IDENTIFICATION OF STATINS IN RICE FERMENTED WITH MONASCUS SPECIES FROM THE MUSEUM OF NATURAL HISTORY
Jude Carlo J. Muca1, Reigna S. Romero1, Ricardo R. Santos², Roberto Z. Yuseco3, Florence M. Blanco1, Lei Anne C. Carolino1, Engkhuan Chew1, Ericka Joy B. De Guzman1, Jordan Carlo S. Galang1, Jin-Gu Lee1, Lawrence Y. Maliwat1, Marixie Ann Q. Manarang1, Jeshua Caleb B. Miole1, Michael Henry B. Piano1, Lou Anthony S. Sico1, Mark Lester I. Tolenada1, Mellanie B. Victoria1

1 Medical Student, Angeles University Foundation, Angeles City 2009;
²Head, Department of Biochemistry, School of Medicine, Angeles University Foundation, Angeles City 2009; 3 Faculty, Department of Biochemistry, School of Medicine, Angeles University Foundation, Angeles City 2009;

ABSTRACT
Pharmaceutical intervention through statin drugs is the most common way of slowing down the adverse effects of heart disease due to cholesterol deposition by regulating the activity of HMG-CoA reductase which catalyzes conversion of HMG-CoA to mevalonate. In order to seek alternatives for commercially available statin drugs, this study idnetified the statins that can be produced by fermentation of rice by Monascus strains available at the Museum of Natural History (MNH). It also shall serve as a set-off point for further studies regarding the use of fermentation products for treatment of certain ailments, such as heart disease. A total of eight (8) isolates available at the Museum of Natural History (MNH) of the University of the Philippines – Los Baños (UPLB) were used for the production of angkak rice. Among these eight (8) strains, six (6) strains were able to produce the three commercially available statins used as standards, namely: simvastatin, rosuvastatin and atorvastatin. Ethanolic extracts of the angkak rice were measured through UV spectrophotometer and plotted against a standard curve. All the strains were able to produce the said statins; three out of six strains namely Monascus anka Nakazawa and Sato 2105, Monascus anka Nakazawa and Sato 2106, and Monascus purpureus Went 2240 were able to produce the three statins whose concentrations can be derived from the calibration curve. It is concluded that angkak rice can be produced from the different Monascus strains available from the Museum of Natural History. Nonetheless, the calibration curve limits may be extended in further studies, and additional analytical tests can be done to confirm the identity of the compounds.

Abbreviations: Hydroxymethylglutaryl-coenzyme A (HMG-CoA), Museum of Natural History (MNH), Ultraviolet spectrophotometer (UV Spec)
Keywords: angkak, atorvastatin, Monascus, Monascus anka Nakazawa and Sato, Monascus purpureus Went, rosuvastatin, simvastatin

INTRODUCTION

The 2003 statistics of the 6th National Nutrition Survey by the Food and Nutrition Research Institute of the Department of Science and Technology determined that one of the three leading illnesses in the Philippines is heart disease. More often than not, the reasons for the growing number of Filipinos suffering from such disease are due to unhealthy diet, such as those with high saturated fat and high cholesterol, and poor lifestyle2.

Pharmaceutical intervention through statin drugs is the most common way of slowing down the adverse effects of heart disease due to cholesterol deposition3. They inhibit cholesterol synthesis by regulating the activity of HMG-CoA reductase which catalyzes conversion of HMG-CoA to mevalonate13. Nevertheless, current researches are focusing on controlling cholesterol with diet Red yeast rice, a product of rice fermentation using Monascus spp., was found to have the ability to reduce blood-lipid levels in both animal models and humans14 as it forms naturally occurring HMG-CoA reductase inhibitors, primarily Monacolin K or lovastatin15,18.

This study aimed to extract and determine the amount of statins present in cultures of Monascus strains available at the Museum of Natural History (MNH) of the University of the Philippines – Los Baños. Specifically, the study aimed: (1) to produce angkak rice using Monascus strains available at the Museum of Natural History (MNH); (2) to extract statins produced in the angkak rice preparations; and (3) to determine the statins produced by the different strains using UV Spectrophotometry. This study served to identify the statins that can be produced by fermentation of rice by Monascus strains available at the Museum of Natural History (MNH), and to identify the strains which are able to produce the higher amounts of statins than do others. This served as a set-off point for further studies regarding the use of fermentation products with regards to treatment of certain ailments, such as heart disease due to abnormal deposition of cholesterol in the body.

The research focused on identifying the different statins extracted from the different angkak preparations using different Monascus strains available only at the Museum of Natural History (MNH). Due to budget constraints, screening was done only through UV spectrophotometry; hence, only a crude estimation of the statins was done.

