Additional monacolins in red yeast rice prepared from Thai glutinous rice (Oryza sativa L.) cv. sanpatong1(spt1) using Monascus Purpureus CMU 001

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1 Additional monacolins in red yeast rice prepared from Thai glutinous rice (Oryza sativa L.) cv. sanpatong1(spt1) using Monascus Purpureus CMU 001 EM ON CHAIROTE *, SAMUCH TAWEEKASEMSOMBAT AND GRIANGSAK CHAIROTE Faculty of Science and Agricultural Technology, Rajamangala University of Technology Lanna Chiang Mai, Chiang Mai, 50300, Thailand or *Corresponding author. ABSTRACT: - Red yeast rice which is a product of solid fermentation was prepared from the most suitable raw material Thai glutinous rice (Oryza sativa L.) cv. Sanpatong1(SPT1). Monascus purpureus CMU 001 was used as the fermentation starter. The rice was incubated at 30 C for 2 weeks in the red yeast rice fermentation process. Dry red yeast rice was ground to a powder and used for extraction of monacolins. The extract was submitted to two steps silica gel column chromatography. Separated monacolins were studied by HPLC, LC-MS and IR-spectroscopy. We could find 11 monacolins with 4 more Dihydromonacolin L, Monacolin X acid form, Monacolin X and Monacolin L in addition to our previous study. The unknown monacolin with the molecular weight of 358 was studied with IR-spectroscopy. The result showed the lack of hydroxyl group with some differences in the IR-spectrum of C-H vibration regions compared to monacolin K. The molecular weight of 56 was less than monacolin K (Mwt. 404) and may correlate to the loss of H 2 O(18) and the change of the structure of monacolin K to lose C 2 H 4 (28) part of the molecule. Key-words: - Glutinous rice, Monascus purpureus, Red yeast rice, IR-spectrum 1 Introduction Red yeast rice or more precisely red mold rice that has been used in Chinese cuisine and medicinal food to promote blood circulation for centuries is a product of rice fermented by using Monascus purpureus. In other Asian countries, red yeast rice is a dietary staple and is used to make rice wine, as a flavoring agent, and to preserve the flavor and color of fish and meat. The medicinal properties of red yeast rice favorably impact lipid profiles of hypercholesterolemia (Li and Wang, 2005[1] and Segura 2003[2]). Chinese red yeast rice is imported into Thailand to be used as a food colorant and flavoring agent. In Thailand two main kinds of rice are grown and consumed by most people in different areas. They are non- glutinous rice and glutinous rice. The amylopectin content in glutinous rice is higher (95 %) than in non-glutinous rice (70-90 %). The latter contains about 10-30% of amylose. Some varieties of glutinous rice have a very small amount of amylose or without amylose (Insomphun, 2003) [3]. The different in their main composition may affect the content of useful compounds in fermentation products. The preparation of red yeast rice from Thai glutinous rice using Monascus purpureus CMU001 has already been studied (Chairote et al, 2007[4], 2008a[5] and 2008b[6]). Various Thai glutinous rice as well as normal non-glutinous rice varieties were used for the studies while solid fermentation with and without addition of soybean milk were done. The presence of monacolins was confirmed and the amount of monacolins content was determined. The determination of the quantity of citrinin which is a mycotoxin damaging the human kidney was also carried out. The comparative results on the content of both monacolins and citrinin present in different fermentation conditions red yeast rice were obtained. In spite of the highest amount of monacolin K (mevalonin) obtained using Thai glutinous rice, Sanpatong 1(SP1), some compounds seemed to be more dominant among the monacolins. These compounds may have some inter-conversion with monacolin K making the decrease of monacolin K content. It is our interest to find other monacolins and gain more information about their structure. The structure elucidation should be done in order to know the relationship and inter-conversion between these compounds and monacolin K. The study will provide important information useful for controlling metabolic pathway subsequently maintaining a high amount of monacolin K. In this work, the extract of monacolins was prepared from the fermentation of Thai glutinous rice, Sanpatong 1(SPT1). The presence of additional known monacolins was confirmed. The unidentified compound with dominant peak in HPLC chromatogram was selected to study. In another work(chairote et al, 2008b[6]), the presence of a monacolin having a related structure to monacolin K was reported; therefore, this work was focused on the study on the basic information supporting the structure elucidation of the compound. 2 Materials and Methods 2.1 Materials Thai glutinous rice, Oryza sativa L. cv. Sanpatong 1(SPT1), used for preparing red yeast rice was purchased from a local rice supplier in Chiang Mai province, Thailand. Monascus purpureus CMU001 isolated from commercial Chinese red yeast rice was kindly provided from the laboratory of microbiotechnology, Chiang Mai University, Thailand, with best regards from Professor Saisamon Lumyong. ISBN:

