Available online at ScienceDirect. Procedia Chemistry 9 (2014 )

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1 Available online at ScienceDirect Procedia Chemistry 9 (2014 ) International Conference and Workshop on Chemical Engineering UNPAR 2013, ICCE UNPAR 2013 The Influence of Temperature and Ethanol Concentration in Monacolin K Extraction from Monascus Fermented Rice Marlia Singgih a *, Vienna Saraswaty b, Diah Ratnaningrum b, Sri Priatni b, Sophi Damayanti a a Pharmacochemistry Research Group, School of Pharmacy, Bandung Institute of Technology, Labtek VII, Jl. Ganesha 10, Bandung 40132, Indonesia b Research Centre for Chemistry, Indonesian Institute of Sciences,Jl. Cisitu Sangkuriang Gd.50, Bandung, 40135, Indonesia Abstract One of the major causes of death is coronary heart disease which is usually caused by hypercholesterolemia. HMG-CoA reductase is a rate-limiting enzyme that plays important role in cholesterol biosynthetis. The enzyme acts a prime target for pharmacology intervention. Monacolin K is known as an inhibitor of HMG-CoA reductase. It was produced as a fungal metabolite either from Monascus sp. or Aspergillus terreus strain. The compound had been proven to be anticholesterol and antihypotensive agent. Red Yeast Rice or angkak is a traditional food that fermented by Monascus sp. Traditional form of angkak resulting specific smell and taste. Extraction of angkak will make a better form with more acceptance by consumer. During extraction process, the type of organic solvent and temperature are critical factor for compound extraction. The aim of this study was to investigate the influence of ethanol concentration and temperature in Monacolin K extraction process from Monascus Fermented Rice (MFR). Investigation of extraction process was carried out using variable of ethanol concentration i.e. 40, 50, 60, 70, 80 and 95% (v/v). The effect of temperature during extraction was also investigated through two condition i.e. room temperature and 60 o C. The amount of Monacolin K, which contained in crude extract, was measured using spectrophotometric and High Performance Liquid Chromatography (HPLC) method. The results showed that the highest amount of Monacolin K in crude extract was found in extraction using 95% v/v of ethanol (MFR95) whereas effect of heating procedure also showed that the amount of Monacolin K in crude extract was increased in 60 o C. HPLC analysis showed that the yield of Monacolin K in crude of MFR95 extract was 1760 g g -1. In conclusion, ethanol 95% v/v and heated in 60 o C were the best choice for extraction of Monacolin K from MFR The The Authors. Authors. Published Published by by Elsevier Elsevier B.V. B.V. This is an open access article under the CC BY-NC-ND license ( Selection and peer-review under responsibility of the Organizing Committee of ICCE UNPAR Peer-review under responsibility of the Organizing Committee of ICCE UNPAR 2013 * Corresponding author. Tel.: /73. address:marlia@fa.itb.ac.id, The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( Peer-review under responsibility of the Organizing Committee of ICCE UNPAR 2013 doi: /j.proche

2 Marlia Singgih et al. / Procedia Chemistry 9 ( 2014 ) Keywords : Monascus sp.; extraction; ethanol; temperature; Monacolin K Nomenclature g µg.g -1 o C rpm v/v weight (gram) yield (microgram per gram) Temperature in celcius Speed (rotary per minute) Ratio of concentration (volume/volume) 1. Introduction Coronary Heart Disease (CHD) is one of main caused death in developing countries especially in the low and middle-income countries 1. There is a strong correlation between total cholesterol, Low Density Lipoprotein (LDL) and CHD 2. LDL is a major of cholesterol carrier in blood. It is a strong CHD predictor 3. A high level of LDL indicates a high risk of CHD. In an effort to lower cholesterol levels, there are several type of cholesterol lowering agent i.e. Niemann-Pick C1 like 1 (NPC1L1) competitor, Intestinal acyl CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors, 3-hydroxy-3-methylglutaryl (HMG-CoA) reductase inhibitors, LDL receptor up-regulators, bile acid reabsorption inhibitors, cholesterol-7 -hydroxylase (CYP7A1) activators, and plasma cholesteryl ester transporting protein (CETP) inhibitors 4. HMG-CoA reductase is an enzyme that mediates the rate limiting reaction of cholesterol biosynthesis 4. This type of medication is usually an option for CHD treatment. A strong HMG-CoA reductase inhibitor that had been widely known among people was statin drug such as Lovastatin and Simvastatin (See Fig.1) 5. Lovastatin or Monacolin K, can be found either as synthesis drug or as natural product It had been investigated that Monacolin K was active as oral cholesterol lowering agent in the dog after 3 weeks treatment of 8 mg/kg weight per day 8. Monacolin K was found as fungal secondary metabolite from Aspergillus terreus, Monascus purpureus, Monascus sp Red Yeast Rice or Angkak is one of Asian traditional medicine which used for alternative statin treatment 14. Angkak was made from solid state fermentation of Monascus sp. Recent study had been proved that rice which is fermented by Monascus sp. producing Monacolin K as one of its major secondary metabolite 4,9-11, Analysis of anti cholesterol agent from Angkak by use of male white rat of Sprague Dawley strain also proved that it could reduce blood cholesterol 17. Figure 1. Structure of Statin Drug (Monacolin K and Simvastatin) Consumer satisfication is one of important factor in product development. Actually, Angkak had no taste but sour in smell. Chinese people like to make it as brew-wine. In traditional use, Angkak was infused then filtered before

3 244 Marlia Singgih et al. / Procedia Chemistry 9 ( 2014 ) drink. Extraction method of angkak will give a benefit in customer satisfication. Condition of angkak that are less favored by consumer will be removed and the extract can be made in several forms of product which adapted to consumer desire. Citrinin is a toxin that found in angkak. The allowance of Citrinin in angkak is 200 ng/g in Japan but its authorized limit is still under consideration in China and Europe 18. Therefore, monitoring of citrinin level should be performed in angkak extraction. This research investigated the influence of ethanol concentration and temperature for Monacolin K extraction from angkak or Monascus Fermented Rice (MFR). Levels of Monacolin K, citrinin and pigment were monitored by use of spectrophotometric method. 2. Material and Method 2.1 Material Monascus purpureus HD001 was taken from collection of Microbiology Laboratory, School of Pharmacy, Bandung Institute of Technology and activated in broth medium before fermentation. Rice was bought from general market and sterilized in autoclave 121 o C for 15 minutes before use. Organic solvent i.e. ethanol is technical grade which were destillated before use. Phosphoric acid, acetonitrile and methanol were bought from Merck with pro analysis (p.a) grade. Monacolin K or Mevinolin as standard was bought from SIGMA. Yeast, peptone, and malt extract were from Pronadisa and dissolved in Aqua destilata before sterilized. Aquabidestilata was used as source of water for HPLC analysis. 2.2 Fermentation About 30 ml of activated Monascus sp. broth (2 x 24 hours) was inoculated aseptically to 300 g of sterile rice in a bottle under Laminar Air Flow (LAF). The inoculated rice were shaken until homogen and incubated for 14 days in incubator of 30 o C. Monascus Fermented Rice (MFR) was moisturized using sugar solution (@4 ml) every 24 hours for the first of 7 days. The shakes of MFR were performed once a day until incubation time completed. On the 14 th day MFR were harvested and dried in oven of 50 o C for 24 hours then blended before extraction. 2.3 Extraction About 1 g of blended dried MFR were put in an Erlenmeyer of 50 ml then added by 20 ml of ethanol solution. Concentration of ethanol solution used was varied i.e. 40, 50, 60, 70, 80, and 95% (v/v). After that MFR was agitated in a shaker incubator (New Brunswick Scientific, Edison M.J, USA) for 2 hours, 60 o C, 110 rpm. The filtrates were separated under filtered vacuum and analyzed using spectrophotometer. For extraction without heat, the MFR in Erlenmeyer was put for 2 hours in shaker incubator, 110 rpm, RT and directly separated under filtered vacuum before analyzed using spectrophotometry. 2.4 Spectrophotometric Analysis Spectrophotometric analysis was performed in optimum wavelength of Monacolin K, Citrinin and Pigment i.e. at 238, 330 and 500 nm respectively by use of HITACHI U-2800 Spectrophotometer. 2.5 High Performance Liquid Chromatography (HPLC) Analysis Presence of Monacolin K in sample was confirmed by using HPLC method. Column was Nucleosil C-18. Detection was by UV detector at 238 nm. Sample was dissolved in methanol p.a and filtered before injection. The mobile phase for analysis were acetonitrile:water (ph was adjusted to 2.5 with H 3 PO 4 p.a) at ratio of 45:55 (by volume) and flow rate was 1.0 ml/min. Mevinolin from Sigma was used as standard.

4 Marlia Singgih et al. / Procedia Chemistry 9 ( 2014 ) Results and Discussion 3.1 The influence of Temperature in Monacolin K Extraction Temperature usually played important role in extraction especially for sensitive natural compound. Previously, heating procedure to the level of temperature at 175 o C showed the degradation of Monacolin K and also increased the proportion of Citrinin. However, heating procedure at temperature of below 140 o C did not give significant effect for Monacolin K degradation. Condition temperature of 65 o C and 30% of ethanol solution had been determined as basal condition for MFR extraction. Due to level of Citrinin decreased significantly by treatment of dry heat at 65 o C for one hour 10. The influence of lower temperature but longer time of extraction can be seen in Table 1. Temperature control strategy showed that extraction by use of RT and two hours contact time almost gave similar M/C ratio with extraction under heat of 65 o C, 1 hour contact time with M/C ratio of 3.0. However, by use of heat at temperature of 60 o C, the yield of Monacolin K increased about two fold greater than extraction in RT. Moreover, by use of 60 o C degree, 110 rpm and 2 hours extraction, the ratio of M/C reached value of 3.7, which in this experiment was the best ratio of M/C. By exposure above, temperature of 60 o C degree, 110 rpm, and 2 hours extraction with 95% v/v ethanolic solution gave a better condition for Monacolin K extraction from MFR. Table 1. Effect of Temperature for Monacolin K Extraction from MFR Temperature Control Strategy Absorbance M/C o C, 110 rpm, 2 hours 0,544 0,148 0, RT, 110 rpm, 2 hours 65 o C, 1 hour 0,294 0,098 0, Data shown was the results of average (n=3), RT : Room Temperature, 3.2 The Influence of Ethanol Concentration in Monacolin K Extraction Previous study had explained that Monacolin K could be extracted using several organic solvents such as ethyl acetate, ethanol, acidified ethanol, and also alkaline solvent i.e. sodium bicarbonate 7,10. Ethanolic solution was selected for Monacolin K extraction from MFR because the polarity met criteria of Monacolin K solubility. Figure 2 showed that different of ethanol concentration caused a significant effect in MFR extraction. From the level of Monacolin K and pigment in MFR crude extract, it can be seen that increase of ethanol concentration in extraction process resulted a higher yield of Monacolin K and pigment. However, trend of Citrinin concentration in MFR crude extract almost similar. It means that by use of ethanol concentration in the range of 40% to 95% v/v showed no significant effect to citrinin extraction level. Absorbance 0,600 0,500 0,400 0,300 0,200 0,100 0,000 Monacolin K Citrinin Red Pigment MFR95 MFR80 MFR70 MFR60 MFR50 MFR40 Ethanol concentration (%v/v) Figure. 2. Influence of Ethanol concentration in Monacolin K extraction process

5 246 Marlia Singgih et al. / Procedia Chemistry 9 ( 2014 ) The ratio of Monacolin K and Citrinin in each of MFR crude extract was calculated. Concentration of citrinin is one of important factor in optimization of extraction because Citrinin is regarded as toxic compound in MFR. After comparing the result, the use of 80% and 95% v/v ethanol concentration for MFR extraction gave the highest M/C ratio (see Table 2.). In Table 2, the study was compared with previous investigation 10, we found that extraction method by use of 95% and 80% v/v ethanol, 2 hours extraction and under temperature of 60 o C is better than previous condition that use of 30% v/v ethanol, 1 hour extraction and temperature of 65 o C. The longer extraction time gave the better yield. Although by use of 80% and 95% v/v ethanol concentration gave the similar of M/C ratio, but the highest yield of Monacolin K was found by use of 95% v/v ethanol solution (MFR95) (See Fig.2). It might happened because of the level polarity from ethanol 80% v/v is higher than 95% v/v and Monacolin K is more soluble in ethanolic solution therefore the higher of ethanol concentration increased the yield of Monacolin K. Table 2. Ratio of Monacolin K and Citrinin (M/C) after Extraction with Different Ethanol Concentration Sample M/C MFR MFR MFR MFR MFR MFR MFR HPLC Analysis Confirmation of Monacolin K concentration in MFR95 crude extract was performed using HPLC method. By use of 200 g of MFR for extraction, the yield of Monacolin K was measured. The result showed that our MFR95 crude ethanol extract contained about 1760 g g -1 of Monacolin K. The amount of Monacolin K in solid state fermentation was influenced by the type or species of microorganism used for fermentation. We compared the concentration of Monacolin K in our MFR95 crude extract that fermented by Monascus sp. of ITB collection (Monascus purpureus HD001) with another MFR that fermented by Aspergillus sp.and Monascus sp.. The results showed that yield of Monacolin K in rice that fermented by Monascus purpureus HD001 was two folds higher than rice that fermented by Aspergillus terreus and another species of Monascus purpureus (See Table 3). However, different type of strain will also give fluctuative yield of Monacolin K content. This was because of each microorganism had its own optimum condition and need different strategy of extraction and fermentation process. Table 3. Yield of Monacolin K in Solid State Fermentation Medium (Rice) Using Different Type of Microorganism Microorganism Monacolin K ( g.g -1 ) Experiment/ Reference Aspergillus terreus JPM Ref 6 Aspergillus terreus JPM Ref 6 Aspergillus terreus JPM Ref 6 Aspergillus terreus MTCC Ref 6 Monascus purpureus HD001 1,760.0 In experiment Monascus purpureus NTU Ref 19 Monascus purpureus NTU Ref 19

6 Marlia Singgih et al. / Procedia Chemistry 9 ( 2014 ) Conclusion In conclusion, research on influence of ethanol concentration and temperature in extraction process of Monacolin K from solid state fermentation (MFR) by use of Monascus sp. had been successfully carried out. The best yield of Monacolin K could be obtained by use of ethanol 95% v/v, heat treatment at 60 o C and two hours contact time. Acknowledgements Thanks to Ministry of Research and Technology for funding year of InSinas References 1. Gaziano, TA, Bitton A, Anand S, Abrahams-Gessel S, and Murphy A, Growing Epidemic of Coronary Heart Disease in Low- and Middle- Income Countries, Curr Probl Cardiol 2010;35(2): Corsini A, Maggi FM, and Catapano L, Pharmacology of Competitive Inhibitor of HMG-CoA Reductase, Pharm. Res. 1995;31(1): Howard BV, Robbins DC.,Sievers ML, Lee ET, Rhoades D, Devereux RB, Cowan LD, Gray RS, Welty TK, Go OT, Howard WMJ, LDL Cholesterol as a Strong Predictor of Coronary Heart Disease in Diabetic Individuals With Insulin Resistance and Low LDL, The Strong Heart Study, Arterioscler Thromb Vasc Bio. 2000;20: , 4. Chen ZY, Ma KY, Liang Y, Peng C, Zuo Y, Role and Classification of cholesterol-lowering functional food, J of Func. Foods, 2011;3: Ahmad A, Panda BP, Khan S, Ali M, and Javed S, Downstreaming and Purification of Lovastatin from Monascus purpureus culture (Original Research Article), Thai J. Pharm. Sci., 2009;33: Jaivel N and Marimuthu P, Optimization of Lovastatin Production in Solid State Fermentation by Aspergillus terreus, Int. J. of Eng. Sci. and Tech., 2010;2(7): Pansuriya RC, and Singhal RS, Supercritical Fluid Extraction of Lovastatin from The Wheat Bran Obtained after Solid State Fermentation, Food Technol. Biotechnol. 2009;47(2): Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, Rothrock J, Lopez M, Joshua H, Harris E, Patchett A, Monaghan R, Currie S, Stapley E, Albers-Schonberg G, Hensens O, Hirshfield J, Hoogsteen K, Liesch J, and Springer J, Mevinolin: A Highly Potent Competitive Inhibitor of Hydroxymethylglutaryl-coenzyme A reductase and a Cholesterol-lowering Agent., Proc. Natl. Acad. Sci. USA, 1980;77(7): Sun JL, Zou XZ and Xiao TF, Elevated Yield of Monacolin K in Monascus purpureus by Fungal elicitor and Mutagenesis of UV and LiCl, Biol. Res., 2011;44: Lee CL, Chen WP, Wang JJ, and Pan TM, A simple and rapid Approach for removing Citrinin while Retaining Monacolin K in red Mold Rice, J. Agric. Food. Chem. 2007;55(26): Pattanagul P, Pinthong R, Phianmongkhol A, and Tharatha S, Mevinolin, Citrinin and Pigments of Adlay Angkak Fermented Monascus sp., Int. J. of Food Microbiol. 2008;126; Chen LC, Lai YK, Wu SC, Lin CC, Guo JH, Production by Clonostachys compactiuscula of a lovastatin esterase that converts lovastatin to Monacolin J Original Research Article, Enzyme and Microbial Tech., 2006;39(5): Hirama M and Iwashita M, Total synthesis of (+)-Monacolin K (Mevinolin), Original Research Article, Tetrahedron Lett. 1983;24(17): Li XM, Shen XH, Duan ZW, and Guo SR, Advances on Pharmacological Effects of Red Yeast Rice, Chin. J. of Nat. Med. 2011;9(3): Chen F and Hu X, Study on red fermented Rice With High Concentration of Monacolin K and Low Concentration of Citrinin, Int. J. of Food Microbiol. 2005;103: Ou HP, Wang CCR, and Lai LS, Thermal degradation Kinetic Analysis of Monacolin K in Monascus fermented products, LWT-Food Sci. and Tech., 2009;42: Kasim E, Kurniawati Y, and Nurhidayat N, Use of Local Isolate of Monascus purpureus for Reducing Blood Cholesterol in Sprague Dawley Rat (Article in Indonesian), Biodiversitas, 2006;7(2): Mandt M, Legal opinion on the Use of Red Fermented Rice (Angkak) in Food. Paper collection for a symposia of Monascus culture and Applications, held from July 8 th to 10 th, 1998 in Toulouse, France 19. Chun LL, Jyh JW, Shing LK, and Tzu MP, Monascus fermentation of dioscorea for increasing the production of cholesterol-lowering agentmonacolin K and antiinflamation agent- Monascin, Appl. Microbiol Biotechnol.,2006;72: