Appl Microbiol Biotechnol (2007) 73:11971202

DOI 10.1007/s00253-006-0578-0

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Isolation and characterization of dihydromonacolin-MV

from Monascus purpureus for antioxidant properties
Mohan A. Dhale & S. Divakar & S. Umesh Kumar &
G. Vijayalakshmi

Received: 8 May 2006 / Revised: 26 June 2006 / Accepted: 10 July 2006 / Published online: 17 October 2006
# Springer-Verlag 2006

Abstract The methanolic extract of Monascus purpureus

cultivated by solid-state fermentation on rice showed strong
2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
activity and better yield as compared to other polarity based
extracted fractions. It was selected for further purification
of the antioxidant. The activity-guided repeated fractionation of methanolic extract on a silica gel column
chromatography yielded a compound that exhibited strong
antioxidant activity. Based on the spectroscopic analysis by
UV, IR, 1H NMR, 13C NMR, 2D-HSQCT NMR, and MS,
the antioxidant isolated was elucidated as a derivative of
dihydromonacolin-K, where the ester group is 2-methyl
propionate, designated as dihydromonacolin-MV. The
DPPH radical was significantly scavenged by the dihydromonacolin-MV (IC50 20T1 g mlj1). The dihydromonacolin-MV showed strong inhibition of lipid peroxidation in a
liposome model with an IC50 value of 5.71T0.38 g mlj1
and superoxide radical scavenging activity with an IC50
value of 163.97T2.68 g mlj1.
Keywords Monascus purpureus . Dihydromonacolin-MV .
Antioxidant . DPPH lipid peroxidation .
Superoxide radical

M. A. Dhale : S. U. Kumar : G. Vijayalakshmi (*)

Department of Food Microbiology,
Central Food Technological Research Institute,
Mysore 570020, India
e-mail: vij19_99@yahoo.com
S. Divakar
Department of Fermentation Technology and Bioengineering,
Central Food Technological Research Institute,
Mysore 570020, India

Introduction
Free radicals are highly reactive, short-lived, toxic molecules that have one or more unpaired electrons and can
damage DNA, proteins, lipids, and carbohydrates (Baskar
et al. 2004) within the tissue. The main cause of mortality
and morbidity in the western world is atherosclerosis, the
accumulation of oxysterol, cholesterol, and peroxide lipids
in arteries, generated by free radicals which lead to heart
attack. Hence, there has been an increased interest in the
application of antioxidants to medical treatment as information are constantly gathered, linking the development of
human diseases to oxidative stress (Vaya and Aviram 2001)
that leads to the generation of free radicals. The effect of
dietary antioxidants on the development of human atherosclerosis is also controversial and a number of contradictory
examples have been published. Most of the research on the
role of antioxidants in cardiovascular diseases has focused
on testing pure compound to prevent lipid peroxidation by
examining its ability to scavenge free radicals. However,
antioxidant contribution in vivo goes far beyond scavenging
free radicals. Moreover, single antioxidants are usually not
present alone in biological systems but act in combination
with other antioxidants. Hence the protective effect of a diet
is not equivalent to the protective effect of antioxidants in it
(Helliwell 2000).
Korantzopoulos (2004) reported that antioxidant effects
of statins might extend beyond atherosclerosis and potential
benefit for atrial fibrillation and heart failure. Red yeast rice
obtained by fermentation with M. purpureus produces
various secondary metabolites and is a common food item
found in China. It was used for many centuries to enhance
the colour and flavour of food, as well as a traditional
medicine for digestive and vascular functions (Ma et al.
2000). The monacolins from Monascus sp (Heber et al.

1198

1999) and dihydromevinolin (Albers-Schonberg et al. 1981)

are reported to exhibit a cholesterol lowering action by
inhibiting the HMG-CoA reductase as similar to the
commercial statin drug. Till date, fourteen different types
of monacolins compounds have been identified in Monascus
sp (Li et al. 2004). In this paper, we describe the isolation
and characterization of dihydromonacolin-MV, a new
monacolin metabolite, as a potent antioxidant from
M. purpureus.