2 2.2 Preparation of red yeast rice Inoculation and cultivation of isolated M. purpureus CMU001 were done using Thai glutinous rice Oryza sativa L. cv. Sanpatong 1 (SPT1). The controlled condition with appropriate weight, culture age, inoculation volume, temperature, humidity and ph according to Boonsangsom et al. (2004) [7] and Chairote et al (2008a)[5] was used. A sample of 3 weeks cultivation was finally obtained and used for further study. Glutinous rice grains were immersed in water for 6 hours followed by steaming for 20 minutes. After cooling, 50 g of steam rice was put in 250 ml flask and sterile at 15 psi and 121 o C for 15 minutes. One week old pre-cultured M. purpureus CMU001 was used as inoculums. The inoculated rice was incubated at 30 o C for 3 weeks. The product was dried in the oven at 65 o C for 6 hours to obtain dried red yeast rice. 2.3 Sample extraction An extraction of the sample was carried out using 0.5 g of ground rice put into a 20 ml centrifugal tube followed by adding 10 ml of 75% HPLC grade ethanol. The mixture was degassed in an ultrasonic bath for 60 min. The supernatant was collected after centrifugation using 3,000 rpm speed at 4 o C (Li, et al., 2004) [8]. The extraction was repeated three times and all of the extracts were mixed together and made up to 50 ml using 75% ethanol. Finally, the extract was filtered through a 0.2 μm membrane and kept in a vial before being analyzed. 2.4 Separation by column chromatography The extracts were evaporated under a vacuum rotary evaporator. The concentrated crude extract was separated further by chromatography using silica gel column as a stationary phase. Elution with a stepwise solvent gradient of hexane 100% to hexane/dichloromethane of 90%:10%, to dichloromethane/ethylacetate of 90%:10% and to ethylacetate/methanol of 60%:40% was performed. Several separated fractions from silica gel column were collected. Fractions showing similar TLC chromatography were combined and separated again by flash column chromatography using hexane:dichloromethane:ethylacetate (70%: 20%: 10%) isocratic system as a mobile phase. The second column fractions were filtered using a 0.2 µm nylon membrane and kept in separated vials for further monacolins analysis. 2.5 High performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC- MS) The presence of monacolins in appropriate fractions samples were confirmed by HPLC (Agilent HP 1100) with Inertsil ODS-3 column (5 µm 4.6x150 mm 6BI85146) and photodiode array detector. The chromatography was performed using acetonitrile as a solvent A and 0.1% trifluoroacetic acid (TFA) as a solvent B. Linear gradient elution (0.5 ml/min) from 35% to 75% A in 30 min and keeping at 75% A from 30 to 40 min was applied with total analysis time of 50 min. The chromatogram was detected at 237 nm with sample injection volume of 10 µl. The samples containing monacolins were analyzed by LC-MS (HP 1100 Binary/G1946A). The mass spectrometer with the electro-spray ionization (API-EI) source was operated in the positive ion mode. The mobile phase, injection volume and flow rate were performed the same as for HPLC analysis. 2.6 Infrared spectroscopic study of unknown monacolin The infrared spectrum of a highly purified monacolin which was separated from others was obtained by using Tensor 27 infrared spectrometer (Bruker,USA) in the rage of cm Results and discussion 3.1 Separation by column chromatography The concentrate crude extract was separated by the first silica gel column chromatography with gradient solvents elution (hexane/dichloromethane ethylacetate/methanol) as described above. The separated fractions that represent most of monacolins in crude extract were recombined. The combined fraction was subjected to HPLC before being separated again in the second column. The chromatogram of combine fraction is shown in figure 1. The labeled peaks were analyzed using reference monacolins and LC-MS technique. The compounds found as well as their molecular weight are shown in table 1. Among the monacolins; dihydromonacolin L, monacolin X acid form, monacolin X and monacolin L are additional monacolins found in red yeast rice prepared using Monascus purpureus CMU 001 and Thai glutinous rice Sanpatong 1(SPT 1). The compound of peak number 8 showing the molecular weight of 358 is one of the dominant peaks. This compound seems to be related to Monacolin K and differs by the lack of OH group by losing H 2 O with the formula weight of 18. The loss of another 28 formula weight may be due to the loss of alkyl group of two carbon atoms or two methyl groups. The purified compound can be further analyzed by Nuclear Magnetic Resonance (NMR) using several techniques such as, 1 H NMR, 13 C-NMR and 2D NMR (2-dimentional or COSY-NMR) to confirm the structure. However, the results from mass spectrum and IR spectrum can contribute to the analysis. In this study, determination of NMR spectrum was not done due to the insufficient purity of this compound causing the presence of the impurity signal in 1 H NMR spectrum. The structure of monacolins which were found in previous studies had been analyzed using several techniques. Monacolin K was identified by Endo (1979)[9] by mass spectrometry, ISBN:

3 infrared spectroscopy and 13 C-NMR. Endo et al. (1985a[10], 1985b[11],1986a[12] and 1986b[13]) identified monacolin J, monacolin L, dihydromonacolin L, monacolin X and monacolin M by these techniques and confirmed the structure of compactin and monacolin K by 2D NMR. In addition, Nagamura et al. (1990)[14] isolated 3-α-hydroxy-3, 5-dihydromonacolin L. In order to obtain more structural information of the compound, the infrared spectroscopic technique was used. The IRspectrum of the highly purified compound number 8 after the separation by a second column is shown in figure 2. The lack of OH group is confirmed by the absence of a broad band centered near 3300 cm -1 due to hydrogen-bonded OH stretching of OH group. Although the IR spectrum shows the different characteristic spectrum of C-H stretching in cm -1 region and anti symmetric deformation of HCH angle of a CH 3 group that gives rise to very strong IR absorption in the cm -1 region(lambert et al,1998)[15], this cannot be used to confirm the different alkyl group compared to monacolin K. To compare the IR spectrum of monacolin number 8 with monakolin K, the IR spectrum of monacolin K studied( Endo, 1979) is shown in figure 3 As described in the previous study, the loss of ß-hydroxyl group may decrease the HMG-CoA reductase inhibition activity of monacolin K (Chairote et al, 2008a). The further study on the relevancy of the compound number 8 and monacolin K is of interest. In order to understand more about the structure, another technique, especially, NMR, should be used; therefore,the sufficient amount and purity of the compound separated is required. References: [1] Li Y.G, Liu H., and Wang Z.T. A validatedstability-indicating HPLC with photodiode array Detector (PDA) method for the stress tests of Monascus purpureus fermented rice, red yeast rice..j Pharm Biomed Anal 2005; 39: [2] Segura B. Red yeast rice: An easy way to lower cholesterol. Nutrition Bytes 2003; 9(1), Article 5. [3] Insomphun S. Rice (Oryza sativa L.). Faculty of Agriculture, Chiang Mai University, Thailand, Available at http//agronomy.agri.cmu.ac.th/elearning/agron 313/rice.doc. Accessed August [4] Chairote E, Chairote G, Wongpornchai S, et al. Preparation of red yeast rice using various Thai glutinous rice and Monascus purpureus CMU001 isolated from commercial Chinese red yeast rice sample. KMITL Sci. Tech. J. 2007; 7(S1): 28. [5] Chairote E, Chairote G, Niamsup H, et al. The presence and the colontent of monacolins in red yeast rice prepared from Thai glutinous rice. World J. Microbiol. Biotechnol. 2008a; 24: [6] Chairote E, Chairote G and Lumyong S. Red yeast rice prepared from Thai glutinous rice and the antioxidant activities. Chiang Mai J.Sci. 2008b; 36(1): [7] Boonsangsom J, Pinthong R, and Raviyan P. Effect of Monascus fungi on the amount of citrinin in red yeast rice. Chiang Mai. Thailand. Research Full Report, Department of Food Science and Technology, Faculty of Agro- Industry, Chiang Mai University, Thailand (in Thai) [8] Li YG, Zhang F, Wang ZT, et al. Identification and chemical profiling of monacolins in red yeast rice using highperformance liquid chromatography with photodiode array detector and mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. 2004;35: [9] Endo A. Monacolin K, a new hypocholesterolemic agent produced by a Monascus species. J Antibiot. 1979;32: [10] Endo A, Hasumi K, Nakamura T, et al. Dihydromonacolin L and monacolin X, new metabolites those inhibit cholesterol biosynthesis. J Antibiot.1985a;38: [11] Endo A, Hasumi K and Negishi S. Monacolins J and L, new inhibitors of cholesterol biosynthesis produced by Monascus ruber. J Antibiot. 1985b;38: [12] Endo A, Hasumi K, Yamada A, et al. The synthesis of compactin (ML-236B) and monacolin K in fungi. J Antibiot. 1986a;39: [13] Endo A, Komagata D and Shimada H. Monacolin M, a new inhibitor of cholesterol biosynthesis. J Antibiot. 1986b;39: [14] Nakamura T, Komagata D, Murakawa S, et al. Isolation and biosynthesis of 3-α-hydroxy-3,5- dihydromonacolin L. J Antibiot. 1990;43: [15] Lambert JC, Shurvell HF, Lightner DA, et al. Organic Structural Spectroscopy: New Jersey. Prentice Hall; ISBN:

4 Table 1 Monacolins identified from red yeast rice extract. Peak No. Compounds MW Reference 1 Dihydromonacolin L 306 Endo et al., Monacolin X acid form Unknown Endo et al., Monacolin X 418 Endo et al., Monacolin K acid form 422 Endo et al., Monacolin L 304 Endo et al., Monacolin K 404 Endo, Unknown Compactin 390 Endo, Monacolin M 406 Li et al., Dehydromonacolin K 386 Ma et al., 2000 Fig. 1 HPLC chromatogram of monacolins in combined fraction of the first separation Fig. 2 IR spectrum of the compound of peak number 8 after separation in a second column. ISBN:

5 Fig. 3 IR spectrum of monacolin K ISBN: