A Curcumin Degradation Product, 7-Norcyclopentadione, Formed by

Dec 18, 2018 - Evidence that anti-inflammatory and other biological effects of curcumin may at least in part be mediated by its metabolites underscore...
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A Curcumin Degradation Product, 7‑Norcyclopentadione, Formed by Aryl Migration and Loss of a Carbon from the Heptadienedione Chain Akil I. Joseph, Paula B. Luis, and Claus Schneider* Department of Pharmacology, Division of Clinical Pharmacology, and Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232, United States

J. Nat. Prod. 2018.81:2756-2762. Downloaded from pubs.acs.org by UNIV DE BARCELONA on 01/11/19. For personal use only.

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ABSTRACT: Evidence that anti-inflammatory and other biological effects of curcumin may at least in part be mediated by its metabolites underscores the importance of identifying novel transformation products. Spontaneous degradation of curcumin in buffer pH 7.5 results mainly in dioxygenated products with a characteristic cyclopentadione ring composed of carbons 2 through 6 of the former heptadienedione chain. When analyzing degradation reactions of 4′-O-methylcurcumin, a product was identified missing one of the terminal carbons of the heptadienedione moiety while containing a cyclopentadione ring and adjacent hydroxy group typical of curcumin degradation products. Analysis of curcumin autoxidation reactions showed formation of an analogous compound, 7norcyclopentadione, a degradation product exhibiting net loss of a carbon and gain of an oxygen atom. Removal of the carbon is proposed to occur via a peroxide-linked curcumin dimer in conjunction with radical-mediated 1,2-aryl migration of a guaiacol moiety. Oxidation reactions of demethoxycurcumin gave demethoxy-7-norcyclopentadione, whereas an analogous product was not observed from bis-demethoxycurcumin. Incubation of RAW264.7 macrophage-like cells with curcumin showed the presence of 7-norcyclopentadione, the formation of which was not increased upon activation of the cells with 12-O-tetradecanoylphorbol13-acetate . 7-Norcyclopentadione is a novel type of degradation product that is most likely formed via autoxidative processes when cells are incubated with curcumin.

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The spontaneous chemical degradation of curcumin is an autoxidation resulting in a variety of products, characterized mainly by a modified heptadienedione chain.29,30 The products carry various types of oxygen substitutions at C-1 and C-7 of the C7 linker between the two guaiacol rings. A second characteristic feature is the cyclization involving C-2 and C-6 of the heptadienedione unit to a cyclopentadione in most of the degradation products (Chart 1). The degradation of curcumin is initiated by formation of a phenoxyl radical via a sequential proton loss−electron transfer process from the phenolic hydroxy group,31 triggering a series of radical reactions that result in cyclization and incorporation of oxygen.32 A detailed mechanism for product formation in curcumin autoxidation has been proposed.32 While there is no evidence so far for biological effects of bicyclopentadione as the major stable oxidation products of curcumin,33,34 it appears that unstable reaction intermediates are bioactive.35 For example, neither curcumin nor a stable analogue (4′,4″-O-dimethylcurcumin) or the final bicyclopentadione oxidation product inhibited topoisomerase-mediated

urcumin is the major orange-yellow pigment in turmeric root that gives the spice mix curry its characteristic color.1 Curcumin is also the major bioactive component of turmeric preparations used in traditional Asian medicine and in dietary supplements.2 Health benefits ascribed to curcumin or turmeric include antioxidant, anti-inflammatory, antimicrobial, antiproliferative, and cancer chemopreventive activities.3−7 These activities were established using cultured cells and animal models of disease.8−12 Recent human clinical trials, albeit some conducted with limited numbers of subjects,6,13−19 and epidemiological observations have provided additional evidence for biological and possibly therapeutic effects of curcumin exerted in vivo.20−22 Interest in metabolic and other chemical transformations of curcumin comes from the near absence of curcumin as the free aglycone in plasma of mice and humans upon oral administration23−25 as well as the chemical instability in buffer at physiological pH.26,27 The former invokes a contribution of in vivo metabolites to effects observed in animals and humans. The latter suggests that even nonenzymatic transformation products of curcumin may be bioactive,28 especially when curcumin is tested in cultured cells under conditions that enable or promote its degradation.29 © 2018 American Chemical Society and American Society of Pharmacognosy

