Secondary Metabolites from the Mycelia of the Fungus Monascus pilosus BCRC 38072 

Monascus has been used in oriental fermented foods

for thousands of years.

1)

_{Red mold rice fermented with}

Monascus spp. produces bioactive metabolites such as

g-aminobutyric acid (GABA), polyketides monacolin K, and

some pigments, which, respectively, function as an

anti-hy-pertension agent,

2)

_{a cholesterol-lowering drug,}

3—5)

_and

pos-sess antibacterial activity.

6)

_{Monascus pigments, secondary}

metabolites possessing mainly azaphilone skeletons, have

traditionally been used as natural food colorants.

1)

Many

other metabolites have also been reported in previous

re-search,

7—15)

_{most of them isolated from red mold rice}

ob-tained from solid fermentation. In contrast, the metabolites

contained in the mycelia pellets from submerged cultures

have rarely been investigated. The antioxidant effects

exhib-ited in the methanol extract fraction of the mycelia pellets

of M. pilosus BCRC 38072 were monitored using the

2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging

method.

16,17)

_{Subsequent chemical examination of the methanol}

extract of mycelia from M. pilosus led to the isolation of

two new phenylacetic acid derivatives, monaspilosin (1)

and monaspiloindole (2), and one new pyranoindole alkaloid,

monaspyranoindole (3), along with twelve known

com-pounds. This paper reports on the isolation, structural

eluci-dation and the DPPH free radical scavenging activity of these

fungal metabolites.

Results and Discussion

Extensive chromatographic purification of the

EtOAc-sol-uble fraction of the MeOH extract of the pellets of M. pilosus

afforded fifteen compounds. The new compound 1, isolated

as colorless oil, was assigned the molecular formula

C

16

H

16

O

3

Na by ESI-MS ([M

Na]

, m/z 279) and

HR-ESI-MS. IR absorptions were observed at 3403, 1712, 1612,

1514 cm

1

pointing to the presence of hydroxyl, an ester

car-bonyl group, and a benzene ring. The UV spectrum showed

maximum absorption at 277 nm, and a bathochromic shift in

alkaline solution indicated the presence of a phenol

deriva-tive.

18)

_{This was confirmed by the}

1

_{H-NMR spectrum, which}

showed one proton at d

_H

4.90 (1H, br s) assigned to OH-15,

which disappeared upon addition of D

2

O. The

1

H-NMR

spectrum of 1 showed a mono-substituted phenyl moiety at

d

H

7.09 (5H, m, H-2—6). An AA

XX pattern at d

H

6.72,

7.00 (each 2H, d, J

8.8 Hz, H-14, 16 and H-13, 17)

sug-gested a 1,4-disubstituted benzene ring in 1. In addition, the

1

H-NMR and HSQC spectra revealed the presence of a

ben-zylic methylene group [d

H

3.60 (2H, s)]. An oxymethylene

unit [d

_H

4.26 (2H, t, J

7.0 Hz, H-10)] split into a triplet due

to coupling with another methylene group [d

_H

2.84 (2H, t,

J

7.0 Hz, H-11)]. The above signals and the COSY

spec-trum established the presence of the partial substructures:

fragments, 1a, 1b, and 1c, for compound 1. The entire

skele-ton of 1 was constructed from the HMBC spectrum (Fig. 2).

The

2

J and

3

J correlations of the signal at

d

_H

3.60 (H-7) and

d

H

4.26 (H-10), with the carbon signal at d

C

171.6 (C-8),

helped to establish the connections of fragments 1a and 1b

with the carbonyl group at C-8. That is, fragments 1a and 1b

can be connected to produce 1d. In addition, the cross peak

between

d

H

2.84 (H-11) and d

C

130.0 (C-13, 17), as well as

d

_H

4.26 (H-10) and d

_C

129.6 (C-12), suggest that fragments

1c and 1d were linked together at C-11. The structure was

further confirmed by

13

C-NMR, DEPT, COSY, NOESY (Fig.

1), HSQC, and HMBC (Fig. 2) experiments. Thus, the

struc-ture of 1 was determined to be a phenylacetic acid

2-(4-hy-droxyphenyl)ethyl ester, and was given the name

monaspi-losin.

