z range of
50−500 and processed with Xcalibur software (Thermo Scientific, Waltham, MA). ESI
conditions: spray voltage, 5.4 kV; sheath gas, 15 arbitrary units; auxiliary gas, 5 arbitrary units;
heated capillary temperature, 275 °C; capillary voltage, 27 V; and tube lens, 100 V.
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Results
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Curcuminoid Metabolism by Human Intestinal Bacteria
The curcuminoid mixture metabolized by three different mixed cell cultures exhibited
different patterns of curcuminoid metabolism (see Figure S1). While two cultures produced
putative reduced curcuminoid metabolites, the MS analysis identified demethylated metabolites
from one of the mixed cell cultures. The culture with demethylation activity contained Blautia
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sp. MRGꢀPMF1, for which demethylation activity was reported previously,
thereby
confirming that curcuminoids could be metabolized by the MRGꢀPMF1 strain. Most
curcuminoids were metabolized within 2 d, and three metabolites were identified from HPLC
chromatograms (Figure 2). Metabolite A disappeared in 24 h, but metabolite B remained even
after 6 d of incubation. When each curcuminoid, curcumin (1), demethoxycurcumin (2), and
bisdemethoxycurcumin (3), was reacted with culture of the MRGꢀPMF1 strain, only
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curcuminoids 1 and 2 were metabolized. Bisdemethoxycurcumin (3) was not metabolized (see
Figure S2). The substrate curcumin (1) at the retention time of 14.42 min started to form the
metabolite A at the retention time of 12.48 min after 3 h, and metabolite C appeared at the
retention time of 10.80 min after 6 h. As metabolite A decreased over 30 h, the formation of
metabolite C increased rapidly (Figure 3A). These results indicated that the metabolic pathway
for curcumin (1) proceeded from 1 → A → C. For demethoxycurcumin (2), metabolite B found
at the retention time of 12.25 min started to form after 3 h of incubation, and most metabolism
was completed in 2 d (Figure 3B). This result indicated that the metabolic pathway for
demethoxycurcumin (2) proceeded from 2 → B.
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The metabolites A and C were identified as demethylcurcumin (4) and bisdemethylcurcumin
(5), respectively, and metabolite B was identified as demethyldemethoxycurcumin (6), based on
the UV and MS spectroscopic data (see Figure S3). The maximum UV absorptions of
metabolites A and C were found at 428 nm, the same as curcumin (1), whereas the UV
absorption of metabolite B was found at 423 nm, the same as demethoxycurcumin (2) (Figure 2).
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The ESIꢀMS analysis of metabolite A resulted in the [MꢀH] peak of demethylcurcumin (4) at m/z
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= 352.95, smaller than the [MꢀH] peak of curcumin by 14 Da due to the loss of a methyl group.
The ESIꢀMS analysis of metabolite C showed a peak corresponding to the demethylated 4 at m/z
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= 338.94, and which was identified as bisdemethylcurcumin (5). The [MꢀH] peak of metabolite
B was found at m/z = 322.84, 14 Da less than demethoxycurcumin (2), and identified as
demethyldemethoxycurcumin (6). The metabolites 4 – 6 were confirmed later by comparison of
HPLC retention time and UV absorption with an authentic references which were obtained
commercially.
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Proposed Metabolic Pathway of Curcumin and Demethoxycurcumin
Curcumin (1) and demethoxycurcumin (2) were metabolized by the fecal samples and Blautia
sp. MRGꢀPMF1. Based on the HPLC chromatographic changes (Figure 2 and 3), it was clear that
the metabolism was performed mainly by Blautia sp. MRGꢀPMF1. Curcumin (1) was converted
to bisdemethylcurcumin (5) via demethylcurcumin (4) by successive demethylation.
Demethoxycurcumin (2) was directly metabolized to demethyldemethoxycurcumin (6) (Figure
4). Since new curcuminoid metabolism in the human intestine was identified in this work,
curcuminoids 1 – 6, as well as the reduced curcuminoinds metabolites, should be included for the
study of the pharmacokinetics of curcuminoids.