Materials and methods

Materials
2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and butylated hydroxy anisole (BHA) were obtained from Sigma Chemicals St. Louis, MO, USA. L-ascorbic acid, nitroblue
tetrazolium (NBT), nicotinamide adenine dinucleotide
(NADH), phenazin methosulphate (PMS) were obtained
from Sisco Research Laboratories, Mumbai, India. Tris
(hydroxymethyl) amino-methane was obtained from
Qualigens Fine Chemicals, Mumbai, India. All the
solvents used for elution of polyketide were of analytical
grade. HPLC grade methanol was obtained from Ranbaxy
Fine Chemicals Limited, New Delhi, India. The culture
medium for cultivation of M. purpureus such as potato
dextrose agar (PDA) was obtained from Hi-Media Laboratories, Mumbai, India. The rice used was from the local
market.
Preparation of red yeast rice
Monascus purpureus: 410 strain used in this study was
obtained from Microbial Type Culture Collection, Institute
of Microbial Technology, Chandhigar, India. Solid-state
medium made with 10 g rice 20 ml of distilled water
sterilized for 20 min at 115-C was inoculated with M.
purpureus spore suspension prepared in 0.85% NaCl. The
culture was incubated at 30-C [Adolf Khuner Therm-Lab,
Birsfelden (Basel), Switzerland] for 11 days with intermittent mixing of rice by shaking.

Appl Microbiol Biotechnol (2007) 73:11971202

scavenging activity were maximum in methanolic extract,

the bioactive compound was isolated from it.
Fractionation of crude methanol extract
Ten gram methanol extract was subjected to column
chromatography using silica gel (60120 mesh) and eluted
stepwise with a linear gradient of hexane, chloroform and
ethyl acetate (100:0; 100; 25:75 v/v). About 20 fractions
measuring approximately 100150 ml were collected
depending upon color intensity. The fractions were concentrated by flash evaporation pooled together after analysis by
TLC (Silica gel 60 F254 plates 2020 cm, Merck, Germany)
and assayed for DPPH radical scavenging activity. For
purification, the fraction was rechromatographed as above
(Fig. 1).
Thin layer chromatography
The fractions collected were spotted on silica gel TLC
plates. The plates were developed in ascending direction of
12 to 15 cm height. Different proportions of dichloromethane and ethyl acetate (7:3, 8:2 and 6:4 v/v) and
dichloromethane, ethyl acetate, and methanol (6:4:0.5,
9:0.5:0.5 and 8:1:1 v/v) were used as a mobile phase
solvent systems. Dichloromethane, ethyl acetate and methanol (8:1:1 v/v) showed best separation. The plates were airdried and exposed to iodine to locate the spots.
High performance liquid chromatography
The purified fraction after lyophilization was dissolved in
methanol and it_s purity was checked by HPLC, using
Shimadzu Liquid Chromatograph LC-10A (Shimadzu,
Japan) fitted with Supelco C18, 5 m, 25 cm 4.6 mm
column (Supelco Park, 595, North Harrison Road PA.
16823-0048). For HPLC, methanol and 0.01% phosphoric
acid (20:80 v/v) were used as solvents for mobile phase after
injecting 10 l samples using for 20 l sample loop.
Gradient elution was carried out at a flow rate of 0.8 ml
min1. Shimadzu diode array detector detected elution profile
at 280 nm.

Isolation of bioactive compound

The fermented red rice dried at 4850-C and powdered to
6080 mesh was used to extract the pigments with series of
solvents of increasing polarity viz. hexane, chloroform,
ethyl acetate, acetone, methanol, and water. After removing
the solvent and water by flash evaporation and lyophilization, the samples were assayed for DPPH radical scavenging activity. Since the yield and DPPH free radical

Identification of bioactive compound by analytical

methods
UV Visible spectrometry
UVVisible spectrum of the isolated bioactive compound in
HPLC grade methanol was recorded using Schimadzu
160A UV-Visible Spectrophotometer.

Appl Microbiol Biotechnol (2007) 73:11971202

1199

Fig. 1 Purification of
dihydromonacolin-MV from
M. purpureus by silica gel
column chromatography using
different solvent systems

Fourier transformer infrared (FTIR) spectrometry

Determination of antioxidant activity

FTIR spectrum was recorded using Nicolet 5700 (Thermo

Electron, Madison, WI, US) spectrometer at room temperature. The bioactive compound was dissolved in dimethylsulfoxide (DMSO) and scanned in the range of 4,000400 cmj1.

DPPH radical scavenging activity were assayed using the

purified compound. The compound was dissolved in
ethanol at the concentration of 1 mg mlj1. All the
experiments were carried out in triplicate.

Mass spectrometry

DPPH radical scavenging activity

Mass spectrum was obtained using a Q-TOF Waters Ultima

instrument (Q-TOF GAA 082, Waters, Manchester, UK)
fitted with an electron spray ionization source. A software
version 4.0 was used for the data acquisition. The positive
ion mode using a spray voltage at 3.5 kV at a source
temperature of 80-C was employed for recording the
spectra. Mass spectra were recorded under electron impact
ionization at 70 eV energy. The sample was prepared in the
concentration range of 0.20.5 mg/ml and injected by flow
analysis at a flow rate of 10 l min1. The recorded mass of
sample was in the range of 1001,000.