Compound 2, also a colorless oil, was assigned the

molec-ular formula C

₁₈

H

₁₇

NO

₂

, as deduced by ESI-MS and

HR-ESI-MS. The presence of an ester group was revealed by an

394 Vol. 56, No. 3

Secondary Metabolites from the Mycelia of the Fungus Monascus pilosus

BCRC 38072

Ming-Jen C

HENG

,

a

Ming-Der W

U

,

a

Ih-Sheng C

HEN

,

b,#

and Gwo-Fang Y

UAN

*

,a

a_{Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute; Hsinchu 300,}

Taiwan, Republic of China: and b_{College of Pharmacy, Kaohsiung Medical University; Kaohsiung 807, Taiwan, Republic}

of China. Received November 12, 2007; accepted December 11, 2007; published online December 13, 2007

Three new compounds, including two phenylacetic acid derivatives, monaspilosin (1) and monaspiloindole (2), and one pyranoindole alkaloid, monaspyranoindole (3), were isolated from the EtOAc-soluble fraction of the MeOH extract of the mycelia of Monascus pilosus BCRC 38072. Twelve known compounds were also obtained in this study. The structures were elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. This is the first report of Monascus metabolites with an indole ring. All isolates were also evaluated for their scavenging properties toward the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in TLC autographic and spectroscopic as-says.

Key words Monascus pilosus; Eurotiaceae; phenylacetic acid; pyranoindole; 2,2-diphenyl-1-picrylhydrazyl Chem. Pharm. Bull. 56(3) 394—397 (2008)

Notes

© 2008 Pharmaceutical Society of Japan ∗ To whom correspondence should be addressed. e-mail: [email protected]

IR absorption at 1716 cm

1

and by a resonance signal in the

13

_{C-NMR spectrum at d}

C

171.3. The presence of a NH group

in the molecule was revealed by a band at 3410 (br) cm

1

in

the IR spectrum, which was confirmed by the signal at d

_H

8.00 (1H, br s), which disappeared upon addition of D

2

O.

The UV spectrum showed a maximum absorption at 280 nm,

indicating the presence of a phenylacetic acid skeleton.

18)

The

1

H-NMR spectrum of compound 2 was similar to the

above compound, monaspilosin (1), except that the

substitu-tent at C-11 in 2 was a 1H-indol-3-yl moiety in place of the

p-hydroxyphenyl group in 1. Signals for the indol-3-yl

moi-ety appeared at d

_H

7.13 (1H, br t, J

7.8 Hz, H-18), 7.20 (1H,

br t, J

7.8 Hz, H-17), 7.36 (1H, br d, J7.8 Hz, H-16), and

7.61 (1H, br d, J

7.8 Hz, H-19), suggesting four coupling

aromatic protons along with a nitrogen-bearing olefinic

pro-ton at d

_H

6.93 (1H, t, J

1.8 Hz, H-13). The above

assign-ments were verified by NOESY correlations between d

H

3.09

(H-11)/d

_H

6.93 (H-13) and d

_H

7.61 (H-19), respectively. The

olefinic proton signal at d

_H

6.93 (H-13) was correlated with a

methylene carbon at d

_C

24.7 (C-11), as well as d

_H

4.38

(H-10) and d

C

112.0 (C-12) from the HMBC spectrum,

suggest-ing that the indole moiety linked at C-11. The structure was

further confirmed by

13

C-NMR, DEPT, COSY, NOESY (Fig.

2), HSQC, and HMBC (Fig. 3) experiments. Thus, the

struc-ture of 2 was determined to be a phenylacetic acid

2-(1H-indol-3-yl)ethyl ester, and designated monaspiloindole.

Com-pound 2 was first isolated from a natural source, although it

has been mentioned by Yamamoto as a reactant for

synthe-sizing a new class of anti-methicillin-resistant

Staphylococ-cus aureus (anti-MRSA) and anti-vancomycin-resistant

en-terococci (anti-VRE) agents.

