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Isolation and structure elucidation oftwo new cytotoxic metabolites fromred yeast riceInes Ferse a , Marina Langlitz a , Karin Kleigrewe a , SebastianRzeppa a , Marlies Lenczyk a , Henning Harrer a , Benedikt Cramera & Hans-Ulrich Humpf aa Institute of Food Chemistry, Westfälische Wilhelms-Universität,Corrensstrasse 45, D-48149 Münster, GermanyVersion of record first published: 02 Dec 2011.

To cite this article: Ines Ferse , Marina Langlitz , Karin Kleigrewe , Sebastian Rzeppa , MarliesLenczyk , Henning Harrer , Benedikt Cramer & Hans-Ulrich Humpf (2012): Isolation and structureelucidation of two new cytotoxic metabolites from red yeast rice, Natural Product Research:Formerly Natural Product Letters, 26:20, 1914-1921

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Natural Product ResearchVol. 26, No. 20, October 2012, 1914–1921

Isolation and structure elucidation of two new cytotoxic metabolites

from red yeast rice

Ines Ferse, Marina Langlitz, Karin Kleigrewe, Sebastian Rzeppa, Marlies Lenczyk,Henning Harrer, Benedikt Cramer and Hans-Ulrich Humpf*

Institute of Food Chemistry, Westfalische Wilhelms-Universitat, Corrensstrasse 45, D-48149Munster, Germany

(Received 15 July 2011; final version received 5 September 2011)

In this study, 10 already described secondary metabolites and 2 unknownmetabolites were identified in an extract of Monascus purpureus by high-performance liquid chromatography–diode array detection. The unknownmetabolites were isolated and their chemical structures were elucidated. Thenew metabolites possess the molecular formulas C21H27NO4 and C23H31NO4.They were named monascopyridines E and F due to their pyridine backbone.The cytotoxicity of the new compounds was studied using immortalised humankidney epithelial cells displaying IC50 values in the micromolar range.

Keywords: Monascus; metabolite; red yeast rice; monascopyridine; pigments

1. Introduction

Red yeast rice, also known as angkak or koji, is obtained by the fermentation of rice(Oryza sativa) with species of the fungus Monascus, especially Monascus purpureus. Due toits colour, red yeast rice has been applied in Asian countries, for example, China, Taiwanand Japan, for centuries as a food colorant and food preservative in fish, meat andsoybean products and in rice wine production. Benefits from consuming red yeast rice havebeen described in Chinese folk medicine, where it is used for health promotion processessuch as digestion, blood circulation and spleen strengthening (Ma et al., 2000). Severalsecondary metabolites of diverse structures are known; the most important of which aresix major pigments as well as organic acids, monacolins, monankarins, citrinin andmonascodilone (Blanc et al., 1994, 1995; Endo, Hasumi, & Negishi, 1985; Hossain,Okuyama, & Yamazaki, 1996; Juzlova, Martinkova, & Kren, 1996; Wild, Toth, & Humpf,2002). Monascus pigments belong to the group of azaphilones and cover a range ofcolours. Alongside, the red pigments rubropunctamine (2) and monascorubramine (7), theyellow pigments monascin (5) and ankaflavin (11) and the orange pigments rubropuncta-tin and monascorubrin have been isolated from Monascus cultures (Chen, Manchand, &Whalley, 1971; Fielding et al., 1960; Haws, Holker, Kelly, Powell, & Robertson, 1959;Inouye et al., 1962; Lin, Yakushijin, Buchi, & Demain, 1992). Monacolin K (also knownas mevinolin and lovastatin) and its related compounds inhibit the cholesterol biosynthe-sis, thus belonging to the group of HMG–CoA reductase inhibitors (Endo, 1979).The daily consumption of red yeast rice was found to reduce blood cholesterol in humans

*Corresponding author. Email: humpf@uni-muenster.de

ISSN 1478–6419 print/ISSN 1478–6427 online

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(Heber et al., 1999). In recent years, further pigments and secondary metabolites have beenidentified. The first metabolites with a pyridine structure, therefore named monascopyr-idines, were characterised in 2003 and 2006 (Knecht, Cramer, & Humpf, 2006; Wild, Toth,& Humpf, 2003) (Figure 1). Nowadays, extracts of red yeast rice have gained more andmore interest in Western countries as food additives or dietary supplements because oftheir natural origin (Chen, Jiao, & Ma, 2008; Fink-Gremmels, Dresel, & Leistner, 1992).However, various biological activities of red yeast rice metabolites have been described,such as embryotoxicity, teratogenicity and cytotoxicity (Eisenbrand, 2006; Knecht &Humpf, 2006; Lin, Wang, Lee, & Su, 2008; Martinkova, Juzlova, & Vesely, 1995; Su, Lin,Lee, & Ho, 2005). Furthermore, toxicological effects of known and unknown metaboliteshave not been evaluated yet or are unclear. Therefore, the identification and character-isation of Monascus metabolites is necessary for the risk versus benefit assessment in orderto fully evaluate the safety of red yeast rice products.

We herein report the isolation and structure elucidation of two new M. purpureusmetabolites with a pyridine ring as characteristic structural element and the determinationof their cytotoxic potential.

2. Results and discussion

2.1. Preparative isolation of new metabolites

The methanol-extract from ground rice fermented withM. purpureus (DSM 1379) was pre-purified using C-18 cartridges. Stepwise elution using an increasing methanol gradientyielded different fractions that were analysed by high-performance liquid chromatogra-phy–diode array detection (HPLC–DAD). Metabolites eluting with 80% methanol werefurther separated into 10 fractions by means of semi-preparative HPLC–UV (Figure S1,supplementary material). Screening of these fractions with HPLC–DAD and HPLCcoupled to Fourier transform mass spectrometry (HPLC–FTMS) revealed several alreadyknown secondary metabolites such as the red pigments rubropunctamine (2) andmonascorubramine (7), the yellow pigments monascin (5) and ankaflavin (11),monascopyridines A (3) and B (9) as well as monascopyridines C (1) and D (8) and thefluorescent metabolites monasfluors A (4) and B (10). These metabolites were identified bycomparing their distinctive UV-spectra with those from the literature as well as by theirexact masses. Alongside these known compounds, an unknown metabolite (12) could be

Figure 1. Structures of secondary metabolites of M. purpureus: red pigments rubropunctamine (2)and monascorubramine (7), monascopyridines A (3) and B (9), monascopyridines C (1) and D (8),monasfluors A (4) and B (10) and yellow pigments monascin (5) and ankaflavin (11).

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isolated from the red yeast rice extract. Furthermore, a second unknown metabolite 6 was

co-eluting with monascorubramine. Isolation of the unknown compound 6 was achieved

by further HPLC separation using a gradient of acetonitrile and 1% formic acid. The use

of this isolation method led to yields ranging from 1–50mg depending on the metabolite

obtained from 90 g rice. In the case of the unknown metabolites, compounds 6 and 12,

11.0 and 46.0mg, respectively, were isolated.

2.2. Structure elucidation of the new metabolites

HPLC–FTMS measurements of compound 12 revealed the exact mass of

[MþH]þ¼ 386.23237. The molecular formula was suggested to be C23H31NO4. For

structure elucidation, nuclear magnetic resonance (NMR) spectroscopy was performed.

1D NMR experiments such as 1H, 13C, 1D nuclear Overhauser enhancement (1H,1H-

NOE) as well as distortionless enhancement by polarisation transfer experiments using a

90� decoupler pulse (DEPT-90) and a 135� one (DEPT-135) were performed. Furthermore,

2D spectra such as 1H,1H-correlation (1H, 1H-COSY), heteronuclear multiple bond

correlation (1H, 13C-HMBC) and heteronuclear multiple quantum correlation (1H, 13C-HMQC) were recorded. 13C-NMR spectroscopy showed signals at 208.9, 199.1 and

165.8 ppm, characteristic for CO, conjugated ketone and ester groups. Signals at 159.7,

145.9, 137.2 and 121.4 ppm were assigned to quaternary sp2 carbon atoms, whereas five

more signals in the sp2 range of DEPT-90 experiments revealed the presence of five CH

groups. In the sp3 range, the DEPT-135 spectrum exhibited 4 CH3 and 7 CH2 groups,

resulting in a total of 23 carbon atoms. 1H-NMR spectroscopy featured methine signals at

6.47 and 6.85 ppm and a methyl signal at 1.93 ppm, showing coupling in the 1H, 1H-COSY

spectrum and accordant carbon atoms at 130.5, 135.5 and 18.8 ppm in the HMQC

experiment, thus leading to the assumption of a propenyl substituent. The couplingconstant of J¼ 15.5Hz indicated a trans-(E)-configured double bond. Methylene signals at

1.26 ((CH2)4), 1.56 and 2.59 ppm and a methyl signal at 0.84 ppm, showing multiplet,

multiplet, triplet and triplet multiplicities and their linked carbon atoms displaying

negative signals as well as a CH3 group at 14.2 ppm in the DEPT-135 experiment and 1H,1H-COSY correlations starting from 0.84 ppm allowed the identification of a CH3(CH2)6-

moiety. A singlet methyl signal at �¼ 3.89 ppm and corresponding CH3 signal from

DEPT-135 experiment indicated a proximate oxygen atom. In addition, the characteristic

ester signal in 13C-NMR revealed a methyl ester substituent. The HMQC spectrum enables

assignments of direct C–H bonds, whereas the HMBC spectrum illustrates longer rangecouplings of protons with carbons. HMBC response from H-9 and H-10 with C-2 showed

the position of the propylene moiety at the pyridine ring, while the cross-peak at 165.8/

9.10 ppm indicated that the methyl ester is linked to pyridine as well. Figure 2 shows the

molecular structure of the unknown metabolite 12 named monascopyridine F due to its

pyridine ring moiety. Figure 3 illustrates relevant HMBC-couplings and NOE-responses in

the NMR. NOE experiments showed a response at 2.48 ppm (H-24) when irradiating with

the frequency at 8.09 ppm (H-12) and vice versa as well as a response at 3.24 ppm (H-14)

when irradiating with the frequency at 7.23 ppm (H-3) and vice versa. This indicated thedouble bond between positions C-12 and C-13 to be trans-configured. The unknown

metabolite 6 revealed the exact mass of [MþH]þ¼ 358.20136, 28 mass units lower than

monascopyridine F. Several Monascus metabolites show the characteristic to occur

pairwise possessing an alkyl-moiety consisting of a C7H15 or a C5H11-side chain,

respectively. This indicated that metabolite 6 is a lower homologue of monascopyridine F.

Comparison of their UV-spectra suggested the same chromophore since their absorption

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does not differ. NMR data confirmed the assumption of compounds 6 and 12 differing in

the alkyl chain. The unknown metabolite 6 was named monascopyridine E (Figure 2).MSn-fragmentation experiments were carried out to further confirm the structure

derived from NMR data. Both compounds resulted in the same fragments. The first

occurring product ion with m/z of 260.12814 is generated by the neutral loss of the alkyl

moiety as hexylketene and butylketene, respectively. Thus, all occurring fragments do not

differ although generated from precursor ions with different m/z signals. Product ion m/z

228.10193 is generated from m/z 260.12814 as well as from the molecule ion due to the loss

of methanol. The cleavage of CO leads to the product ion m/z 200.10686. The loss of the

methoxy group as ethenone from m/z 228.10193 leads to m/z 186.09116. The postulated

fragmentation pattern of monascopyridine F is shown in Figure S2 (supplementary

material).Regarding the methyl ester moiety in monascopyridines E and F, we considered the

possibility that the methyl ester is an artefact due to the use of methanol during the

isolation procedure. Therefore, the protocol of extraction was modified by eliminating any

methanol and using exclusively acetonitrile and ethanol, respectively. From HPLC–FTMS

measurements, we could show that monascopyridines E and F were not present in the

acetonitrile and ethanol extracts. However, in the case of the ethanol extract, a novel peak

with the exact mass of [MþH]þ¼ 400.24779 was detected. The proposed molecular

formula of C24H33NO4 revealed a structure 14 mass units higher than that of

monascopyridine F. This finding indicated the formation of an ethyl ester in ethanolic

environment instead of a methyl ester in methanolic environment. MS2-fragmentation

experiments verified the existence of the ethyl ester. Product ions with m/z 274.1, 228.0,

200.1 and 186.1 occurred as illustrated in Figure S3. While the latter product ions are in

agreement with those obtained during the fragmentation process of the newly found

monascopyridines, the first product ion comprises a mass difference of 14 mass units.