The DPPH radical scavenging activity (Blois 1958) of the

dihydromonacolin-MV was measured according to the
method of Moon and Terao (1998). The IC50 value of
bioactive compound dissolved in ethanol (1 mg mlj1) was
determined using different concentrations (1025 g ml1)
of the compound. To 1.0 ml DPPH (500 M in ethanol),
the compound dissolved in ethanol was added and the
reaction mixture was made to 2.0 ml with TrisHCl buffer
(100 mM, pH 7.4). The mixture was shaken vigorously and
incubated at room temperature for 30 min. The absorbance
of the resulting solution was measured at 517 nm. Blanks
contained no DPPH.

Two-dimensional heteronuclear single quantum coherence

transfer spectroscopy (2D-HSQCT-NMR)

Lipid peroxidation assay

The 1H and 13C NMR spectra were recorded on Bruker

DRX500 NMR instrument operating at 500 MHz for 1H at
room temperature. The region from 0 to 12 ppm for 1H and 0
200 ppm for carbon was employed for scanning. Signals were
referred to tetramethylsilane within T0.01 ppm. About 10 mg
of sample dissolved in 0.5 ml of CDCl3 was used for the
recording the spectra.

Lipid peroxidation inhibitory activity of dihydromonacolinMV was measured according to the method of Kulkarni et
al. (2004). Egg lecithin (3 mg mlj1 in phosphate buffer,
pH 7.4) was sonicated (Hielscher GmbH UP 50H ultrachallprozessor sonicator) for 30 min to obtain small
membrane liposome vesicles. Different concentrations of
dihydromonacolin-MV (515 g mlj1) were added to

1200

Appl Microbiol Biotechnol (2007) 73:11971202

Table 1 Yield and DPPH radical scavenging activity of different

solvent extracts of M. purpureus
Solvent

Yield (%)

DPPH radical scavenging

activity (%)

Hexane
Chloroform
Ethyl acetate
Acetone
Methanol
BHA

5.630.18
3.490.24
1.210.08
1.080.09
13.070.17

13.910.33
9.261.22
19.170.20
8.670.63
59.781.66
79.910.51

Concentration of sample was 100 g ml1.

0.5 ml of liposome mixture. Lipid peroxidation was

induced by adding 10 l of 400 mM FeCl3 and 10 l of
200 mM L-ascorbic acid. After 60 min reaction at 37-C
(Buchi Heating-bath B-490, Flawil, Switzerland) the reaction was stopped by the addition of 1 ml 0.25N HCl
containing 15% TCA and 0.375% TBA and incubation in a
boiling water bath for 15 min. The absorbance of the
supernatant was measured at 532 nm after centrifugation at
10,000 rpm. Blank and control were maintained without
liposome and sample, respectively.
Superoxide radical scavenging activity
Superoxide radicals were generated in 1.0 ml of 0.02M,
TrisHCl buffer (pH 8.3) containing 0.1 mM NADH,
0.1 mM NBT, 10 M PMS, and dihydromonacolin-MV
(50200 g mlj1). The reaction kinetics was measured for
2 min at 560 nm (Liu et al. 1997).

Results
The sequential solvent extraction followed by repeated
silica gel column chromatography of M. purpureus
powder yielded a potential antioxidant compound. Higher
Fig. 2 HPLC elution profile of
dihydromonacolin-MV
purified from M. purpureus

DPPH radical scavenging activity of the methanol extract

(Table 1) selected the fraction for further purification by
activity-guided repeated column chromatography. Elution
with solvents (Fig. 1) of increasing polarity, yielded a
bioactive antioxidant compound in chloroform ethyl
acetate (75:25 v/v) fraction.
Purity of the eluted fractions when analyzed by TLC and
HPLC showed a single major spot and a peak respectively
(Fig. 2). Strong DPPH (500 M) free radical scavenging
antioxidant activity of the fraction resulted in the structure
elucidation by different spectroscopic methods.
UV spectrum of the compound as absorption at 228 nm
indicating pp* transition of the olefinic group. IR data
showed OH stretching at 3,445 cmj1 carbonyl stretching
at 1,660 cmj1 and C=C stretching at 1,435 cmj1. Direct
mass spectrum of the sample showed the parent ion (M+) at
406.
The identity of the compound was deciphered from the
2D-HSQCT NMR data analysis. The presence of 5-CH3
group was detected in the proton and the respective carbon13 correlation (Table 2). Two olefinic protons were detected
at 6.75 and 6.55 ppm respectively with characteristic cis
coupling constant of 10.5 Hz. The 13C signal at 176.15 ppm
indicated the presence of a carbonyl group. The region
between 1.2 to 2.7 ppm pointed to the presence of several
CH and magnetically nonequivalent CH2 groups. Three
signals at 65.8 ppm (17-CH), 65.4 ppm (3-CH), and
70.9 ppm (15-CH) suggested the presense of carbon next
to oxygen atom in the form of esters, lactone, and OH
groups. Thus, a derivative of dihydromonacolin-K was
identified as the structure of the bioactive compound.
Presence of 2-methyl propionate as the ester group at
position 3 instead of 2-mehyl butyrate found in dihydromonacolin-K. Hence, the bioactive compound characterized
in this study was designated as dihydromonacolin-MV.
Dihydromonacolin-MV isolated from the solid-state
cultures of M. purpureus was a potent DPPH radical
scavenger that is known to abstract the liable hydrogen

Appl Microbiol Biotechnol (2007) 73:11971202

1201

Table 2 2D-HSQCT NMR Spectral data of dihydromonacolin-MV

Assignments

11-CH3
12-CH3
23-CH3, 24-CH3
20-CH3
13-CH2
22-CH
6-CH
14-CH2
4-CH2
5-CH2
10-CH
18-CH2
1C
2-CH
9-CH
16-CH2
17-CH
3-CH
15-CH
8-CH
7-CH
19-CO
21-CO
a

Chemical shift
Carbon-13

Protona

18.9
19.1
22.5
23.1
27.2
29.8
30.9
35.2
36.3
36.1
42.3
40.9
42.3
43.4
44.3
46.9
65.8
65.4
70.9
117.2
121.5
171.5
176.1

1.21 (s)
1.25 (s)
1.18 (d, 6.5 Hz), 1.15 (d, 6.5 Hz)
1.27 (d, J=6.3 Hz)
1.26, 1.43 (m)
1.3 (J=3.3 Hz, septet)
1.33 (m)
1.92 (m)
1.89 (m)
1.85 (m)
2.68 (m)
2.65, 2.51 (dd, J=3.8, 6.1 Hz)

2.57 (t1)
2.55 (m)
2.52, 2.68 (m)
4.35 (m)
4.63 (m)
3.8 (m)
6.75 (d, 10.5)
6.55 (m, 10.5)

Some of the assignments are interchangeable.

atom. It also strongly inhibited the peroxidation of lipids.

The IC50 value to scavenge DPPH radical was found to be
20T1 g mlj1, whereas that of the synthetic antioxidant
BHA was 15.21T0.78 g mlj1 and methanolic extract
showed 50% radical scavenging activity at 100 g mlj1.
The lipid peroxidation inhibitory activities of methanol
extract and dihydromonacolin-MV are shown in Table 3.
The IC50 value for dihydromonacolin-MV, methanol extract
and BHA were found to be 5.71, 36.16 and 32.41 g mlj1,

respectively. Superoxide, the most important source of

initiating radicals in vivo, is produced in mitochondria
during electron chain transfer and it regularly leaks
outside of the mitochondria. The superoxide radical
scavenging activity of dihydromonacolin-MV (IC50=
163.97T2.68 g mlj1) is significant since it can attenuate
the formation of harmful hydroxyl radicals.

Discussion
M. purpureus and A. terreus that produce secondary
metabolite known as monacolins and dihydromevinolin
(Ma et al. 2000; Albers-Schonberg et al. 1981) are known
to inhibit the enzyme HMG-CoA reductase. The statins play
important role in atherosclerosis as antioxidant by preventing
the oxidation of low-density lipoprotein during oxidative
stress (Rosenson 2004). However, there is no evidence for
the antioxidant or radical scavenging activity of monacolins
from M. purpureus. Thus, the report on characterization of
dihydromonacolin-MV isolated from M. purpureus for free
radical scavenging appears to new literature (Fig. 3).
Dihydromonacolin-MV strongly scavenged DPPH by
donating electrons to free radicals. Free radicals are highly
reactive, toxic molecules due to the presence of one or more
impaired electrons. Within tissue they damage DNA,
proteins, lipids and carbohydrates (Baskar et al. 2004).
The reactivity of didhydromonacolin-MV by donating its
electrons to the free radicals and its natural occurence in the
fungus suggested its use as antioxidant.
The results showed that dihydromonacolin-MV chelating
metal ions and inhibiting oxidation of lipids by breaking the
chain reaction due to Fe+3. Atherosclerosis is characterized
by the accumulation of cholesterol, lipid peroxides and
oxysterols in the arterial wall and it is the main cause of
heart attack and stroke (Vaya and Aviram 2001). Dual
activity of dihydromonacolin-MV to inhibit lipid peroxidation and scavenge free radical showed this statin characterized from M. purpureus for application to prevent
atherosclerosis.

