Microbial conversion of curcumin into colorless hydroderivatives by the endophytic fungus Diaporthe sp. associated with Curcuma longa

Article abstract of DOI:10.1248/cpb.59.1042

We investigated the microbial conversion of curcumin (1) using endophytic fungi associated with the rhizome of Curcuma longa (Zingiberaceae). We found that Diaporthe sp., an endophytic filamentous fungus, converts curcumin (1) into four colorless derivatives, namely (3R,5R)-tetrahydrocurcumin (2), a novel (3R,5S)-hexahydrocurcumin (3) named neohexahydrocurcumin, (3S,5S)- octahydrocurcumin (4) and meso-octahydrocurcumin (5).

Full text of DOI:10.1248/cpb.59.1042

1042
Note
Chem. Pharm. Bull. 59(8) 1042—1044 (2011)
Vol. 59, No. 8
Microbial Conversion of Curcumin into Colorless Hydroderivatives by the
Endophytic Fungus Diaporthe sp. Associated with Curcuma longa
Shoji MAEHARA,a Michiteru IKEDA,a Hiroyuki HARAGUCHI,b Chinami KITAMURA,a Tetsuro NAGOE,c
a
,a
*
Kazuyoshi OHASHI, and Hirotaka SHIBUYA
b
a Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University; Faculty of Life Science and Biotechnology,
Fukuyama University; 1 Sanzo, Gakuen-cho, Fukuyama, Hiroshima 729–0292, Japan: and c Rohokeimeido Co., Ltd.;
579–25 Kusugawa, Kamiyaku-cho, Kumage-gun, Kagoshima 891–4206, Japan.
Received February 15, 2011; accepted May 30, 2011; published online June 3, 2011
We investigated the microbial conversion of curcumin (1) using endophytic fungi associated with the rhizome
of Curcuma longa (Zingiberaceae). We found that Diaporthe sp., an endophytic filamentous fungus, converts
curcumin (1) into four colorless derivatives, namely (3R,5R)-tetrahydrocurcumin (2), a novel (3R,5S)-hexahydro-
curcumin (3) named neohexahydrocurcumin, (3S,5S)-octahydrocurcumin (4) and meso-octahydrocurcumin (5).
Key words microbial conversion; curcumin; endophytic fungus; Diaporthe species; Curcuma longa
Curcuma longa L. (Zingiberaceae) is a well-known istered as AB537342 in DDBJ/EMBL/GeneBank).
turmeric rhizome whose main chemical constituent curcumin
(1) has a potent antioxidant effect.1) However, it also pos- peptone medium. After cultivation at 28 °C with rotary shak-
sesses a distinct yellow color, which limits its use. ing at 90 rpm for 7 d, a methanol solution of curcumin (1)
CLO-13 was inoculated into 2% glucose-yeast extract-
We have been studying a microbial conversion of natural was added to the culture, which was further cultivated for
products2—4) by hypothesizing that the enzyme system in 2 d. The cultivation medium including the fungal bodies was
cells of endophytic microbes5,6) can be activated to convert homogenized and extracted with chloroform. The chloro-
the chemical constituents of the host plant since endophytic form-soluble portion was separated by silica gel column
microbes live near the chemical constituents of the plant. In chromatography to give a colorless curcuminoid fraction,
this paper, we report on the conversion of curcumin (1) by which was purified by reversed-phase HPLC to afford the
Diaporthe sp., an endophytic fungus isolated from the rhi- following products in their respective yields: 2, 18%; 3, 27%;
zome of Curcuma longa, into four colorless hydroderivatives 4, 8%; and 5, 17%.
including a novel substance, named neohexahydrocurcumin
(3).
Compounds 4 and 5 were white powders and showed a
quasi-molecular ion peak at m/z 375 corresponding to
Thirteen endophytic filamentous fungi (CLO-1—CLO-13) C21H27O6 by negative FAB-MS. From the physicochemical
were obtained from the rhizome of Curcuma longa (Zingi- properties obtained by IR, UV, 1H-NMR, 13C-NMR measure-
beraceae) using a previously reported procedure7,8) and sub- ments, both 4 and 5 were identified as be 1,7-bis(4-hydroxy-
jected to the base sequence analysis of the internal tran- 3-methoxyphenyl)-heptane-3,5-diol (octahydrocurcumin).9)
scribed spacer-1 (ITS-1), 5.8S ribosomal DNA (rDNA), and Furthermore, the optical rotation data showed that absolute
ITS-2 region. The analysis showed that the fungi comprised structure of 4 was (3S,5S)-octahydrocurcumin ([a]D ꢀ8.3°,
three Diaporthe spp. (CLO-3, CLO-4, and CLO-13), five lit.9) [a]D ꢀ7.4°) and 5 was meso-octahydrocurcumin ([a]D
Phomopsis spp. (CLO-6, CLO-9, CLO-10, CLO-11, and ꢁ0°).