Received: October 5, 2018 Published: December 18, 2018 2756

DOI: 10.1021/acs.jnatprod.8b00822 J. Nat. Prod. 2018, 81, 2756−2762

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Chart 1. Structures of Curcumin, Its Main Bicyclopentadione Oxidation Product, and the Spiroepoxide Reaction Intermediatea

Identical carbons are labeled with the same numbers in all schemes and figures.

a

DNA cleavage.33 Topoisomerase inhibition by curcumin required the addition of an oxidizing agent to achieve poisoning of the enzyme.33,36 The requirement for oxidative transformation of curcumin to reveal the active agent(s) was also shown for the anti-inflammatory effects of curcumin.35 Inhibition of the transcription factor NF-κB by curcumin and synthetic analogues correlated well with the ability of the compounds to undergo degradation, i.e., to form reactive electrophilic products.35,37 Inhibition of NF-κB was proposed to involve covalent binding of electrophilic intermediates to cysteine residues of regulatory proteins within the pathway, for example, the upstream kinase IKKβ.35,38 This hypothesis was supported by testing the HPLC-isolated spiroepoxide reaction intermediate and showing that it had activity similar to curcumin.35 Here we describe a product of curcumin autoxidation that is formed by the unexpected loss of the terminal carbon from the heptadienedione moiety and show that the product is formed when RAW264.7 macrophage-like cells are incubated with curcumin.

Figure 1. Oxidative transformation of 4′-O-methylcurcumin, 1. (A) LC-MS analysis of an oxidation reaction of 4′-O-methylcurcumin (1) showing ion traces for 1 (m/z 381), novel product 2 (m/z 385), and dioxygenated products (m/z 413). Chromatograms were acquired in negative ion mode. (B) Structures of 1, 2, and 4′-O-methylbicyclopentadione.

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side of the molecule, i.e., between C-2 and the arene, consistent with the presence of C-1 and location of the ring in a β-position relative to C-2. The 1-hydroxy group and absence of C-7 accounted for the change in molecular weight of 2 relative to 1 observed in the MS analyses. Accordingly, 2 was identified as 4″-O-methyl-7-norcyclopentadione [5-[(3,4dimethoxyphenyl)(hydroxy)methyl]-3-hydroxy-4-(4-hydroxy3-methoxyphenyl)cyclopent-2-en-1-one] (Figure 1B). [The chemical name for the compound requires a change in the assignment of the carbons of the cyclopentadione in comparison to the numbering in the structure shown.] A possible isomer that would carry the 3′,4′-dimethoxyphenyl moiety attached to C-6 rather than C-1 was not detected. Attempts to achieve resolution of 2 into stereoisomers using a chiral-phase Chiralpak AD-RH HPLC column were unsuccessful. Autoxidation reactions of curcumin produced an analogous product 3, which was detected based on its predicted molecular ion (m/z 371) in LC-MS analyses (Figure 2A). HR-MS analysis indicated loss of a carbon and addition of an oxygen atom (m/z 371.1134, calcd for C20H19O7, 371.1136). Absence of C-7 of the heptadienedione chain was confirmed by NMR analyses of the HPLC-purified compound (Table 1), identifying 3 as 7-norcyclopentadione [3-hydroxy-5-[hydroxy(4-hydroxy-3-methoxyphenyl)methyl]-4-(4-hydroxy-3methoxyphenyl)cyclopent-2-en-1-one; The chemical name for the compound requires a change in the assignment of the carbons of the cyclopentadione in comparison to the numbering in the structure shown] (Figure 2B). Autoxidation of a 1:1 mixture of d0- and d6-curcumin (with the label placed in the methoxy groups)40 gave a ratio of d0-3 (m/z 371) to d6-