19)

Compound 3 was also isolated as colorless oil. The

HR-ESI-MS spectrum gave a molecule ion [M

Na]

at

m/z 224.3139, consistent with a molecular formula of

C

13

H

15

NONa. UV spectrum showed maximum absorption at

288 nm, indicating the presence of an indole skeleton.

18)

Its

IR spectrum revealed NH absorption at 3320 cm

1

. Analysis

of the

1

_{H-NMR spectrum of 3 revealed four typical mutually}

coupling aromatic protons of indole alkaloid at d

_H

7.11 (1H,

td, J

7.6, 1.0 Hz, H-7), 7.17 (1H, td, J7.6, 1.0 Hz, H-6),

7.33 (1H, dd, J

7.6, 1.0 Hz, H-5), 7.49 (1H, dd, J7.6,

1.0 Hz, H-8) and one NH group at d

_H

7.66 (1H, br s,

ex-changeable with D

2

O). In addition, the appearance of a set of

A

2

X

2

pattern signals at d

H

2.80 (2H, t, J

5.4 Hz, H-4) and

4.05 (2H, t, J

5.4 Hz, H-3), accompanied by a singlet of two

methyl groups at d

_H

1.57 (6H, s, CH

₃

-9, 10). The HMBC

correlations (Fig. 3) from d

_H

1.57 (CH

3

-9, 10) to d

C

138.9

(C-1a); d

_H

2.80 (H-4) to d

_C

106.9, 138.9 (C-4a, 1a), and d

_H

4.05 (H-3) to d

C

106.9 (C-4a) verify the junction of the

1,1-dimethyldihydropyrano ring to the indole moiety at C-1a, and

4a. The other key correlations of HMBC are illustrated in

Fig. 3. Based on the above data, the structure of 3, named

monaspyranoindole, was elucidated as

1,1-dimethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole, which was further confirmed

by

13

C-NMR, COSY, NOESY (Fig. 2), HSQC and HMBC

(Fig. 3) experiments. Compound 3 was first isolated from a

natural source, though it has since been synthesized.

20)

The other known isolates, b-sitosteryl stearate,

21)

_a

mix-ture of b-sitosterol and stigmasterol,

22)

ergosterol,

23)

p-hy-droxybenzoic acid,

24)

methylparaben,

25)

trans-caffeic acid,

26)

linoleic acid,

27)

_cyclo-(

L

-Pro-

L

-Tyr),

28)

5-(hydroxymethyl)fur-fural,

29)

_{(Z)-pulchellalactam,}

30)

_and

(4R,5S)-5-hydroxyhexan-4-olide,

31)

were readily identified by comparison of their

physico-chemical, spectroscopic, and mass-spectrometric

data with the literature. Except for linoleic acid, this is the

first time any of the compounds described above have been

isolated from Monascus spp. The radical-scavenging

proper-ties of the fifteen compounds were evaluated against the

DPPH radical.

16)

_{By using DPPH as a TLC spray reagent,}

p-hydroxybenzoic acid and trans-caffeic acid (5, 10

mg)

ap-peared as strong yellow spots against a purple background,

while compound 1 displayed moderate yellow spots, and

March 2008 395

Fig. 1. New Compounds Isolated from Monascus pilosus

Fig. 2. NOESY Interactions of Monaspilosin (1), Monaspiloindole (2), and Monaspyranoindole (3)

other compounds did not react with the radical. The phenol

derivatives p-hydroxybenzoic acid and trans-caffeic acid

were more active than 1 in the above concentration range.

The free radical scavenging effects of the isolates (compound

1, p-hydroxybenzoic acid and trans-caffeic acid),

correspond-ing to the intensity of quenchcorrespond-ing of the DPPH radical, were

evaluated by spectroscopic assay. At a concentration of

50

m

M

, the test compounds showed moderate DPPH radical

scavenging activity, with 40, 65, and 87% inhibition for

com-pound 1, p-hydroxybenzoic acid and trans-caffeic acid,

re-spectively, with trans-caffeic acid the most active compound

in this study.

In summary, most of recent studies on secondary

metabo-lites from Monascus have investigated red mold rice and

whole broth. These metabolites are azaphilone,

15)

fura-noisophthalides,

15)

amino acid,

13,15)

polyketides,

1)

and fatty

acids.

1)

_{Nevertheless, the chemical characteristics, as well as}

the biological activities, of many Monascus metabolites still

remain unclear. In this study we focus on the secondary

metabolites appearing in the mycelia of Monascus

sub-merged culture, which has seldom been reported on. The

three metabolites 1, 2 and 3, phenyl ester, indole, and

pyra-noindole alkaloid found in this study are new, naturally

oc-curring compounds. Interestingly, this is the first report of an

indole alkaloid isolated from this Monascus spp. Compound

1, p-hydroxybenzoic acid and trans-caffeic acid were found

to have significant antioxidant properties, as determined by

experiments with 2,2-diphenyl-1-picrylhydrazyl (DPPH),

which suggests their ability to efficiently scavenge free

radi-cals. Compounds 1 and 2 have similar structures, but the

lat-ter does not show the ability to efficiently scavenge free

radi-cals, which implies that the presence of a phenol group is

crucial for activity. The hydrogen donating ability was

asso-ciated with the radical scavenging effects of antioxidants on

the DPPH radical. Structures containing phenolic hydroxyl

and carboxylic acid moieties have long been recognized to

function as electron or hydrogen donors. Thus, the higher

DPPH radical scavenging activity of p-hydroxybenzoic acid

and trans-caffeic acid may be related to the phenolic and

car-boxylic acid groups present in the molecules. These results

suggest that Monascus has distinct and diverse metabolites

which arise under different fermentation conditions. It may

therefore be possible to find more new bioactive natural

products by cultivating Monascus under different conditions.

Experimental

General All melting points were determined on a Yanaco micro-melting point apparatus and were uncorrected. Optical rotations were measured on a Jasco P-1020 digital polarimeter, UV spectra were obtained on a Jasco UV-240 spectrophotometer in MeOH, and IR spectra (KBr or neat) were taken on a Perkin-Elmer System 2000 FT-IR spectrometer. 1D (1_H, 13_{C, DEPT)} and 2D (COSY, NOESY, HSQC, HMBC) NMR spectra using CDCl3and CD3OD as solvent were recorded on a Varian Unity Plus 400 (400 MHz for 1_{H-NMR, 100 MHz for} 13_{C-NMR) and Varian INOVA-500 (500 MHz for} 1_{H-NMR, 125 MHz for} 13_{C-NMR) spectrometer. Chemical shifts were} inter-nally referenced to the solvent signals in CDCl3(

1_H,

d 7.26; 13_C, d 77.0) with Tetramethylsilane (TMS) as the internal standard. Low-resolution ESI-MS spectra were obtained on an API 3000 (Applied Biosystems) and high-resolution ESI-MS spectra on a Bruker Daltonics APEX II 30e spectrometer. Low-resolution EI-MS spectra were recorded on a Quattro GC/MS spec-trometer having a direct inlet system. Silica gel (70—230, 230—400 mesh) (Merck) was used for column chromatography, and silica gel 60 F-254 (Merck) was used for TLC and prep. TLC. For radical scavenging TLC au-tographic assay, DPPH (Sigma) was used as spray reagent.

Microorganism Monascus pilosus BCRC 38072 was used throughout

this study, and specimens deposited at the Bioresource Collection and Re-search Center (BCRC) of the Food Industry ReRe-search and Development In-stitute.

396 Vol. 56, No. 3

Table 1. 1_{H- and} 13_{C-NMR Data for Compounds 1—3 in CDCl}

3(400 MHz)

1 2 3

No.

dC dH(mult; J, Hz) dC dH(mult; J, Hz) dC dH(mult; J, Hz)

