APPLIED MICROBIAL AND CELL PHYSIOLOGY

Isolation and characterization of dihydromonacolin-MVfrom 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 purpureuscultivated by solid-state fermentation on rice showed strong2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavengingactivity and better yield as compared to other polarity basedextracted fractions. It was selected for further purificationof the antioxidant. The activity-guided repeated fraction-ation of methanolic extract on a silica gel columnchromatography yielded a compound that exhibited strongantioxidant activity. Based on the spectroscopic analysis byUV, IR, 1H NMR, 13C NMR, 2D-HSQCT NMR, and MS,the antioxidant isolated was elucidated as a derivative ofdihydromonacolin-K, where the ester group is 2-methylpropionate, designated as dihydromonacolin-MV. TheDPPH radical was significantly scavenged by the dihydro-monacolin-MV (IC50 20T1 g ml

j1). The dihydromonaco-lin-MV showed strong inhibition of lipid peroxidation in aliposome model with an IC50 value of 5.71T0.38 g ml

j1

and superoxide radical scavenging activity with an IC50value of 163.97T2.68 g mlj1.

Keywords Monascus purpureus . Dihydromonacolin-MV.

Antioxidant . DPPH lipid peroxidation .

Superoxide radical

Introduction

Free radicals are highly reactive, short-lived, toxic mole-cules that have one or more unpaired electrons and candamage DNA, proteins, lipids, and carbohydrates (Baskaret al. 2004) within the tissue. The main cause of mortalityand morbidity in the western world is atherosclerosis, theaccumulation of oxysterol, cholesterol, and peroxide lipidsin arteries, generated by free radicals which lead to heartattack. Hence, there has been an increased interest in theapplication of antioxidants to medical treatment as infor-mation are constantly gathered, linking the development ofhuman diseases to oxidative stress (Vaya and Aviram 2001)that leads to the generation of free radicals. The effect ofdietary antioxidants on the development of human athero-sclerosis is also controversial and a number of contradictoryexamples have been published. Most of the research on therole of antioxidants in cardiovascular diseases has focusedon testing pure compound to prevent lipid peroxidation byexamining its ability to scavenge free radicals. However,antioxidant contribution in vivo goes far beyond scavengingfree radicals. Moreover, single antioxidants are usually notpresent alone in biological systems but act in combinationwith other antioxidants. Hence the protective effect of a dietis not equivalent to the protective effect of antioxidants in it(Helliwell 2000).

Korantzopoulos (2004) reported that antioxidant effectsof statins might extend beyond atherosclerosis and potentialbenefit for atrial fibrillation and heart failure. Red yeast riceobtained by fermentation with M. purpureus producesvarious secondary metabolites and is a common food itemfound in China. It was used for many centuries to enhancethe colour and flavour of food, as well as a traditionalmedicine for digestive and vascular functions (Ma et al.2000). The monacolins from Monascus sp (Heber et al.

Appl Microbiol Biotechnol (2007) 73:11971202DOI 10.1007/s00253-006-0578-0

M. A. Dhale : S. U. Kumar :G. Vijayalakshmi (*)Department of Food Microbiology,Central Food Technological Research Institute,Mysore 570020, Indiae-mail: vij19_99@yahoo.com

S. DivakarDepartment of Fermentation Technology and Bioengineering,Central Food Technological Research Institute,Mysore 570020, India

1999) and dihydromevinolin (Albers-Schonberg et al. 1981)are reported to exhibit a cholesterol lowering action byinhibiting the HMG-CoA reductase as similar to thecommercial statin drug. Till date, fourteen different typesof monacolins compounds have been identified in Monascussp (Li et al. 2004). In this paper, we describe the isolationand characterization of dihydromonacolin-MV, a newmonacolin metabolite, as a potent antioxidant fromM. purpureus.

Materials and methods

Materials

2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and butylated hy-droxy anisole (BHA) were obtained from Sigma Chem-icals St. Louis, MO, USA. L-ascorbic acid, nitrobluetetrazolium (NBT), nicotinamide adenine dinucleotide(NADH), phenazin methosulphate (PMS) were obtainedfrom Sisco Research Laboratories, Mumbai, India. Tris(hydroxymethyl) amino-methane was obtained fromQualigens Fine Chemicals, Mumbai, India. All thesolvents used for elution of polyketide were of analyticalgrade. HPLC grade methanol was obtained from RanbaxyFine Chemicals Limited, New Delhi, India. The culturemedium for cultivation of M. purpureus such as potatodextrose agar (PDA) was obtained from Hi-Media Labo-ratories, Mumbai, India. The rice used was from the localmarket.