RESULTS AND DISCUSSION An unknown product was detected in degradation reactions conducted with 4′-O-methylcurcumin, 1. Compound 1 was autoxidized or enzymatically oxidized using horseradish peroxidase/H2O2 to catalyze formation of a phenoxyl radical,39 giving indistinguishable product profiles. Product formation was analyzed by LC-MS using direct injection in order to avoid potential loss of highly polar products during extraction. LCMS ion traces showed the presence of known dioxygenated bicyclopentadione products (m/z 413; negative ion mode)32 as well as unreacted 1 (m/z 381) (Figure 1A). A prominent but unknown product 2 with m/z 385 was detected in both autoxidation and enzymatic oxidation of 1. The increase of 4 mass units in 2 suggested that the transformation included loss of a carbon and incorporation of oxygen compared to starting compound 1. For structural identification 2 was isolated using RP-HPLC from a large-scale autoxidation reaction of 1. HR-MS analysis of purified 2 confirmed loss of a carbon and incorporation of oxygen (m/z 385.1299, calcd for C21H21O7, 385.1293). 1H NMR analysis (Table 1) showed that C-1 of the former heptadienedione chain carried a hydroxy group (δ 5.26), while C-2 and C-6 were involved in the formation of the typical cyclopentadione moiety present in oxygenated transformation products of curcumin, cf. Chart 1.32 The absence of a signal for H-7 in the 1H NMR spectrum suggested that C-7 was missing from the molecule. This was supported by HMBC analysis that showed lack of a signal for C-7 and cross-peaks between H-6 and C-1″ and -2″ of the directly adjacent aromatic ring. Equivalent HMBC cross-peaks were not observed on the other 2757

DOI: 10.1021/acs.jnatprod.8b00822 J. Nat. Prod. 2018, 81, 2756−2762

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Table 1. NMR Spectroscopic Data (600 MHz, d4-Methanol) for Norcyclopentadiones 2 and 3 2 position

δC, typea

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″ 2″ 3″ 4″ 5″ 6″ 7″

70.5, CH 63.3, CH 204.9, C 104.4, CH 206.3, C 50.6, CH 137.0, C 109.5, CH 148.6, C 148.1, C 111.2, CH 117.9, CH 54.9, CH3 55.1, CH3 134.7, C 111.7, CH 143.8, C 147.2, C 114.5, CH 119.1, CH 54.6, CH3

δH (J in Hz)

3 HMBCb

δC, typea

5.26, d (2.6) 2.62, dd (2.6)

2′ 3

5.07, s

2

3.50, d (2.6)

1″, 2′′, 5

6.99, d (1.9)

1, 4′, 6′

6.89, 6.97, 3.74, 3.80,

1′, 3′ 4′, 6′ 3′ 4′

70.6, CH 63.3, CH 205.1, C 104.5, CH 206.4, C 50.5, CH 135.7, C 109.4, CH 147.4, C 144.8, C 114.3, CH 118.1, CH 54.9, CH3

d (8.2) dd (8.5, 1.9) s s

6.08, d (1.9)

4″,6,6′′

6.52, d (8.1) 6.28, dd (8.2, 2.0) 3.58, s

1″, 3′′ 2″, 4′′ 3″

134.7, C 111.7, CH 143.8, C 147.2, C 114.4, CH 119.0, CH 54.7, CH3

δH (J in Hz)

HMBCb

5.24, d(2.6) 2.62, dd (2.6)

2′ 3

5.07, s

2

3.51, d (2.5)

1″, 2′′, 5

6.96, d (1.8)

1, 4′, 6′

6.73, d (8.1) 6.97, dd (8.1, 1.3) 3.76, s

1′, 3′ 4′, 6′ 3′

6.06, d (1.9)

4″, 6, 6′′

6.53, d (8.2) 6.31, dd (8.2, 2.0) 3.60, s

1″, 3′′ 2″, 4′′ 3″

δC and type were determined from HSQC and HMBC experiments. bFrom proton (position) to the indicated carbon(s).