CLO-12), two Neofuicoccum spp. (CLO-1 and CLO-8), one
Compound 2, a white powder, showed a quasi-molecular
Botryosphaeria sp. (CLO-7), one Gibberella sp. (CLO-2), ion peak at m/z 371 corresponding to C21H23O6 by negative
and one Sarcinomyces sp. (CLO-5). Then, endophytic mi- FAB-MS. Its IR spectrum indicated the presence of hydroxyl
crobes for the microbial conversion of 1 were selected and aromatic functional groups and its UV spectrum showed
through several preliminary experiments, which led to the se-
lection of the endophytic fungus Diaporthe sp. CLO-13 (reg-
Fig. 2. HPLC Profile of Microbial Conversion Products [2 d after Adding
Curcumin (1)]
HPLC conditions: column, Capcell Pack C18 UG120 (4.6ꢂ250 mm); mobile phase,
CH3CN/H2Oꢃ22 : 78; flow rate, 1.0 ml/min; column temperature, 40 °C; wavelength,
260 nm.
Fig. 1. Chemical Structures of Curcumin (1) and Its Microbial Conversion
Products 2, 3, 4, and 5
∗ To whom correspondence should be addressed. e-mail: shibuya@fupharm.fukuyama-u.ac.jp
© 2011 Pharmaceutical Society of Japan

August 2011
1043
adenine dinucleotide phosphate (NADPH)-dependent peroxidation of micro-
somal lipid and the NADH-dependent peroxidation of microsomal lipid was
carried out as previously reported.12)
Plant Material Curcuma longa L. (Zungiberaceae) was collected on
Yakushima Island, Japan, in March 2007.
Isolation of Endophytic Fungi from the Rhizome of Curcuma longa
The rhizome of Curcuma longa was cut into small pieces and washed under
tap water. The pieces were then successively treated with 75% ethanol for
1 min, 5.3% sodium hypochlorite for 5 min, and 75% ethanol for 0.5 min,
and cut into two pieces. The cut rhizomes were placed on the plating
medium CMMA containing 0.05 mg/ml chloramphenicol and incubated at
27 °C. After 3—5 d, individual colonies were transferred to potato dextrose
agar (PDA) and cultivated again at 27 °C for 5 d with periodic checks for pu-
rity to obtain 13 isolates of endophytic filamentous fungi (CLO-1—CLO-
13), which were then stored on PDA at ꢀ80 °C.
an absorption pattern due to a conjugated phenyl functional
1
group with a double bond. From the H- and 13C-NMR data,
2 was surmised to be the tetrahydro derivative 1,7-bis(4-hy-
droxy-3-methoxyphenyl)-hepta-(1E,6E)-diene-3,5-diol.10)
Furthermore, the absolute configuration of 2 was shown to
be (3R,5R)-tetrahydrocurcumin, because catalytic reduction
afforded (3S,5S)-octahydrocurcumin (4).
Compound 3, colorless needles, showed a quasi-molecular
ion peak at m/z 373 corresponding to C21H25O6 by negative
FAB-MS. The IR and UV spectra indicated that 3 is a cur-
cumin derivative. The 13C-NMR and distortionless enhance-
ment by polarization transfer (DEPT) spectra revealed the
presence of three methylene carbons (4-C, 6-C, and 7-C),
two hydroxylmethine carbons (3-C and 5-C), two olefinic
Microbial Conversion of Curcumin (1) by Diaporthe sp. The endo-
phytic fungus Diaporthe sp. (CLO-13) was inoculated into 2% glucose–yeast
carbons (1-C and 2-C), two methoxyl carbons (3ꢄ-OCH3 and extract–peptone medium [1.0 g of peptone, 0.20 g of yeast extract, 4.0 g of
1
glucose, 0.10 g each of K2HPO4 and MgSO4·7H2O, 2.0 mg of FeSO4·7H2O,
0.20 g of CaCO3 and 200 ml of tap water] in a 500-ml Erlenmeyer flask (ꢂ5)
and then cultivated with shaking at 90 rpm at 28 °C. After 7 d, curcumin (1,
10 mg in 10 ml of methanol, totally 50 mg) was added into each flask. After
3ꢅ-OCH3) and twelve aromatic carbons. The H-NMR spec-
tra showed the signals of two trisubstituted aromatic rings
bearing a hydroxyl functional group, a methoxyl functional
group and a carbon chain moiety. From these results, com-
bined with the 2D-NMR spectra [1H–1H correlation spec-
troscopy (COSY) and heteronuclear multiple bond correla-
tion (HMBC)], 3 was presumed to be a new colorless hexa-
hydro derivative, named neohexahydrocurcumin.
The absolute structure was determined by two following
methods. Firstly, the catalytic reduction of 3 furnished
(3S,5S)-octahydrocurcumin (4),9) which evidence showed
further cultivation at 28 °C for 2 d, the cultivation medium including the fun-
gal bodies was homogenized and extracted with CHCl3. The CHCl3 layer
was concentrated under reduced pressure to give a colorless curcuminoid
fraction, which was purified by silica gel column chromatography (n-
hexane/CHCl3/EtOHꢃ2 : 18 : 1) and reversed-phase HPLC (Capcell Pak
C18, CH3CN/H2Oꢃ23 : 77) to afford the products 2 (9.0 mg, 18%), 3
(13.5 mg, 27%), 4 (4.0 mg, 8%) and 5 (8.5 mg, 17%).
3 (neohexahydrocurcumin): Colorless needles, mp 157—158 °C from n-
hexane/EtOAc, [a]D ꢀ4.3° (cꢃ1.0, in EtOH at 24 °C). IR (KBr) cmꢀ1
:
3470, 3350, 2837, 1599, 1036. UV (EtOH) nm (e): 269 (15,000), 288 (sh),
the absolute structure of
3 was 1,7-bis(4-hydroxy-3-
1
302 (sh), 313 (sh). H-NMR (500 MHz, CD3OD) d: 1.53—1.69 (4H, m, 4-
methoxyphenyl)-1E-heptene-3R,5S-diol. In addition, the
modified Mosher’s method11) was applied to the 3-keto-5-ol
derivative, which was prepared from 3 by methylation and
manganese oxide oxidation. The Dd values (shown in Fig. 4)
obtained by subtracting chemical shifts of the (R)-2-
methoxy-2-trifluoromethylphenylacetic acid (MTPA) esters
(6b) from those of the (S)-MTPA ester (6a) supported 5S- 41.1 (6-C), 45.8 (4-C), 56.3 (3ꢄ-, 3ꢅ-OCH3), 68.5 (5-C), 70.6 (3-C), 110.5
configuration.
Finally, the colorless products (2—5) inhibited the peroxi-
dation of microsomal and mitochondrial lipids prepared from
H2, 6-H2), 2.49 (1H, ddd, Jꢃ7.3, 8.5, 14.0 Hz, 7-Ha), 2.60 (1H, ddd, Jꢃ6.1,
8.5, 14.0 Hz, 7-Hb), 3.75 (1H, overlapped with the signal of 3ꢅ-OMe, 5-H),
3.75 (3H, s, 3ꢅ-OMe), 3.77 (3H, s, 3ꢄ-OMe), 4.33 (1H, m, 3-H), 5.97 (1H,
dd, Jꢃ6.7, 15.9 Hz, 2-H), 6.34 (1H, d, Jꢃ15.9 Hz, 1-H), 6.53 (1H, dd,
Jꢃ1.8, 7.9 Hz, 6ꢅ-H), 6.58 (1H, d, Jꢃ7.9 Hz, 5ꢅ-H), 6.63 (1H, d, Jꢃ8.2 Hz,
5ꢄ-H), 6.67 (1H, d, Jꢃ1.8 Hz, 2ꢅ-H), 6.71 (1H, dd, Jꢃ1.8, 8.2 Hz, 6ꢄ-H),
6.86 (1H, d, Jꢃ1.8 Hz, 2ꢄ-H). 13C-NMR (125 MHz, CD3OD) d: 32.5 (7-C),
(2ꢄ-C), 113.2 (2ꢅ-C), 116.1 (5ꢄ-C), 116.2 (5ꢅ-C), 120.9 (6ꢄ-C), 121.8 (6ꢅ-C),
130.6 (1ꢄ-C), 130.7 (1-C), 131.2 (2-C), 135.2 (1ꢅ-C), 145.4 (4ꢅ-C), 147.4
(4ꢄ-C), 148.8 (3ꢅ-C), 149.0 (3ꢄ-C). Negative FAB-MS m/z: 373 [MꢀH]ꢀ.
High-resolution negative FAB-MS m/z: 373.1663 (Calcd for C21H25O6:
373.1652 [MꢀH]ꢀ). The physicochemical data for 2, 4 and 5 were identical
rat liver, as shown in Table 1.
to the data previously reported in literatures.9,10)
Experimental
Catalytic Reduction of 2 To a solution of 2 (1.2 mg) in MeOH (0.5 ml)
was added 5% palladium on carbon (3 mg). The mixture was then stirred
under a hydrogen atmosphere at room temperature for 24 h. The catalyst was
The instruments used to determine the physical properties of curcumin
and its microbial conversion products, and the experimental conditions used
for chromatography, were the same as those in our previous paper.4) DNA
extraction, polymerase chain reaction (PCR) amplification, and DNA se-
quencing were carried out using the same primer by the same procedure de-
scribed in our previous paper.7,8) The bioassay for the reduced nicotinamide
Table 1. Antioxidant Activities of Curcumin and Its Microbial Conversion
Products
IC50 (mg/ml)
Mitochondria
Microsome
Curcumin (1)
8.9
6.8
8.9
13.4
13.5
30.1
7.0
12.5
27.6
28.1
2
3
4
5
Fig. 3. 1H–1H COSY and HMBC Correlations for 3 (Neohexahydro-
curcumin)
Fig. 4. Modified Mosher’s Method Results for 6a and 6b
The Dd values [d(S)-MTPA of 6aꢀd(R)-MTPA of 6b] are shown.

1044
Vol. 59, No. 8
removed by filtration, and the filtrate was concentrated in vacuo. The residue
was purified by column chromatography (SiO2 1 g, CHCl3/MeOHꢃ20 : 1) to
afford (3S, 5S)-octahydrocurcumin 4 [1.0 mg, [a]D ꢀ7.5° (cꢃ0.08, in EtOH
at 24 °C)].
Catalytic Reduction of 3 To a solution of 3 (3.0 mg) in MeOH (0.5 ml)
was added 5% palladium on carbon (5 mg). The mixture was then stirred
under a hydrogen atmosphere at room temperature for 12 h. The catalyst was
removed by filtration, and the filtrate was concentrated in vacuo to give a
residue, which was purified by column chromatography (SiO2 1 g,
CHCl3/MeOHꢃ20 : 1) to furnish (3S, 5S)-octahydrocurcumin 4 [2.8 mg,
[a]D ꢀ8.7° (cꢃ0.25, in EtOH at 24 °C)].
H), 6.54 (1H, d, Jꢃ16.0 Hz, 2-H), 6.68 (1H, d, Jꢃ1.8 Hz, 2ꢅ-H), 6.69 (1H,
dd, Jꢃ1.8, 7.9 Hz, 6ꢅ-H), 6.78 (1H, d, Jꢃ7.9 Hz, 5ꢅ-H), 6.88 (1H, d,
Jꢃ8.2 Hz, 5ꢄ-H), 7.03 (1H, d, Jꢃ2.0 Hz, 2ꢄ-H), 7.10 (1H, dd, Jꢃ2.0, 8.2 Hz,
6ꢄ-H), 7.37—7.39 (3H), 7.46 (1H, d, Jꢃ16.0 Hz, 1-H), 7.53—7.56 (2H).
FAB-MS m/z: 617 [MꢆH]ꢆ. High-resolution FAB-MS m/z: 617.2361
(Calcd for C33H36F3O8: 617.2362 [MꢆH]ꢆ). The Dd values [d(S)-MTPA of
6aꢀd(R)-MTPA of 6b] are shown in Fig. 4.
Acknowledgments This work was supported by the Strategic Support
Project of Research Infrastructure Formation for Private Universities from
the Ministry of Education, Culture, Sports, Science and Technology of
Japan.
Preparation of 6a and 6b from 3 A solution of 3 (4.0 mg) in ben-
zene/MeOH (4 : 1, 1.0 ml) was treated with 10% trimethylsilyldiazomethane
in n-hexane (0.5 ml) at room temperature for 1 h. The solvent was evapo-
rated off under reduced pressure to yield a product. To a solution of the
product in 1.0 ml of CH2Cl2 was added active MnO2 (5 mg). The whole mix-
ture was stirred at room temperature for 2 h. The MnO2 was removed by fil-
tration, and the filtrate was concentrated in vacuo. The residue was purified
by column chromatography (SiO2 1 g, CHCl3/MeOHꢃ20 : 1) to afford the 3-
keto-5-ol derivative (3.2 mg). The 3-keto-5-ol derivative (1.0 mg) was treated
with a solution of dicyclohexylcarbodiimide (DCC, 1.9 mg), (S)- or (R)-
MTPA (each 1.7 mg) and 4-dimetylaminopyridine (DMAP, 0.8 mg) in
CH2Cl2 (0.1 ml). The reaction mixture was stirred at room temperature for
8 h and the solvent removed in vacuo. The residue was purified by column
chromatography (SiO2 1 g, n-hexane/EtOAcꢃ3 : 2) to afford the (S)-MTPA
ester (6a, 1.0 mg) and the (R)-MTPA ester (6b, 1.2 mg), respectively.
6a: [a]D ꢆ15.0° (cꢃ0.053, in EtOH at 24 °C). IR (KBr) cmꢀ1: 1746,
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