Production of Optically Active Hexahydrocurcumin by Human Intestinal Bacterium in Vitro

Abstract

A hexahydrocurcumin-producing bacterium named 2a1-2b was isolated from human feces. It was observed that the bacterium had more than 99% similarity with Enterococcus avium ATCC14025^T according to 16S ribosomal DNA (rDNA) sequence. The strain 2a1-2b produced optically active 5R-hexahydrocurcumin (enantiomeric excess (e.e.) > 95%) from tetrahydrocurcumin but not from curcumin. Our results showed that intestine is an important place for producing hexahydrocurcumin.

INTRODUCTION

Curcumin (1, Fig. 1) is a famous ingredient in turmeric. Turmeric is a rhizomatous herbaceous perennial plant (Curcuma longa) of the ginger family that has received much interest due to its use in the wide variety of activities. However, some reports reveal limitations in its activity in vivo because of its low bioavailability.^1,2) Therefore, several studies have been performed to improve curcumin’s bioavailability by chemical modifications.^3,4) Many studies suggest that there is a possibility of modification of chemical compounds in the human body. Intestine is an important organ because of its bacterial metabolism ability.⁵⁾ This has led to an increase in the research on the intestine. Tetrahydrocurcumin (2, Fig. 1) is a well-known curcumin metabolite, and often exhibits a more potent activity compared to that of curcumin.^6,7) Therefore, curcumin metabolites may have an important role for curcumin activity in vivo.

Fig. 1. Considerable Metabolic Pathways for Kazuranol by Bacterial Fraction 1C

Curcumin, tetrahydrocurcumin, hexahydrocurcumin and demethyltetrahydrocurcumin were converted to kazuranol by bacterial fraction 1C. The bacterium 2a1-2b produced 5R-hexahydrocurcumin (3) from tetrahydrocurcumin.

Recently, we reported 3-hydroxy-1,7-bis(3,4-dihydroxyphenyl) heptane incubated with bacteria 1C obtained from human feces as a curcumin metabolite in vitro.⁸⁾ This metabolite was optically active but not under strict selectivity (enantiomeric excess (e.e.) < 90%). To study the curcumin metabolism on the asymmetric reaction, the bacterium that relates to curcumin metabolism was isolated from bacteria 1C.

MATERIALS AND METHODS

Chemicals and Reagents

Curcumin was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). While tetrahydrocurcumin and hexahydrocurcumin were prepared from curcumin as described previously.⁸⁾ Demethyltetrahydrocurcumin was prepared from tetrahydrocurcumin by BBr₃⁹⁾ and trimethylsilyldiazomethane was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Gifu Anaerobic Medium (GAM) and BL agar used were the products of Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan).

Screening of Demethyltetrahydrocurcumin Metabolize Bacterium

As we previously described⁸⁾ in accordance with the Declaration of Helsinki (2018SR028), we have a bacterial fraction named 1C, which produced optically active 3-hydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane. We made colonies from the bacteria on Petri dishes by incubation for two or three days under an anaerobic condition at 37 °C. To isolate a bacterium that relates to curcumin metabolism; this was followed by the incubation of the sample with demethyltetrahydrocurcumin and the EtOAc extracts were analyzed using TLC. Briefly, colonies were seen to have formed by the incubation of diluted 1C on Petri dishes (i.d. 9 cm) plated BL agar. Each distinct colony was placed to a well filled with 8 mL GAM containing 100 µM substrate using 6-well plates. After incubation around three days under anaerobic condition, every medium in the wells (1 mL) were collected into 24-well plate and stored at −80 °C. The residual culture medium in the wells was extracted with 5 mL EtOAc and evaporated under reduced pressure. The extracts were dissolved with 0.3 mL MeOH, and were applied to TLC eluted with a solvent mixture of hexane/EtOAc (1 : 2). The stocked colonies corresponding to those made novel spots on TLC were again plated to make colonies. Repeating the selection, all colonies made from the bacterium showed similar spots on TLC. This isolated bacterium was labeled as 2a1-2b.

Isolation of 2a1-2b-Producing Metabolite from Tetrahydrocurcumin

This bacterial product was isolated using the method mentioned previously.^8,10) The incubation of the bacterium in 2 L of GAM containing commercially available (Sigma) tetrahydrocurcumin (100 µM) was done for three days under anaerobic condition at 37 °C. This is followed by the applying the EtOAc extract to Merck 1 mm-thick TLC plate and eluted with a solvent mixture containing hexane/EtOAc (1 : 2). Finally, the metabolite was purified using preparative HPLC on a 250 × 20 mm i.d. Wakosil-II 5C18HG column (Wako Pure Chemical Industries, Osaka, Japan) eluted with 40% MeOH at a flow rate of 5.0 mL/min at ambient temperature. After these procedures, 10.0 mg (13.4%) hexahydrocurcumin was obtained. The structure was confirmed by the ¹H-NMR.¹¹⁾

Genetic Analysis of Bacterium

Genetic analysis of bacterium was done by TechnoSuruga Laboratory Co., Ltd. (Shizuoka, Japan).

RESULTS AND DISCUSSION

We previously reported a curcumin metabolite in vitro. The metabolite was optically active but the enantio excess was under 90%.⁸⁾ The predominant metabolite was an enantiomer of rubranol, as kazuranol (4, Fig. 1) hereafter. According to this relatively low optical activity, bacterial fraction 1C seems to contain more than two bacteria that relate to the metabolism on the carbonyl reduction. Therefore, the bacterium that makes secondary alcohol was isolated. First, an intermediate between curcumin and kazuranol was searched for. By incubating bacteria 1C in curcumin (100 µM)-containing medium for one day, hexahydrocurcumin was found (data not shown). Indeed, bacteria 1C could produce kazuranol not only from hexahydrocurcumin but also from tetrahydrocurcumin. There was a tentative pathway made, which is indicated by the left arrows in Fig. 1. However, no information was obtained on the demethyl process. This led to the preparation of demethyltetrahydrocurcumin (5) and its application to bacteria 1C. As a result, demethyltetrahydrocurcumin was also metabolized to kazuranol. This result suggested another route as indicated by the right fished hooks in Fig. 1.

Every effort made to isolate hexahydrocurcumin-producing bacterium using curcumin as the substrate proved abortive. We then tried to find a bacterium that metabolizes demethyltetrahydrocurcumin. By selecting a colony, a bacterium named 2a1-2b was isolated. We then performed structural elucidation of the metabolite obtained from demethyltetrahydrocurcumin and the bacterium 2a1-2b. The metabolite had m/z: 345 [M–H]^– on LC-MS(electrospray ionization (ESI)–) analysis using 3200 QTRAP (AB Sciex, Framingham, MA, U.S.A.). The result suggested the reduction of one carbonyl moiety of the substrate and the metabolite was demethylhexahydrocurcumin (6, Fig. 1). Moreover, both the isolated metabolite and hexahydrocurcumin made dimethylhexahydrocurcumin that had m/z: 425 [M + Na]⁺ on LC-MS(ESI+) after treating with trimethylsilyldiazomethane, as a similar manner reported previously.⁸⁾ These results showed that demethyltetrahydrocurcumin (5) was metabolized to demethylhexahydrocurcumin (6) by the bacterium 2a1-2b. The structure was also supported by preparing demethylhexahydrocurcumin from hexahydrocurcumin by BBr₃ as a similar manner for demethyltetrahydrocurcumin synthesis. The ¹H-NMR spectra (400 MHz, acetone-d₆) of the prepared 6 were in a good agreement with those of hirsutanonol,¹²⁾ an optically active demethylhexahydrocurcumin, except to the five D₂O exchangeable peaks. We clearly found the signals of four phenols (δ_H 7.75, 7.71, 7.69, 7.65) and an alcohol (δ_H 3.75, d, J = 4.8 Hz). When tetrahydrocurcumin was applied to bacterium 2a1-2b, hexahydrocurcumin was produced as described in materials and methods section.

To determine the optical activity of hexahydrocurcumin produced by 2a1-2b, an HPLC condition for the separation of hexahydrocurcumin enantiomers was determined. First, CHIRALPAK-OZ-RH column, which was previously used for kazuranol analysis was tried but we could not separate the enantiomers of hexahydrocurcumin clearly. Then the CHIRALPAK-IF3 column was employed and could separate synthetic hexahydrocurcumin more clearly (Fig. 2). The bacterial hexahydrocurcumin had almost one peak on the HPLC (e.e. > 95%). This result showed that one carbonyl of tetrahydrocurcumin has been reduced by bacterium 2a1-2b asymmetrically. We then tried to determine the absolute stereochemistry of hexahydrocurcumin produced by the bacterium. Previously, 5S-hexahydrocurcumin was determined and isolated from ginger.¹³⁾ The optically active hexahydrocurcumin was prepared from a commercially available powder of ginger (Commpro, Tokyo, Japan), which was cultured in India (0.2 mg from 100 g powder), and applied to the HPLC (Fig. 2). The predominant peak was different from hexahydrocurcumin produced by bacterium 2a1-2b. The low optical purity of the sample obtained from ginger was also found in other HPLC conditions, modifying eluent, detection wavelength, or column. There was not enough data on whether the optical purity of hexahydrocurcumin depends on growing conditions for the ginger. However, Fig. 2 indicated that bacterium 2a1-2b produced 5R-hexahydrocurcumin (3, Fig. 1). To confirm the stereochemistry, the optical rotation of the hexahydrocurcumin produced by 2a1-2b was measured ([α]_D²⁵=−13.6° (c 0.1, CHCl₃)) even at lower concentration than the previous conditions for optically active hexahydrocurcumins.^13–15) The observed intensity was not same to any reported data, which are no more than 10°. However, the levorotary power also indicated that 2a1-2b produced 5R-hexahydrocurcumin.^13–15) The stereochemistry of the secondary alcohol does not contradict to that hexahydrocurcumin was an intermediate for kazuranol (4). However further study is required for whether hexahydrocurcumin was a predominant intermediate for kazuranol.

Fig. 2. Chiral Analysis of Hexahydrocurcumins

Three microliter samples (0.5 mg/mL) were subjected to an HPLC using a CHIRALPAK IF-3 column (i.d. 4.6 × 250 mm: Daicel Chemical Industries Ltd., Osaka, Japan) eluted with a solvent mixture of 50 mM phosphate buffer (pH 2.0)/acetonitrile (7 : 3) with 1.0 mL/min at 30 °C.

A few decades ago, Holder et al.¹⁶⁾ reported the production of hexahydrocurcumin from curcumin using rat. Recently, Jude et al.¹⁷⁾ detected hexahydrocurcumin in human serum after oral curcumin supplement. However, the mechanism of hexahydrocurcumin production was not fully established.^18,19) Our isolated bacterium failed to produce hexahydrocurcumin from curcumin itself. However, tetrahydrocurcumin is produced in our body, with intestinal bacteria²⁰⁾ and epithelial cells.¹⁸⁾ Therefore, our result suggested that optically active hexahydrocurcumin could be produced in our intestine with the aid of bacteria.

Finally, the 16S ribosomal DNA (rDNA) sequence (DDBJ accession no. LC577559) of the bacterium 2a1-2b was analyzed (Fig. 3). The results showed that the bacterium has almost the same sequence to that of Enterococcus avium ATC C14025^T. E. avium was originally isolated from human feces in Germany²¹⁾ and may be found around the world.^22,23) Although metabolic activity is not always correlated to phylogeny,²⁴⁾ we think that certain persons can produce optically active hexahydrocurcumin in their intestine.

Fig. 3. Phylogenetic Relationship for Isolated Hexahydrocurcumin-Producing Bacterium, 2a1-2b

Neighbor–Joining tree showing the phylogenetic relationships of strain 2a1-2b with related bacteria based on 16S rDNA sequences. The phylogenetic tree was constructed using MEGA ver. 7.0 program. The scale bar indicates 1% substitutions per site.

Acknowledgments

This work was financially supported by the Japan Food Chemical Research Foundation, and Urakami Foundation for Food and Food Culture Promotion. We wish to thank Daicel for helpful information on the chiral separation of hexahydrocurcumin. We also thank Dr. Sho Morohoshi (TechnoSuruga Laboratory) for the deposition in the DDBJ.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Kurita T, Makino Y. Novel curcumin oral delivery systems. Anticancer Res., 33, 2807–2821 (2013).
• 2) Nelson KM, Dahlin JL, Bisson J, Graham J, Pauli GF, Walters MA. The essential medicinal chemistry of curcumin. J. Med. Chem., 60, 1620–1637 (2017).
• 3) Venkateswarlu S, Ramachandra MS, Subbaraju GV. Synthesis and biological evaluation of polyhydroxycurcuminoids. Bioorg. Med. Chem., 13, 6374–6380 (2005).
• 4) Ravindran J, Subbaraju GV, Ramani MV, Sung B, Aggarwal BB. Bisdemethylcurcumin and structurally related hispolon analogues of curcumin exhibit enhanced prooxidant, anti-proliferative and anti-inflammatory activities in vitro. Biochem. Pharmacol., 79, 1658–1666 (2010).
• 5) Rowland I, Gibson G, Heinken A, Scott K, Swann J, Thiele I, Tuohy K. Gut microbiota functions: metabolism of nutrients and other food components. Eur. J. Nutr., 57, 1–24 (2018).
• 6) Pari L, Murugan P. Antihyperlipidemic effect of curcumin and tetrahydrocurcumin in experimental type 2 diabetic rats. Ren. Fail., 29, 881–889 (2007).
• 7) Morales NP, Sirijaroonwong S, Yamanont P, Phisalaphong C. Electron paramagnetic resonance study of the free radical scavenging capacity of curcumin and its dimethoxy and hydrogenated derivatives. Biol. Pharm. Bull., 38, 1478–1483 (2015).
• 8) Niwa T, Yokoyama S, Mochizuki M, Osawa T. Curcumin metabolism by human intestinal bacteria in vitro. J. Funct. Foods, 61, 103463 (2019).
• 9) Changtam C, de Koning HP, Ibrahim H, Sajid MS, Gould MK, Suksamrarn A. Curcuminoid analogs with potent activity against Trypanosoma and Leishmania species. Eur. J. Med. Chem., 45, 941–956 (2010).
• 10) Niwa T, Yokoyama S, Matsugasaki N, Inomata E, Taira A, Osawa T. Stereochemical determination of O-desmethylangolensin produced from daidzein. Food Chem., 171, 153–156 (2015).
• 11) Uehara SI, Yasuda I, Akiyama K, Morita H, Takeya K, Itokawa H. Diarylheptanoids from the rhizomes of Curcuma xanthorrhiza and Alpinia officinarum. Chem. Pharm. Bull., 35, 3298–3304 (1987).
• 12) Ohta S, Aoki T, Hirata T, Suga T. The structures of four diarylheptanoid glycosides from the female flowers of Alnus serrulatoides. J. Chem. Soc. Perkin 1, 1635–1642 (1984).
• 13) Kikuzaki H, Usuguchi J, Nakatani N. Constituents of Zingiberaceae. I. Diarylheptanoids from the rhizomes of ginger (Zingiber officinale ROSCOE). Chem. Pharm. Bull., 39, 120–122 (1991).
• 14) Itokawa H, Morita H, Midorikawa I, Aiyama R, Morita M. Diarylheptanoids from the rhizome of Alpinia officinarum HANCE. Chem. Pharm. Bull., 33, 4889–4893 (1985).
• 15) Sabitha G, Srinivas C, Reddy TR, Yadagiri K, Yadav JS. Synthesis of gingerol and diarylheptanoids. Tetrahedron Asymmetry, 22, 2124–2133 (2011).
• 16) Holder GM, Plummer JL, Ryan AJ. The metabolism and excretion of curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) in the rat. Xenobiotica, 8, 761–768 (1978).
• 17) Jude S, Amalraj A, Kunnumakkara AB, Divya C, Löffler BM, Gopi S. Development of validated methods and quantification of curcuminoids and curcumin metabolites and their pharmacokinetic study of oral administration of complete natural turmeric formulation (Cureit™) in human plasma via UPLC/ESI-Q-TOF-MS spectrometry. Molecules, 23, 2415 (2018).
• 18) Ireson CR, Jones DJL, Orr S, Coughtrie MWH, Boocock DJ, Williams ML, Farmer PB, Steward WP, Gescher AJ. Metabolism of the cancer chemopreventive agent curcumin in human and rat intestine. Cancer Epidemiol. Biomarkers Prev., 11, 105–111 (2002).
• 19) Hoehle SI, Pfeiffer E, Sólyom AM, Metzler M. Metabolism of curcuminoids in tissue slices and subcellular fractions from rat liver. J. Agric. Food Chem., 54, 756–764 (2006).
• 20) Hassaninasab A, Hashimoto Y, Tomita-Yokotani K, Kobayashi M. Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism. Proc. Natl. Acad. Sci. U.S.A., 108, 6615–6620 (2011).
• 21) Collins MD, Jones D, Farrow JAE, Kilpper-Bälz R, Schleifer KH. Enterococcus avium nom. rev., comb. nov.; E. casseliflavus nom. rev., comb. nov.; E. durans nom. rev., comb. nov.; E. gallinarum comb. nov.; and E. malodoratus sp. nov. Int. J. Syst. Bacteriol., 34, 220–223 (1984).
• 22) Kubota H, Tsuji H, Matsuda K, Kurakawa T, Asahara T, Nomoto K. Detection of human intestinal catalase-negative, gram-positive cocci by rRNA-targeted reverse transcription-PCR. Appl. Environ. Microbiol., 76, 5440–5451 (2010).
• 23) Shin NR, Moon JS, Shin SY, Li L, Lee YB, Kim TJ, Han NS. Isolation and characterization of human intestinal Enterococcus avium EFEL009 converting rutin to quercetin. Lett. Appl. Microbiol., 62, 68–74 (2016).
• 24) Maini Rekdal V, Bess EN, Bisanz JE, Turnbaugh PJ, Balskus EP. Discovery and inhibition of an interspecies gut bacterial pathway for Levodopa metabolism. Science, 364, eaau6323 (2019).