Discussion
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Study of the metabolic pathway of dietary polyphenols in the human intestine is important for
human health. It can not only lead to new pharmaceutical compounds, but also provide insights
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into disease etiology and prevention. Metabolic pathways of plant polyphenols by human gut
microbiota have been extensively studied by us and others. Generally, intestinal metabolism of
polyphenols includes hydrolysis of glycosides and esters, reduction of nonꢀaromatic alkenes, and
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cleavage of the skeletons.
The biotransformation of turmeric curcuminoids, 1 – 3, by human
gut microbiota reported here is reminiscent of Sꢀequol production from the soy isoflavone,
daidzein (Figure 5). For example, repetitive reduction of curcuminoids 1 – 3 is known to produce
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a series of reduced curcumin metabolites.
Similarly, stereospecific reduction of daidzein
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When three different mixed cultures were reacted with curcuminoids, it was found that
curcuminoids were metabolized differently depending on the fecal microflora. Similar to
daidzein metabolism leading to Sꢀequol formation, it appears that curcuminoid demethylation
does not occur in everybody. Recently, Lou et al. also reported curcumin (1) demethylation by
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the fecal sample of a Chinese male.
However, it is still an open question whether dietary
polyphenolics can alter human gut microbiota diversity or not.
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Further study of one of the fecal samples which contained Blautia sp. MRGꢀPMF1 identified
three new metabolites, 4 – 6, arising from the different biotransformation of curcuminoids. From
the biotransformation of each curcuminoid, the metabolic pathways for 1 and 2 were established
as 1 → 4 → 5 and 2 → 6, respectively. It is noteworthy that the potential pharmaceutical
properties of the metabolites 4 – 6 had previously been studied as chemical derivatives of
curcumin. For example, demethoxycurcumin (4) showed more potent cytotoxicity than curcumin
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(1) against human HCT116 colon cancer cells, and the synthetic demethylated curcuminoids
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were reported to have better neuroprotective and antiꢀinflammatory properties.
Interestingly, the recently reported synthetic curcuminoids with preventative properties against
Alzheimer’s disease also included the metabolites 4 – 6. The reported colonic metabolites 4 – 6,
were reported to activate neprilysin, an endogenous amyloidꢀβ peptides degrading enzyme.
Especially, metabolites 4 – 5 were reported to increase neprilysin mRNA expression in the brain.
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In conclusion, our results support the thesis that the demethylated curcuminoids metabolites
with strong pharmacological activities and better bioavailability than curcuminoids can be
produced by human gut microbiota.
Supporting Information
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Curcuminoid biotransformation by the three different mixed cell cultures (Figure S1),
bisdemethoxycurcumin (3) biotransformation by Blautia sp. MRGꢀPMF1 (Figure S2), and ESIꢀ
MS spectra of curcuminoids and the metabolites (Figure S3). This Supporting Information is
available free of charge on the ACS Publications website at DOI:
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This work was supported under the framework of international cooperation program
managed by the National Research Foundation of Korea (NRFꢀ2015K2A1A2068137) and by
the Korean government (MSIP) (NRFꢀ2015R1A1A3A04001198).
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The authors declare no competing financial interest.
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FIGURE CAPTIONS
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Figure 1. Molecular structures of curcuminoids.
Figure 2. HPLC chromatogram changes (425 nm) of the curcuminoid mixture metabolism by
Blautia sp. MRGꢀPMF1. The insets show UV spectra of the compounds.
Figure 3. HPLC chromatogram changes (425 nm) of curcumin (1) (a) and demethoxycurcumin
(2) (b) metabolisms by Blautia sp. MRGꢀPMF1.
Figure 4. Proposed metabolic pathway of curcuminoids by human gut microbiota.
Figure 5. Established colonic metabolism of curcuminoids and daidzein. Sequential reduction of
curcuminoids produces dihydroꢀ, tetrahydroꢀ, and hexahydroꢀcurcuminoids. Dehydroxylation of
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2
the metabolite was also reported. Analogously, stereospecific reduction of daidzein results in
the formation of Sꢀequol via dihydrodaidzein and tetrahydrodaidzein.
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