a

3 (m/z 377) similar to that observed for d0/d6-bicyclopentadione (m/z 399 and m/z 405) (Figure 2C). This indicated that both methoxy groups were present in 3, providing additional evidence that carbon loss had occurred at a different site. Attempts to achieve further resolution of 3 into stereoisomers were unsuccessful. 7-Norcyclopentadione 3 is a novel oxidation product of curcumin. The known degradation products formed at physiological pH in vitro are cyclopentadiones (C-2 through C-6) with various oxygen substitutions at C-1 and C-7 of the former heptadienedione moiety of curcumin. The most abundant product is a mixture of bicyclopentadione diastereomers exhibiting fused bicyclic cyclopentadione and tetrahydrofuran moieties (Chart 1).32 Additional degradation products that involve some form of a carbon bond cleavage reaction are diguaiacol and vanillin, both of only minor abundance.32,41 Formation of ferulic acid and feruloylmethane was reported in an early study of aqueous degradation products of curcumin27 but could not be confirmed in subsequent analyses.32,41 An autoxidation reaction of curcumin was conducted in buffer containing 50% H218O in order to elucidate the origin of oxygen in the 1-hydroxy group of 3 (Figure 3). LC-MS analysis confirmed the previously established incorporation of 18O from H218O into the major oxidation product bicyclopentadione, detected as a ∼1:1 ratio of m/z 399 and 401 when the reaction was conducted in H218O-enriched buffer (Figure 3A,B).32 Incorporation of 18O into bicyclopentadione occurs as an exchange of water during transformation of a spiroepoxide intermediate to a vinyl ether compound, an immediate precursor to the final product (Figure 3C).32 In contrast, 3 (m/z 371) did not incorporate 18O, suggesting that the 1hydroxy group was derived from molecular oxygen rather than H2O and that no further exchange of oxygen occurs (Figure 3). A proposed mechanism for loss of C-7 in 2 and 3 is via 1,2aryl migration.42,43 Aryl migration may be initiated by

formation of a C-6 carbon-centered radical that in turn may result from addition of a curcumin peroxy radical to the Δ6(7)double bond (Scheme 1). This provides a dimer in which two curcumin subunits are linked via a peroxy group, similar to the peroxy-mediated dimerization and subsequent carbon chain fragmentation of unsaturated fatty acids.44,45 Aryl migration entails the C-6 radical forming a bond with C-1″; rearomatization is achieved by cleavage of the C-7−C-1″ bond. Following aryl migration the peroxy-linked dimer undergoes fragmentation, resulting in loss of C-7, the detailed steps of which were not investigated. The resulting product with the guaiacol ring attached to C-6 retains the radical (C-6•) that reacts with the Δ2(3)-double bond in a 5-exo cyclization to provide the cyclopentadione and locates the radical at C-1. Oxygen addition to the carbon-centered radical and reduction afford the C-1 hydroxy group. Addition of O2 to the carboncentered radical is consistent with the reactions carried out in H218O-enriched buffer that did not show incorporation of labeled oxygen from water (Figure 3). Water could potentially have been introduced upon formation of a quinone methide and subsequent Michael addition of water. Aryl migration and loss of C-7 were less abundant with curcumin but are otherwise identical to the loss of the respective carbon from a curcumin derivative that carries additional methyl groups at C2 and C-6 of the heptadienedione chain.46,47 The fact that in oxidation reactions of asymmetric 1 only regioisomer 2 was detected with the guaiacol group attached at C-6 but not at C-1 may indicate a preference in the dimerization for addition of the peroxyl radical to the double bond adjacent to the guaiacol ring. Alternatively, the 4′-Omethyl-substituted ring may be less prone to aryl migration, and only binding of the peroxyl to the Δ6(7)-double bond yields a norcyclopentadione product. Formation of a 7-nor product was next analyzed in oxidation reactions of the natural curcumin isomers, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC). Oxidation was 2758

DOI: 10.1021/acs.jnatprod.8b00822 J. Nat. Prod. 2018, 81, 2756−2762

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Figure 3. Origin of oxygen in 7-norcyclopentadione 3 and bicyclopentadione formed by oxidation of curcumin. Partial LCmass spectra (negative ion mode) of curcumin (cur, m/z 367), 3 (m/ z 371), and bicyclopentadione (BCP, m/z 399) formed in (A) buffer pH 7.4 and (B) the same buffer containing 50% H218O. (C) Lack of an ion at m/z 373 in 3 (B, middle panel) shows that H218O is not incorporated, indicating that the 1-hydroxy group (red) is derived from O2. In bicyclopentadione the ethereal oxygen (blue) is derived from H2O, and the oxygen of the tetrahydrofuran ring (red) is from O2.32