1 133.9 — 134.1 — 71.8 — 1a — — — 138.9 — 2 129.3 7.09 (m) 129.3 7.30 (m) — 3 127.1 7.09 (m) 127.0 7.30 (m) 60.5 4.05 (t, 5.4) 4 128.5 7.09 (m) 128.5 7.30 (m) 22.4 2.80 (t, 5.4) 4a — — — 106.9 — 5 127.1 7.09 (m) 127.0 7.30 (m) 110.8 7.33 (dd, 7.6, 1.0) 5a — — — 127.0 — 6 129.3 7.09 (m) 129.3 7.30 (m) 121.7 7.17 (td, 7.6, 1.0) 7 41.4 3.60 (s) 41.5 3.63 (s) 119.6 7.11 (td, 7.6, 1.0) 8 171.6 — 171.6 — 118.3 7.49 (dd, 7.6, 1.0) 8a — — — 135.7 — 9 — — — 27.9 1.57 (s) 10 65.6 4.26 (t, 7.0) 65.0 4.38 (t, 7.0) 27.9 1.57 (s) 11 34.1 2.84 (t, 7.0) 24.7 3.09 (td, 7.0, 1.8) — — 12 129.6 — 112.0 — — — 13 130.0 7.00 (d, 8.8) 122.0 6.93 (t, 1.8) — — 14 115.3 6.72 (d, 8.8) — — — 15 154.2 — 136.1 — — — 16 115.3 6.72 (d, 8.8) 111.1 7.36 (br d, 7.8) — — 17 130.0 7.00 (d, 8.8) 122.1 7.20 (br t, 7.8) — — 18 — — 119.4 7.13 (br t, 7.8) — — 19 — — 118.8 7.61 (br d, 7.8) — — 20 — — 127.4 — — — OH-15 — 4.90 (br s) — — — NH — — 8.00 (br s) — 7.66 (br s)

Media The inoculum’s medium contained: malt extract, 3 g; yeast extract, 3 g; glucose, 5 g; agar, 1.5 g; and distilled water 1 l. The initial pH of the medium was 8. The synthetic culture medium contained: glucose, 20 g; monosodium L-glutamate (MSG), 10 g; K2HPO4, 5 g; KH2PO4, 5 g; MgSO4· 7H2O, 1.0 g; KCl, 0.5 g; ZnSO4· 7H2O, 0.01 g; FeSO4· 7H2O, 0.01 g; and MnSO4· H2O, 0.003 g per liter of distilled water. The initial pH of the medium was adjusted to 5.5.

Cultivation Methods The slant culture was kept on PDA (potato dex-trose agar) Difco. Spores of strains were prepared by growth on PDA slants for 14 d at 28 °C. Spores were washed with sterile water. A suspension of 107_{spores was used to incubate a 5 l Erlenmeyer flask containing 2 l} inocu-lum medium, which was incubated at 28 °C on a rotary shaker for 3 d. This inoculum was then transferred to a 50 l fermentor (B. Braun, Germany) con-taining 30 l of synthetic medium, operated at 100 rpm and 30 °C, with an aeration rate of 0.3 vvm. After 14 d of cultivation, the pellet mycelia har-vested from the culture broth were used as samples for further extraction.

Extraction and Isolation The dried mycelia of the M. pilosus BCRC 38072 (2 kg) were extracted three times with MeOH at room temperature. The methanol syrup extract was partitioned between EtOAc and H2O (1 : 1) to afford EtOAc (2.5 g) and H2O (10.2 g) soluble fractions. The EtOAc-solu-ble fraction (2.5 g) was chromatographed over silica gel (75 g, 70—230 mesh), eluting with n-hexane and enriched with EtOAc to produce ten frac-tions (A1—A10). Fraction A4 (0.56 g) was subjected to a silica gel (18 g) chromatography by eluting with n-hexane–EtOAc (100 : 1), enriched with EtOAc, to furnish 20 fractions (A4-1—A4-20). Fraction A4-11 (21.0 mg) was purified by preparative TLC (n-hexane–EtOAc, 5 : 1) to give monaspyra-noindole (3) (1.3 mg), (4R,5S)-5-hydroxyhexan-4-olide (6.2 mg), and ergo-sterol (2.9 mg). Fraction A5 (125.5 mg) was subjected to a silica gel (3 g) chromatography by eluting with n-hexane–EtOAc (10 : 1), enriched with EtOAc, to furnish 8 fractions (A5-1—A5-8). Fraction A5-4 (7.5 mg) was pu-rified by preparative TLC (n-hexane–EtOAc, 15 : 1) to furnish monaspilosin (1) (2.0 mg). Fraction A5-5 (12.5 mg) was purified by preparative TLC (n-hexane–EtOAc, 5 : 1) to yield b-sitosteryl stearate (4.0 mg), and linoleic acid (9.8 mg). Fraction A6 (214.3 mg), eluting with n-hexane–acetone (20 : 1), was further separated using silica gel column chromatography and prepara-tive TLC (n-hexane–EtOAc (5 : 1)) and gave monaspiloindole (2) (2.7 mg), (Z)-pulchellalactam (21.7 mg), and a mixture of b-sitosterol and stigmasterol (8.8 mg). Fraction A7 (312.4 mg) was repeatedly chromatographed over sil-ica gel and purified by preparative TLC to afford p-hydroxybenzoic acid (4.2 mg), 5-(hydroxymethyl)furfural (14.1 mg), and methylparaben (2.5 mg). Fraction A9 (290.3 mg) was chromatographed on a silica gel (9 g) column, eluting with CHCl3–EtOAc (20 : 1) to yield 12 fractions (A9-1—A9-12). Fraction A9-1 (26.2 mg) was subjected to further silica gel column chro-matography and purified by preparative TLC to afford trans-caffeic acid (3.2 mg) and cyclo-(L-Pro-L-Tyr) (4.1 mg).

Monaspilosin (1): Colorless oil. 1_{H-NMR (CDCl}

3, 400 MHz) and 13 C-NMR (CDCl3, 100 MHz): see Table 1. IR (Neat) cm1: 3403 (OH), 1712 (CO), 1612, 1514 (aromatic ring CC stretch). UV lmax (MeOH) nm (loge): 277 (3.42). HR-ESI-MS m/z 279.0997 [M
Na]
_{(Calcd for} C16H16O3Na, 279.0995). ESI-MS m/z 279 [M
Na]
.

Monaspiloindole (2): Colorless oil. 1_{H-NMR (CDCl}

3, 400 MHz) and 13 C-NMR (CDCl3, 100 MHz): see Table 1. IR (Neat) cm1: 3410 (OH), 1716 (CO), 1617, 1517 (aromatic ring CC stretch). UV lmax (MeOH) nm (loge): 280 (3.74). HR-ESI-MS m/z 302.1159 [M
Na]
(Calcd for C18H17NO2Na, 302.1157). ESI-MS m/z 302 [M
Na]
.

Monaspyranoindole (3): Colorless oil. 1_{H-NMR (CDCl}

3, 400 MHz) and 13_{C-NMR (CDCl}

3, 100 MHz): see Table 1. IR (Neat) cm1: 3320 (NH). UV lmax(MeOH) nm (loge): 288 (3.60). HR-ESI-MS m/z 224.3137 [M
Na]
(Calcd for C13H15NONa, 224.3139). ESI-MS m/z 224 [M
Na]
.

Reduction of DPPH Radical After developing and drying, TLC plates were sprayed with a 0.2% DPPH (Aldrich-Sigma) solution in MeOH. The plates were examined 30 min after spraying. Compounds showing a yellow-on-purple spot were regarded as having antioxidant qualities.16)_{The intensity} of the yellow color depends upon the amount and nature of radical scavenger present in the sample.

Determination of the Scavenging Effect on DPPH Radical The radi-cal scavenging activity of the test compounds was examined with the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, as described previously.17) a-Toco-pherol (vitamin E) (Sigma) was used as control. Fifty microliters of a solu-tion containing the different compounds (final concentrasolu-tion was 50mM) to be tested was added to 5 ml of a 1.0104MMeOH solution of DPPH. The

reaction mixture was shaken vigorously, and its absorbance at 517 nm was determined after 30 min incubation in a dark area. Decreasing DPPH solu-tion absorbance indicates an increase in DPPH radical-scavenging activity. The DPPH solution, without sample solutions, was used as a control. All tests were run in triplicate and averaged. This activity is given as % DPPH radical-scavenging and is calculated in the equation: % DPPH radical-scav-enging(control absorbancesample absorbance/control absorbance)100.

Acknowledgements Support from the Ministry of Economic Affairs, Taiwan ROC, (Grant No. 94-EC-17-A-17-R7-0563) to the Food Industry Research and Development Institute (FIRDI) is appreciated.

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