METHODOLOGY

Monascus Strains
A total of eight (8) isolates (Table 1) available at the Museum of Natural History (MNH) of the University of the Philippines–Los Baños (UPLB) were used for the production of angkak rice. Stock cultures were kept in PDA (Potato Dextrose Agar) medium containing infusion of potatoes (20%), dextrose (2%), and agar (2%), and were subcultured monthly19.

Preparation of Seed Culture
Seed cultures were prepared by transferring a loopful of spore from PDA agar plate into 500 mL Erlenmeyer flask containing 100 mL basal medium (100 g dextrose, 10 g peptone, 2 g KNO3, 2 g NH4H2PO4, 0.5 g MgSO4.7H2O, 0.1 g CaCl2 dissolved in 1000 mL distilled water, pH 6.0). The culture was incubated at 30oC for 48 h22. After incubation 5% seed culture was inoculated for preparation of angkak rice (solid state fermentation).

Preparation of Angkak Rice
Rice was soaked in distilled water at room temperature for 8 h, removing excess water after soaking. The soaked rice was autoclaved for 20 min at 121°C in an autoclave. Thirty (30) grams of sterile cooked rice were placed in each plate, with three plates per strain. The substrates were then cooled and then inoculated with 5% seed culture of the Monascus strains, and the inoculated substrates was incubated at 30°C for 10 days22.

Preparation of Angkak Rice Extracts
Presence of an intense red color in the rice preparations indicates the successful fermentation of the Monascus strains. Four grams of rice per strain was mixed with 40 mL of sterilized distilled water and boiled for 4 h at 100°C. The extract was then filtered through a Buchner funnel.

Preparation of Samples for Analysis
Aqueous extracts of the angkak rice were adjusted to a pH of 6.5. One gram of extract per strain was diluted (5 mL) with ethyl alcohol (90% v/v) and filtered through Whattman filter paper No.1.

Preparation of Standards Commercially available statin drugs atorvastatin, rosuvastatin, and simvastatin were used as standards. Standard solutions of the reference drugs were prepared1. Standard stock solution was prepared by dissolving 10.0 mg of reference drug in 100mL of 90% ethanol. The solutions were then filtered. The linearity for the spectrophotometric method was established in the concentration of 5, 10, 15, 20 and 25g/mL.

Quantitative Analysis of Statins The samples were read at different wavelengths, namely: 244 nm for rosuvastatin10; 255 nm for simvastatin12; and 245 nm for atorvastatin17. The values obtained were plotted against the calibration curves obtained.

RESULTS AND DISCUSSION

This chapter features the results, analyses and interpretations of data obtained from the experiment performed. Tables will be presented. The researchers prepared Potato Dextrose Agar (PDA) to which the different Monascus strains were sub cultured. A seed culture was prepared and inoculated on the non-glutinous rice. Growth of Monascus species were noted and angkak rice were extracted using ethanol. The ethanolic extracts were read using a UV spectrophotometer at wavelengths based on the different standards.

Fermentation of ang-kak rice was done for 10 days at 30OC, and results of the growth of the strains are presented in Table 2. After the incubation period, only six out of eight strains were able to produce an intense red color in the rice preparation, indicating growth. Monascus species produce an intense red pigment as well as other metabolic by-products when cultivated on cooked non-glutinous rice16,20,23.

The concentrations of the statins present in the ethanolic extracts from the angkak rice preparations based on their absorbance at specific wavelengths are illustrated in Table 3. Concnetrations were computed using the Beer-Lambert’s Law (See Appendices). All six strains were able to produce the three commercially available statins which were used as standards. However, it can be noted that they vary in concentrations. Three out of six strains namely Monascus anka Nakazawa and Sato 2105, Monascus anka Nakazawa and Sato 2106, and Monascus purpureus Went 2240 were able to produce the three statins whose concentrations can be derived from the calibration curve (See Appendix Figures). On the other hand, Monascus purpureus Went 2108 and Monascus sp. 2196 only produced atorvastatin whose concentrations can be derived from the calibration curve; the other two statins were also produced but were nonetheless beyond the curve’s limits. Monascus purpureus Went 2107 produced the three statins but the concentrations cannot be obtained from the calibration curve.