lack of a product with predicted molecular ion at m/z 311 and a corresponding product at m/z 319 from d8-BDMC (data not shown). This further underscored the requirement for the guaiacol unit in aryl migration and carbon loss. The formation of 2 and 3 was analyzed in RAW264.7 macrophage-like cells, a widely used cellular model to analyze anti-inflammatory effects of curcumin. RAW264.7 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (PMA) to induce oxidative activation, or left untreated, and incubated with curcumin or 1, respectively, for 30 min. Products were extracted and analyzed by LC-MS in the SRM mode employing specific ion transitions for detection of curcumin, 1, 2, and 3. Both 2 and 3 were detected in the supernatants of cells treated with their respective precursors, but there was no increase in their formation upon activation of the cells with PMA (Figure 5). Based on ion intensities, 2 and 3 were of about 5% and 2% abundance, respectively, relative to their precursors. This indicated that formation of 7-norcyclopentadiones was likely due to autoxidation and that enzymatic oxidation by peroxidases catalyzing radical formation at the phenolic group30 was negligible. This was different from oxidative metabolism of curcumin and its glucuronide by isolated human leukocytes, in which PMA activation resulted in a 3−4-fold increase in formation of the respective bicyclopentadione products.39 Detection of 3 under standard cell culture conditions indicates that the transformation of curcumin to the novel metabolite 3 is functionally relevant. Since 3 was produced with a small yield in vitro as well as in cultured cells, it was not feasible to isolate the product in an amount required to

Figure 2. Identification of 7-norcyclopentadione 3 formed by autoxidation of curcumin. (A) LC-MS ion traces for curcumin (m/z 367), novel product 3 (m/z 371), and known dioxygenated products (m/z 399). Chromatograms were acquired in negative ion mode. (B) Structure of 3. (C) MS1 spectrum of 3 and coeluting bicyclopentadione formed in the autoxidation of a 1:1 mixture of d0- and d6-curcumin. The spectrum was obtained at 1.1−1.2 min retention time, cf. elution in panel A.

performed using horseradish peroxidase/H2O2 since autoxidation of DMC is slow and negligible for BDMC.36 The major oxidation product of DMC is demethoxybicyclopentadione, whereas oxidation of BDMC stops at the bis-demethoxyspiroepoxide, which is stable under the reaction conditions and does not form a bicyclopentadione product.36 The results with DMC and BDMC suggest that the lack of one or two methoxy groups exerts specific effects on rate and course of the oxidation reaction. LC-MS analyses of DMC oxidation indicated formation of demethoxy-7-norcyclopentadione [4 (m/z 341)], which showed identical as well as analogous MS2 fragment ions to 3 (Figure 4). Assignment of MS2 fragment ions was supported by MS2 spectra of d4-4 obtained by oxidation of d4-DMC (Figure 4).40 The MS2 spectra showed that the guaiacol unit was attached to C-1. Thus, aryl migration and carbon loss occurred at the side of the guaiacol moiety rather than the phenol unit. BDMC, in contrast, did not yield a bisdemethoxy-7-norcyclopentadione, as determined from the 2759

DOI: 10.1021/acs.jnatprod.8b00822 J. Nat. Prod. 2018, 81, 2756−2762

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Scheme 1. Proposed Mechanism of Formation of 7-Norcyclopentadione (3) during Autoxidation of Curcumin

Figure 4. LC-MS2 spectra of 7-norcyclopentadiones 3, 4, and d4-4 derived from oxidation of curcumin, demethoxycurcumin (DMC), and d4-DMC, respectively.

aromatic methoxy group, as has been suggested.48−51 Although demethylation provides a ready explanation for carbon loss in many transformation reactions, more complex reactions as described here may also be considered.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were acquired using a Bruker DRX 600 MHz spectrometer equipped with a cryoprobe. Samples were dissolved in 150 μL of methanol-d4 (δ 3.31) using 3 mm sample tubes. Pulse frequencies were taken from the Bruker library. High-resolution mass spectrometry was performed by direct liquid infusion using an Orbitrap mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with an Ion-Max source housing and a standard electrospray ionization probe operated in negative mode at a resolving power of 30 000 (at m/z 400). LC-MS analyses used a Thermo Vantage triple quadrupole LC-MS instrument equipped with a heated electrospray ionization interface operated in the negative ion mode. Chromatography used a Waters Symmetry Shield C18 1.7 μm column (2.1 × 50 mm). The column was eluted with a linear gradient of MeCN in water (both containing 0.1% formic acid) programmed from 15% to 85% in 3 min and then to 95% in 1 min, hold for 1 min before returning to starting conditions at 0.4 mL/ min flow rate. The ion transitions used in selected reaction monitoring were as follows: curcumin m/z 367.0 → 217.0 (10 eV),