Preparation of red yeast rice

Monascus purpureus: 410 strain used in this study wasobtained from Microbial Type Culture Collection, Instituteof Microbial Technology, Chandhigar, India. Solid-statemedium made with 10 g rice 20 ml of distilled watersterilized for 20 min at 115-C was inoculated with M.purpureus spore suspension prepared in 0.85% NaCl. Theculture was incubated at 30-C [Adolf Khuner Therm-Lab,Birsfelden (Basel), Switzerland] for 11 days with intermit-tent mixing of rice by shaking.

Isolation of bioactive compound

The fermented red rice dried at 4850-C and powdered to6080 mesh was used to extract the pigments with series ofsolvents of increasing polarity viz. hexane, chloroform,ethyl acetate, acetone, methanol, and water. After removingthe solvent and water by flash evaporation and lyophiliza-tion, the samples were assayed for DPPH radical scaveng-ing activity. Since the yield and DPPH free radical

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 columnchromatography using silica gel (60120 mesh) and elutedstepwise with a linear gradient of hexane, chloroform andethyl acetate (100:0; 100; 25:75 v/v). About 20 fractionsmeasuring approximately 100150 ml were collecteddepending upon color intensity. The fractions were concen-trated by flash evaporation pooled together after analysis byTLC (Silica gel 60 F254 plates 2020 cm, Merck, Germany)and assayed for DPPH radical scavenging activity. Forpurification, the fraction was rechromatographed as above(Fig. 1).

Thin layer chromatography

The fractions collected were spotted on silica gel TLCplates. The plates were developed in ascending direction of12 to 15 cm height. Different proportions of dichloro-methane and ethyl acetate (7:3, 8:2 and 6:4 v/v) anddichloromethane, ethyl acetate, and methanol (6:4:0.5,9:0.5:0.5 and 8:1:1 v/v) were used as a mobile phasesolvent systems. Dichloromethane, ethyl acetate and meth-anol (8:1:1 v/v) showed best separation. The plates were air-dried and exposed to iodine to locate the spots.

High performance liquid chromatography

The purified fraction after lyophilization was dissolved inmethanol and it_s purity was checked by HPLC, usingShimadzu Liquid Chromatograph LC-10A (Shimadzu,Japan) fitted with Supelco C18, 5 m, 25 cm 4.6 mmcolumn (Supelco Park, 595, North Harrison Road PA.16823-0048). For HPLC, methanol and 0.01% phosphoricacid (20:80 v/v) were used as solvents for mobile phase afterinjecting 10 l samples using for 20 l sample loop.Gradient elution was carried out at a flow rate of 0.8 mlmin1. Shimadzu diode array detector detected elution profileat 280 nm.

Identification of bioactive compound by analyticalmethods

UV Visible spectrometry

UVVisible spectrum of the isolated bioactive compound inHPLC grade methanol was recorded using Schimadzu160A UV-Visible Spectrophotometer.

1198 Appl Microbiol Biotechnol (2007) 73:11971202

Fourier transformer infrared (FTIR) spectrometry

FTIR spectrum was recorded using Nicolet 5700 (ThermoElectron, Madison, WI, US) spectrometer at room tempera-ture. The bioactive compound was dissolved in dimethylsulf-oxide (DMSO) and scanned in the range of 4,000400 cmj1.

Mass spectrometry

Mass spectrum was obtained using a Q-TOF Waters Ultimainstrument (Q-TOF GAA 082, Waters, Manchester, UK)fitted with an electron spray ionization source. A softwareversion 4.0 was used for the data acquisition. The positiveion mode using a spray voltage at 3.5 kV at a sourcetemperature of 80-C was employed for recording thespectra. Mass spectra were recorded under electron impactionization at 70 eV energy. The sample was prepared in theconcentration range of 0.20.5 mg/ml and injected by flowanalysis at a flow rate of 10 l min1. The recorded mass ofsample was in the range of 1001,000.

Two-dimensional heteronuclear single quantum coherencetransfer spectroscopy (2D-HSQCT-NMR)

The 1H and 13C NMR spectra were recorded on BrukerDRX500 NMR instrument operating at 500 MHz for 1H atroom temperature. The region from 0 to 12 ppm for 1H and 0200 ppm for carbon was employed for scanning. Signals werereferred to tetramethylsilane within T0.01 ppm. About 10 mgof sample dissolved in 0.5 ml of CDCl3 was used for therecording the spectra.

Determination of antioxidant activity

DPPH radical scavenging activity were assayed using thepurified compound. The compound was dissolved inethanol at the concentration of 1 mg mlj1. All theexperiments were carried out in triplicate.

DPPH radical scavenging activity

The DPPH radical scavenging activity (Blois 1958) of thedihydromonacolin-MV was measured according to themethod of Moon and Terao (1998). The IC50 value ofbioactive compound dissolved in ethanol (1 mg mlj1) wasdetermined 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 thereaction mixture was made to 2.0 ml with TrisHCl buffer(100 mM, pH 7.4). The mixture was shaken vigorously andincubated at room temperature for 30 min. The absorbanceof the resulting solution was measured at 517 nm. Blankscontained no DPPH.

Lipid peroxidation assay

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

Fig. 1 Purification ofdihydromonacolin-MV fromM. purpureus by silica gelcolumn chromatography usingdifferent solvent systems

Appl Microbiol Biotechnol (2007) 73:11971202 1199

0.5 ml of liposome mixture. Lipid peroxidation wasinduced by adding 10 l of 400 mM FeCl3 and 10 l of200 mM L-ascorbic acid. After 60 min reaction at 37-C(Buchi Heating-bath B-490, Flawil, Switzerland) the reac-tion was stopped by the addition of 1 ml 0.25N HClcontaining 15% TCA and 0.375% TBA and incubation in aboiling water bath for 15 min. The absorbance of thesupernatant was measured at 532 nm after centrifugation at10,000 rpm. Blank and control were maintained withoutliposome 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 for2 min at 560 nm (Liu et al. 1997).

Results

The sequential solvent extraction followed by repeatedsilica gel column chromatography of M. purpureuspowder yielded a potential antioxidant compound. Higher

DPPH radical scavenging activity of the methanol extract(Table 1) selected the fraction for further purification byactivity-guided repeated column chromatography. Elutionwith solvents (Fig. 1) of increasing polarity, yielded abioactive antioxidant compound in chloroform ethylacetate (75:25 v/v) fraction.

Purity of the eluted fractions when analyzed by TLC andHPLC showed a single major spot and a peak respectively(Fig. 2). Strong DPPH (500 M) free radical scavengingantioxidant activity of the fraction resulted in the structureelucidation by different spectroscopic methods.

UV spectrum of the compound as absorption at 228 nmindicating pp* transition of the olefinic group. IR datashowed OH stretching at 3,445 cmj1 carbonyl stretchingat 1,660 cmj1 and C=C stretching at 1,435 cmj1. Directmass spectrum of the sample showed the parent ion (M+) at406.

The identity of the compound was deciphered from the2D-HSQCT NMR data analysis. The presence of 5-CH3group was detected in the proton and the respective carbon-13 correlation (Table 2). Two olefinic protons were detectedat 6.75 and 6.55 ppm respectively with characteristic ciscoupling constant of 10.5 Hz. The 13C signal at 176.15 ppmindicated the presence of a carbonyl group. The regionbetween 1.2 to 2.7 ppm pointed to the presence of several CH and magnetically nonequivalent CH2 groups. Threesignals at 65.8 ppm (17-CH), 65.4 ppm (3-CH), and70.9 ppm (15-CH) suggested the presense of carbon nextto oxygen atom in the form of esters, lactone, and OHgroups. Thus, a derivative of dihydromonacolin-K wasidentified as the structure of the bioactive compound.Presence of 2-methyl propionate as the ester group atposition 3 instead of 2-mehyl butyrate found in dihydro-monacolin-K. Hence, the bioactive compound characterizedin this study was designated as dihydromonacolin-MV.

Dihydromonacolin-MV isolated from the solid-statecultures of M. purpureus was a potent DPPH radicalscavenger that is known to abstract the liable hydrogen

Fig. 2 HPLC elution profile ofdihydromonacolin-MVpurified from M. purpureus

Table 1 Yield and DPPH radical scavenging activity of differentsolvent extracts of M. purpureus

Solvent Yield (%) DPPH radical scavengingactivity (%)

Hexane 5.630.18 13.910.33Chloroform 3.490.24 9.261.22Ethyl acetate 1.210.08 19.170.20Acetone 1.080.09 8.670.63Methanol 13.070.17 59.781.66BHA 79.910.51

Concentration of sample was 100 g ml1.

1200 Appl Microbiol Biotechnol (2007) 73:11971202

atom. It also strongly inhibited the peroxidation of lipids.The IC50 value to scavenge DPPH radical was found to be20T1 g mlj1, whereas that of the synthetic antioxidantBHA was 15.21T0.78 g mlj1 and methanolic extractshowed 50% radical scavenging activity at 100 g mlj1.The lipid peroxidation inhibitory activities of methanolextract and dihydromonacolin-MV are shown in Table 3.The IC50 value for dihydromonacolin-MV, methanol extractand BHAwere found to be 5.71, 36.16 and 32.41 g mlj1,

respectively. Superoxide, the most important source ofinitiating radicals in vivo, is produced in mitochondriaduring electron chain transfer and it regularly leaksoutside of the mitochondria. The superoxide radicalscavenging activity of dihydromonacolin-MV (IC50=163.97T2.68 g mlj1) is significant since it can attenuatethe formation of harmful hydroxyl radicals.