Table 3 Comparative antioxidant activity of methanol extract,

dihydromonacolin-MV, and BHA
Sample

Methanol extract
DihydromonacolinMV
BHA

IC50 value (g mlj1)

DPPH
radical
scavenging
activity

Lipid
peroxidation
inhibition
activity

Super oxide
radical
scavenging
activity

100.78T2.66
20T1

36.16T1.32
5.71T0.38

163.97T2.68

15.21T0.78

32.41T1.49

264T1.6

Fig. 3 Structure of dihydromonacolin-MV isolated from

M. purpureus

1202
Acknowledgement Mohan A. Dhale acknowledge the Council of
Scientific and Industrial Research (CSIR), New Delhi, for providing
Research Fellowship.

References
Albers-Schonberg G, Joshua H, Lopez MB, Hensens OD, Springer JP,
Chen J, Ostrove S, Hoffaman CH, Alberts AW, Patchett AA
(1981) Dihydromevinolin, a potent hypocholesterolemic metabolite produced by Aspergillus terreus. J Antibiot (Tokyo) 34:507
512
Baskar AA, Manoharan S, Manivasagam T, Subramanian P (2004)
Temporal patterns of lipid peroxidation product formation and
antioxidants activity in oral cancer patients. Cell Mol Biol 9:665
673
Blois MS (1958) Antioxidant determinations by the use of a stable
free radical. Nature 181:11991200
Gotto AM (2003) Antioxidants, statins, and atherosclerosis. J Am Coll
Cardiol 41:12051210
Heber D, Yip I, Ashley JM, Elashoff DA, Elashoff RM, Go VLW
(1999) Cholestero-lowering effects of proprietary Chinese red
yeast rice dietary supplement. Am J Clin Nutr 69:231236
Helliwell B (2000) The antioxidant paradox. Lancet 355:11791180
Korantzopoulos P (2004) Letter to the editor. The antioxidant
effects of statins may extend beyond atherosclerosis: potential
benefit for atrial fibrillation and heart failure. Atherosclerosis
175:187

Appl Microbiol Biotechnol (2007) 73:11971202

Kulkarni AP, Aradhya M, Divakar S (2004) Isolation and identification of a radical scavenging antioxidantpunicalagin from pith
and carpellary membrane of pomegranate fruit. Food Chem
87:551557
Liu F, Ooi VEC, Chang T (1997) Free radical scavenging activity of
mushroom polysaccharide extract. Life Sci 60:763771
Li Y, Zhang F, Wang Z, Hu Z (2004) Identification and chemical
profiling of monacolins in red yeast rice using high-performance
liquid chromatography with photodiode array detector and mass
spectrometry. J Pharm Biomed Anal 35:11011112
Ma J, Li Y, Ye Q, Li J, Hua Y, Ju D, Zhang D, Cooper R, Chang M
(2000) Constituents of red yeast rice, a traditional Chinese food
and medicine. J Agric Food Chem 48:52205225
Moon JH, Terao J (1998) Antioxidant effect of caffeic acid and
dihydrocaffeic acid in lard and human low-density lipoprotein.
J Agric Food Chem 46:50625065
Rosenson RS (2004) Statins in atherosclerosis: lipid-lowering
agents with antioxidant capabilities. Atherosclerosis 173:
112
Stahl W, Sies H (1996) Biological activity of carotenoids and their
bioavailability in the human organism. The Royal Society of
Chemistry, Thomas Graham House, Science Park, Milton Road,
Cambridge CB4 4WF, UK
Vaya J, Aviram M (2001) Nutritional antioxidants: mechanisms of
action, analyses of activities and medical applications. Curr Med
Chem Immune Endocr Metabol Agents 18:99117
Wei W, Li C, Wang Y, Su H, Zuh J, Kritchevsky D (2003)
Hypolipidemic and anti-atherogenic effects of long-term Cholestin (Monascus purpureus-fermented rice, red yeast rice) in
cholesterol fed rabbits. J Nutr Biochem 14:314318