Figure 2. Structures of the new metabolites monascopyridines E (6) and F (12).

Figure 3. (A) HMBC-coupling in monascopyridine F (12). (B) HMBC-coupling in monascopyridineF. (C) NOE-responses in monascopyridine F.

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Given the fact that the ester moiety is present in the first appearing product ion whereas aloss of the ester group is observed in all following product ions, this data confirmed theassumed structure. In the case of monascopyridine E, a novel peak with m/z 372.2165 wasdetected in the ethanol extract. As expected, the above-described product ions were alsoobtained in MS2 fragmentation experiments, verifying that monascopyridine E occurs asethyl ester in ethanol solution. The ethyl esters were not identified in samples extractedwith methanol nor acetonitrile. Consequently, these findings indicated that the naturallyoccurring compounds imply a carboxylic acid moiety. The zwitterionic character of thesecompounds is a problem during extraction and might be the cause why we were not able todetect monascopyridines E and F in their free acid forms in the raw extracts.

Regarding the cytotoxic properties of these new compounds, cell culture experimentsusing immortalised human kidney epithelial (IHKE) cells were performed. The newlyidentified monascopyridines E and F caused a reduction of the cell viability in micromolarconcentrations in the CCK-8 assay. The calculated IC50 values from this assay were17.6� 5.2 mM in the case of monascopyridine E and 35.1� 3.4 mM in the case ofmonascopyridine F.

3. Experimental

3.1. Reagents

Methanol and acetonitrile (HPLC grade) were purchased from VWR (Darmstadt,Germany) or Sigma-Aldrich (Steinheim, Germany). Ethanol was obtained from Roth(Karlsruhe, Germany) and formic acid was purchased from Grussing (Filsum, Germany).Water for HPLC analysis was purified with a MilliQ Gradient A 10 system (Millipore,Schwalmbach, Germany).

3.2. Preparation and extraction of red yeast rice

The strain of M. purpureus (DSM 1379) was obtained from the German Collection ofMicroorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Subcultures weremaintained on Salt Nirenberg Yeast medium in slant agar tubes for 2 weeks at 30�C andstored at 4�C. After the addition of 2.5mL sterile water, the resulting suspension was usedfor inoculation of the rice medium. In this method, 90 g rice was grounded, soaked with500mL distilled water, sterilised and portioned on 20 Petri dishes (10 cm i.d.). Cultureswere incubated at 30�C. After 3 weeks, the cover was removed and the mycelium was driedfor 1 week in a drying chamber followed by pulverisation using a laboratory mill. Analiquot of the obtained powder (0.5 g) was extracted with 5mL methanol by sonicationand subsequent centrifugation. Extraction was repeated thrice, and supernatants werecombined. For metabolite accumulation, 20mL of the extract was diluted with 20mLwater, resulting in 40mL extract in methanol/water (1 : 1 v/v). The solution was passedthrough a Strata C18-E cartridge (Phenomenex, Aschaffenburg, Germany) after condi-tioning the sorbent with 20mL methanol and water (4 : 6, v/v). Stepwise elution with70mL of 40%, 60%, 80% and 100% methanol was carried out. Eluates were analysed byHPLC–DAD, evaporated and lyophilised. The fraction eluting with 80% methanol wasused for further preparative isolation. Additionally, extraction of 100mg dried myceliumpowder was performed with 1mL methanol, ethanol and acetonitrile, respectively,followed by centrifugation and direct analysis of the supernatant via HPLC–FTMS.

3.3. Preparative isolation of metabolites

An aliquot of 0.1 g of the lyophilised 80% methanol extract was dissolved in 10mLmethanol and 1% formic acid (4 : 6 v/v) and separated on a 250� 10mm i.d., 5 mm,

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Microsorb 100-5 C18 column (Varian, Darmstadt, Germany) using an isocratic flow of4mL 78% methanol and 22% formic acid (1% in water) delivered by a Varian Pro StartM–210 pump. Injection volume was 1.8mL. Chromatograms were recorded on a VarianPro Star 325 UV-detector at 306 nm wavelength. Fractions were checked by HPLC–DADand HPLC–FTMS.

3.4. HPLC analysis

3.4.1. HPLC–DAD

A Jasco PU-2089 low-pressure pump, an autosampler AS-2057 plus and an MP 201 plusdiode array detector combined with Galaxy software (Jasco, Grossumstadt, Germany)were used. A 250� 4mm i.d., 5 mm Reprosil-Pur C18-AQ column (Dr Maisch,Ammerbuch, Germany) was used with a binary gradient of 1% formic acid (A) andmethanol (B) using the following gradient: 0–2min, 20% B; 2–32min, 20–100% B; 32–40min 100% B. The flow rate was 1mLmin�1. Chromatograms were recorded on a JascoPlus MD-2010 diode array detector between 200 and 600 nm. Separation of monasco-rubramine and monascopyridine E was carried out with a gradient of 1% formic acid (A)and acetonitrile (B) as follows: 0–1min, 68% B; 1–9min, 81% B; 9–10min, 81% B. Theflow rate was 1mLmin�1.

3.4.2. HPLC–FTMS

Extracts were analysed with an Accela LC system. An Accela 60057-60010 pump andautosampler were linked to an LTQ Orbitrap XL mass spectrometer (Thermo FisherScientific, Bremen, Germany). Data acquisition was performed with Xcalibur 2.07 SP1(Thermo Fisher Scientific). Separation was performed on a Gemini C18 110 A 150� 2mmi.d., 5 mm column (Phenomenex, Aschaffenburg, Germany) using a binary gradient ofacetonitrile and water with 1% formic acid. The injection volume was 10 mL and the flowrate was 200 mLmin�1. HPLC was programmed as follows: linear gradient from 40%acetonitrile to 100% acetonitrile in 18min. After each run, HPLC column was washedwith 100% acetonitrile for 2min and equilibrated for 5min at starting conditions. Themass spectrometer was operated in positive-heated electrospray ionisation mode. Furtheradjustments were as follows: capillary temperature: 225�C, ion spray voltage: 3500V;capillary voltage: 16V; tube lens voltage: 127V; multipole 00 offset: �5.25V; lens 0voltage: �4V; multipole 0 offset: �4.5V; lens 1 voltage: �8V; gate lens voltage: �42V;multipole 1 offset: �8V; front lens voltage �5.25V. For structural elucidation viafragmentation, experiments in collision-induced dissociation mode with fragmentationenergy values ranging from 0% to 35% were performed.

3.5. NMR spectroscopy

NMR data were acquired in CDCl3 on a Bruker DPX-400 (Bruker Biospin, Rheinstetten,Germany) NMR spectrometer. Also, 1D NOE spectroscopy data were acquired on aUnity Plus 600 (Varian, Palo Alto, CA) NMR spectrometer. For structural elucidation,2D experiments such as 1H, 1H-COSY, 1H, 13C-HMQC and 1H, 13C-HMBC experimentswere performed.

3.6. Spectral data

3.6.1. Monascopyridine E

HESI–FTMS positive mode m/z 358.20136 [MþH]þ (m/z Calcd for [C21H27NO4þH]þ:358.20128), UV �max (MeOH)¼ 312 nm.

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1H-NMR (400MHz, CDCl3) �: 0.86 (3H, t, J20,19¼ 7.0Hz, H-20), 1.18–1.32 (4H, m, H-18–19), 1.51–1.60 (2H, m, H-17), 1.93 (3H, d, J11,10¼ 6.8Hz, H-11), 2.49 (3H, s, H-22),2.50 (2H, t, J16,17¼ 7.3Hz, H-16), 3.25 (2H, s, H-14), 3.90 (3H, s, H-8), 6.48 (1H, d,J9,10¼ 15.6Hz, H-9), 6.86 (1H, dq, J10,11¼ 6.8Hz, J10,9¼ 15.6Hz, H-10), 7.23 (1H, s, H-3),8.10 (1H, s, H-12) and 9.11 (1H, s, H-6).

13C-NMR (101MHz, CDCl3) �: 14.4 (C-20), 19.2 (C-11), 23.0 (C-19), 24.0 (C-17), 26.0 (C-22), 31.8 (C-18), 41.2 (C-14), 43.8 (C-16), 52.9 (C-8), 120.7 (C-3), 121.7 (C-4), 130.9 (C-9),135.9 (C-10), 137.5 (C-13), 141.5 (C-12), 146.2 (C-5), 152.5 (C-6), 160.0 (C-2), 166.1 (C-7),199.4 (C-21) and 209.2 (C-15).

3.6.2. Monascopyridine F

HESI–FTMS positive mode m/z 386.23237 [MþH]þ (m/z Calcd for [C23H31NO4þH]þ:386.23258). UV �max (MeOH)¼ 312 nm.

1H-NMR (400MHz, CDCl3) �: 0.84 (3H, t, J22,21¼ 6.4Hz, H-22), 1.22–1.30 (8H, m, H-18–21), 1.51–1.61 (2H, m, H-17), 1.93 (3H, d, J11,10¼ 6.8Hz, H-11), 2.48 (3H, s, H-24),2.59 (2H, t, J16,17¼ 7.5Hz, H-16), 3.24 (2H, s, H-14), 3.89 (3H, s, H-8), 6.47 (1H, d,J9,10¼ 15.5Hz, H-9), 6.85 (1H, dq, J10,11¼ 6.8Hz, J10,9¼ 15.5Hz, H-10), 7.23 (1H, s, H-3),8.09 (1H, s, H-12) and 9.10 (1H, s, H-6).

13C-NMR (101MHz, CDCl3) �: 14.2 (C-22), 18.8 (C-11), 22.8 (C-21), 24.0 (C-17), 25.6(C-24), 29.3 (C-18), 29.3 (C-19), 31.8 (C-20), 40.9 (C-14), 43.5 (C-16), 52.5 (C-8), 120.3(C-3), 121.4 (C-4), 130.5 (C-9), 135.5 (C-10), 137.2 (C-13), 141.1 (C-12), 145.9 (C-5), 152.1(C-6), 159.7 (C-2), 165.8 (C-7), 199.1 (C-23) and 208.9 (C-15).

3.7. Cytotoxicity assay

IHKE cells were used for determination of the cytotoxic properties of the newly identifiedsubstances. Therefore, the metabolic activity of the cells was measured using the CCK-8assay according to the previously described protocol (Knecht et al., 2006). The substanceswere added in concentrations ranging from 0.1 to 250 mM, and the incubation time was24 h. Results are presented as mean values from three replicate experiments with threedifferent passages. The calculation of the IC50 values was done using SigmaPlot 11.0.

4. Conclusions

Alongside, 10 already described secondary metabolites and 2 unknown metabolites wereidentified after analysing an extract of M. purpureus by HPLC–DAD. The occurringmetabolites were pre-purified using a C18-cartridge and further isolated by semi-preparativeHPLC. The chemical structures of the two new metabolites were elucidated using FTMSand 1D- and 2D-NMR experiments. The molecular formulas were C21H27NO4 andC23H31NO4, differing only in an alkyl side chain as frequently found for other Monascusmetabolites. Furthermore, the molecules contain a pyridine backbone and were thereforenamed monascopyridines E and F. The methyl ester moiety of both metabolites was shownto be an artefact formed by methanol utilisation during the isolation procedure. Thedetermination of the cytotoxicities of monascopyridines E and F was carried out usingIHKE cells in the CCK-8 assay displaying IC50 values in the micromolar range.

Acknowledgement

The authors thank K. Bergander for NOE-measurements.

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