Figure 5. Formation of 7-norcyclopentadiones 2 and 3 in RAW264.7 cells. RAW264.7 cells were incubated with precursors (A) curcumin and (B) 1 for 30 min followed by extraction and LC-MS analysis in the SRM mode.

investigate its biological activity. Nevertheless, 7-norcyclopentadione 3 represents a new and not readily predictable metabolite of curcumin. The unusual carbon loss in formation of 3 underscores the need for cautious interpretation of MS data for the identification of curcumin metabolites in the absence of supporting NMR analyses. Metabolites or degradation products of curcumin with an apparent loss of a carbon are not necessarily formed by O-dealkylation of an 2760

DOI: 10.1021/acs.jnatprod.8b00822 J. Nat. Prod. 2018, 81, 2756−2762

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1 m/z 380.9 → 149.0 (20 eV), 2 m/z 385.3 → 219.1 (10 eV), and 3 m/z 371.2 → 219.1 (15 eV). HPLC was performed using an Agilent 1200 diode array system equipped with a Waters Atlantis T3 5 μm column (4.6 × 250 mm) eluted at 1 mL/min flow rate with a linear gradient of MeCN/H2O/HOAc from 20/80/0.01 (by vol) to 80/20/ 0.01 (by vol) in 20 min. Synthesis of Curcuminoids. Curcumin, 4′-O-methylcurcumin, DMC, BDMC, d6-curcumin, and d4-DMC were synthesized according to the original method described by Pabon52 with appropriate modifications.30,40 Oxidation Reactions. Analytical-scale reactions were carried out in 1 mL of buffer (20 mM NH4OAc, pH 8) using 4′-Omethylcurcumin (50 μM) and 2.5 × 10−6 U horseradish peroxidase (HRP) (type II, P8250, Sigma). The reaction was initiated by the addition of H2O2 (40 μM) and conducted for 1 h at 37 °C. Products were analyzed by direct injection of an aliquot on LC-MS. Similar reactions were carried out with other curcuminoids. Large-scale reactions were conducted in 500 mL of buffer (20 mM NH4OAc, pH 8). 4′-O-Methylcurcumin (50 μM) was reacted with HRP/H2O2 at 37 °C for 3 h. Curcumin (50 μM; 300 mL of buffer) was reacted in the absence of catalyst at room temperature overnight. Products were extracted using a 5 g Supelco Discovery DSC-18 cartridge and eluted with MeOH/H2O 1:3 (by vol). The eluate was concentrated under a stream of nitrogen gas, and products were isolated using RP-HPLC. Incorporation of 18O was analyzed using 200 μL of NH4HCO3 buffer (20 mM, pH 7.4) made with a 1:1 mixture of H2O and H218O (97%). Curcumin (50 μM) was added and incubated at 37 °C for 1 h. Aliquots of the reaction were analyzed by LC-MS without prior extraction. Cell Culture. RAW264.7 cells were obtained from ATCC. Cells were maintained in Dulbecco’s modified Eagle’s medium containing fetal bovine serum (10%). Cells (400 000 cells/well) were seeded in six-well plates and allowed to attach overnight. Cells were treated with PMA or vehicle (EtOH) for 5 min followed by 4′-O-methylcurcumin or curcumin (10 μM) for 30 min. Supernatants were extracted using Waters HLB cartridges (30 mg), eluted with MeOH, and evaporated to dryness under a stream of N2. The residue was dissolved in MeCN/H2O, 1:1 (50 μL), for LC-MS analysis.

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Disease Research Center supported by NIH grant P30DK058404 Core Scholarship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00822.

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REFERENCES

NMR spectra (1H, COSY, HSQC, HMBC) of products 1 and 3 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 615-3439539. Fax: 615-322-4707. ORCID

Claus Schneider: 0000-0003-4215-967X Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by NIH awards R01AT006896 from the National Center for Complementary and Integrative Health (NCCIH) and the Office of Dietary Supplements (ODS) and R01GM076592 from the National Institute of General Medical Sciences of the National Institutes of Health (NIH). Mass spectrometric analyses were in part performed through Vanderbilt University Medical Center’s Digestive 2761

DOI: 10.1021/acs.jnatprod.8b00822 J. Nat. Prod. 2018, 81, 2756−2762

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