Discussion

M. purpureus and A. terreus that produce secondarymetabolite known as monacolins and dihydromevinolin(Ma et al. 2000; Albers-Schonberg et al. 1981) are knownto inhibit the enzyme HMG-CoA reductase. The statins playimportant role in atherosclerosis as antioxidant by preventingthe oxidation of low-density lipoprotein during oxidativestress (Rosenson 2004). However, there is no evidence forthe antioxidant or radical scavenging activity of monacolinsfrom M. purpureus. Thus, the report on characterization ofdihydromonacolin-MV isolated from M. purpureus for freeradical scavenging appears to new literature (Fig. 3).

Dihydromonacolin-MV strongly scavenged DPPH bydonating electrons to free radicals. Free radicals are highlyreactive, toxic molecules due to the presence of one or moreimpaired electrons. Within tissue they damage DNA,proteins, lipids and carbohydrates (Baskar et al. 2004).The reactivity of didhydromonacolin-MV by donating itselectrons to the free radicals and its natural occurence in thefungus suggested its use as antioxidant.

The results showed that dihydromonacolin-MV chelatingmetal ions and inhibiting oxidation of lipids by breaking thechain reaction due to Fe+3. Atherosclerosis is characterizedby the accumulation of cholesterol, lipid peroxides andoxysterols in the arterial wall and it is the main cause ofheart attack and stroke (Vaya and Aviram 2001). Dualactivity of dihydromonacolin-MV to inhibit lipid peroxida-tion and scavenge free radical showed this statin charac-terized from M. purpureus for application to preventatherosclerosis.

Fig. 3 Structure of dihydromo-nacolin-MV isolated fromM. purpureus

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

Assignments Chemical shift

Carbon-13 Protona

11-CH3 18.9 1.21 (s)12-CH3 19.1 1.25 (s)23-CH3, 24-CH3 22.5 1.18 (d, 6.5 Hz), 1.15 (d, 6.5 Hz)20-CH3 23.1 1.27 (d, J=6.3 Hz)13-CH2 27.2 1.26, 1.43 (m)22-CH 29.8 1.3 (J=3.3 Hz, septet)6-CH 30.9 1.33 (m)14-CH2 35.2 1.92 (m)4-CH2 36.3 1.89 (m)5-CH2 36.1 1.85 (m)10-CH 42.3 2.68 (m)18-CH2 40.9 2.65, 2.51 (dd, J=3.8, 6.1 Hz)1C 42.3 2-CH 43.4 2.57 (t1)9-CH 44.3 2.55 (m)16-CH2 46.9 2.52, 2.68 (m)17-CH 65.8 4.35 (m)3-CH 65.4 4.63 (m)15-CH 70.9 3.8 (m)8-CH 117.2 6.75 (d, 10.5)7-CH 121.5 6.55 (m, 10.5)19-CO 171.5 21-CO 176.1

a Some of the assignments are interchangeable.

Table 3 Comparative antioxidant activity of methanol extract,dihydromonacolin-MV, and BHA

Sample IC50 value (g mlj1)

DPPHradicalscavengingactivity

Lipidperoxidationinhibitionactivity

Super oxideradicalscavengingactivity

Methanol extract 100.78T2.66 36.16T1.32 Dihydromonacolin-MV

20T1 5.71T0.38 163.97T2.68

BHA 15.21T0.78 32.41T1.49 264T1.6

Appl Microbiol Biotechnol (2007) 73:11971202 1201

Acknowledgement Mohan A. Dhale acknowledge the Council ofScientific and Industrial Research (CSIR), New Delhi, for providingResearch Fellowship.

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1202 Appl Microbiol Biotechnol (2007) 73:11971202

Isolation and characterization of dihydromonacolin-MV from Monascus purpureus for antioxidant propertiesAbstractIntroductionMaterials and methodsMaterialsPreparation of red yeast riceIsolation of bioactive compoundFractionation of crude methanol extractThin layer chromatographyHigh performance liquid chromatography

Identification of bioactive compound by analytical methodsUVVisible spectrometryFourier transformer infrared (FTIR) spectrometryMass spectrometryTwo-dimensional heteronuclear single quantum coherence transfer spectroscopy (2D-HSQCT-NMR)

Determination of antioxidant activityDPPH radical scavenging activityLipid peroxidation assaySuperoxide radical scavenging activity

ResultsDiscussionReferences

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