Various studies were already done regarding the determination of metabolites in red yeast rice, especially their statin contents. In the study, three commercially available statins present in the ethanolic extracts of the angkak rice preparations were found to occur in all the samples. Literatures regarding the biosynthesis of atorvastatin, rosuvastatin, and simvastatin by Monascus species are limited, if any. Nevertheless, studies on the synthesis and characterization of the statin-related compounds indicated that several monacolins were obtainable from Monascus strains. Monacolin J and L were isolated and characterized from cultures of a Monascus ruber strain4,5,6. Moreover, Endo has reported dihydromonacolin L and monacolin X production and activity from a mutant strain of M. ruber7. A series of statins were also obtained by chemical modification of the C8 side chain in the lovastatin molecule, which is a byproduct of angkak rice production, and a systematic evaluation of the structure-activity relationships of the obtained compounds was also carried out. One of the obtained molecules, simvastatin was found to be a semi-synthetic molecule with practical applications8,11. This color in the rice preparation, indicating growth. Monascus species produce an intense red pigment as well as other metabolic by-products when cultivated on cooked non-glutinous rice 16,20,23. This study supports biotransformation studies carried out in the lovastatin to obtain new and powerful statin1,11,15.Hence, there is a need for further analysis, determination, and characterization of the possible statin-related compounds which might occur in the angkak extracts.

SUMMARY AND CONCLUSION

Studies show that angkak or red yeast rice, a product of rice fermentation using Monascus spp., have the ability to reduce blood-lipid levels in both animal models and humans as it forms naturally occurring HMG-CoA reductase inhibitors, primarily Monacolin K or lovastatin. Due to the unavailability of a standard reference lovastatin drug, the researchers resorted to determining the presence of other statins, specifically atorvastatin, simvastatin and rosuvastatin in the cultures of Monascus strains available at the Museum of Natural History (MNH), namely: Monascus anka Nakazawa and Sato 2105, Monascus anka Nakazawa and Sato 2106, Monascus purpureus Went 2107, Monascus purpureus Went 2108, Monascus purpureus Went 2239, Monascus purpureus Went 2240, Monascus sp. 2196 and Monascus sp. 2197.

After procuring the strains from MNH, the researchers sub cultured the eight strains of Monascus in Potato Dextrose Agar (PDA). This was followed by the preparation of the seed culture, then by solid state fermentation wherein the seed cultures were inoculated on autoclaved non-glutinous rice for preparation of the angkak rice. After the incubation period, only six out of eight strains were able to produce an intense red color in the rice preparation which indicates growth. These are the following strains which produced the intense red color: Monascus anka Nakazawa and Sato 2105, Monascus anka Nakazawa and Sato 2106, Monascus purpureus Went 2107, Monascus purpureus Went 2108, Monascus purpureus Went 2240 and Monascus species 2196. In contrast, Monascus purpureus Went 2239 and Monascus sp. 2197 did not grow on the angkak rice preparations as evidenced by the absence of an intense red color on the rice preparations.

From the six strains, ethanolic extracts of the angkak rice were prepared, and subjected to UV Spectrophotometry at various wavelengths based on the standards. Three out of six strains namely Monascus anka Nakazawa and Sato 2105, Monascus anka Nakazawa and Sato 2106, and Monascus purpureus Went 2240 were able to produce the three statins whose concentrations can be derived from the calibration curve. On the other hand, Monascus purpureus Went 2108 and Monascus sp. 2196 only produced atorvastatin whose concentrations can be derived from the calibration curve; the other two statins were also produced but were beyond the curve’s limits. Monascus purpureus Went 2107 produced the three statins but the concentrations cannot be obtained from the calibration curve.

From these, the researchers concluded that angkak rice can be produced from the different Monascus strains available from the Museum of Natural History. Three commercially available statins were used as standards, namely: atorvastatin, simvastatin, and rosuvastatin. The said standards were present in the ethanolic extracts of the angkak rice preparations, but occur in different concentrations.

RECOMMENDATIONS

There is only a little number of studies, if any, regarding fermentation of red rice with Monascus species from the Museum of Natural History. The researchers would like to recommend to the future researchers that they should consider using other methods of analysis such as Reversed Phase-High Performance Liquid Chromatography in determining the metabolites and secondary metabolites (specifically rosuvastatin and atorvastatin) present in the angkak rice. Furthermore, the future researchers must consider the usage of a analytical and/or pharmaceutical grade standards with high purity in the analytical methods.

The researchers also suggest the determination of toxic substances found in red yeast rice fermented with Monascus species, paving way for its safety as a functional food in treatment of various diseases. Finally, the future researchers may conduct laboratory animal trials on the various pharmacological activities of the extracts from Monascus fermented rice. This study shall in one way or another lay the foundation for the determination of the presence of the different statins in the angkak rice prepared from Monascus strains obtained from the Museum of National History (MNH).

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