A POSSIBLE ALTERNATIVE FOR STATIN DRUGS
Cyril John Domingo
Department of Food Science and Chemistry, College of Science and Mathematics
Lovastatin, also known as mevinolin or monacolin K, is the major monacolin found in red yeast rice. Monacolin K, one of the first among many monacolins used for humans, belongs to polyketides and the structure thereof shares similarity with HMG-CoA which has been used as a hypocholesterolemic agent. The enzyme HMG-CoA reductase is the target of the widely available cholesterol-lowering drugs known collectively as statins. This study was conducted to screen for Monascus strains which would yield high amounts of lovastatin in rice cultures or angkak to be used for food and perhaps as food supplement. Eight (8) Monascus strains were studied using the submerged fermentation technique. Screening was done by thin layer chromatography (TLC), and the confirmation and quantification of lovastatin was done by RP-HPLC and concentration was confirmed using standard grade lovastatin. Of the eight strains, M. purpureus Went (JCM 6934) produced the highest lovastatin content, while the Philippine strain M. purpureus UPLB-MNH-MCC 2108 followed as second highest. Using the latter, our local strain to inoculate rice, the angkak produced was analyzed for lovastatin by extraction in 75% ethanol and analysis by TLC. Quantification of lovastatin from solid-substrate fermentation can only be done by HPLC using a photodiode array detector which we still have to acquire. Using TLC, with a standard, lovastatin was identified in angkak rice and this showed that this was also a characteristic constituent of angkak rice, aside from that detected in submerged fermentation. Red Yeast Rice or angkak, a product of fermenting rice with Monascus spp., has a long history as a food colorant and a folk medicine in China. In many Asian countries now, it is used in the preparation of cheese, brewing wine and liquor, while in the Philippines angkak has long been used as coloring agent for bagoong and as preservative for meat and fish. The top lovastatin producing Philippine strain from the above experiment was mass produced on rice and was tried as a cookie ingredient in the Food Science test kitchen of this campus. Citrinin content which was analyzed by TLC prior to the sensory evaluation was found to be very low to be detected.
Keywords: Monascus, angkak rice, RYR, lovastatin, hypocholesterolemic
Monascus spp. belongs to the group of Ascomycetes of the Family Monascaceae and is known for its ability to produce three types of pigments, namely: orange, yellow and red. Four species have been isolated from traditional oriental food, however, Monascus purpureus, is the most well studied by Went in 1985. This species is known for its production of the purple angkak pigment which is now used for various purposes in food found in Asian countries. Aside from its coloring properties and of improving the taste of food, Monascus has also been recorded in the ancient Chinese pharmacopoeia by the great pharmacologist Li Shizen of the Ming dynasty as a medical agent for improving digestion, revitalizing the blood and strengthening the spleen and stomach (Hsieh et al., 2008; Liu et al., 2006). Recent studies have shown that angkak can inhibit and prevent increase in total cholesterol, low-density lipoprotein cholesterol (LDL-C) and triglycerides and acts as antioxidative, anticarcinogenic and antimicrobial agent (Erdogrul and Azikar 2005). Monascus colorants have long history of consumption in Asia yet, no reports have shown that it poses hazard to human health. Some researchers, however, reported that some Monascus strains produce citrinin which is a nephrotoxin. This has led to the controversy about the safety of angkak. However, more recent researches conducted by Allok (2004) and Chen, et. al (2009) and Lee et al. 2010 confirmed that angkak poses no threat to human health.
The inhibition of cholesterol synthesis by monacolins is similar to the action of commercial statin drugs (Journod and Jones, 2003). Monacolin K belongs to the polyketides and the structure thereof shares similarity with HMG-CoA. Therefore, monacolin K competitively inhibits cholesterol synthesis with HMg-CoA in producing mevalonate, resulting in the reduction of cholesterol synthesis.
Lovastatin production can be done through solid-state fermentation (SSF) or submerged fermentation. A comparison of both methods on lovastatin production was reported by Guanrong, et al.(2002). For submerged fermentation, average productivity of lovastatin was observed to be highest on the 17th day. For solid-state fermentation, lovastatin production was slow for the first 8 days but a significant yield was obtained in the latter phase, from the 20th day to the 30th day. Several analytical procedures have been developed for the determination of lovastatin, based on thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and Liquid Chrom-Mass spec (LC-MS). A detection method established by Li et al.(2004) used HPLC with a photodioide array and tandem mass spectrometry (MS/MS) where well resolved peaks of the seven main compounds of monacolin were profiled. Studies done by Jaivel and Marimuthu (2010) and Samieeet al.(2003) both made use of TLC as screening method for several fungal strains and HPLC for confirmation of the presence of lovastatin and quantification.
The present study therefore, aimed to find available strains of Monascus which can give a very good yield of lovastatin using the submerged culture method, and produce red yeast rice or angkak from these strains. Further, this study would like to determine if the angkak samples also contain a good yield of the lovastatin, even if they are incorporated in certain foods.
Materials and Methods
The study was conducted at the Department of Food Science and Chemistry, College of Science and Mathematics. Eight (8) Monascus strains, three of which were from the Japan Collection of Microorganisms (JCM) and five from the UPLB-MNH- Microbial Culture Collection were used for the screening. The submerged fermentation was chosen since the conditions are easier to replicate and monitor. Preculture and production conditions were done according to Samiee et al. (2003) by adding 1mL of spore suspension from strains grown on PDA onto a 40 mL seed medium, after which ten percent seed broth was added onto the production medium, consisting of 5% glucose, 2% yeast extract, 3% tomato paste, 2% oatmeal 1% sodium acetate, 0.5% ammonium sulphate, and 0.2% potassium dihydrogen phosphate, The medium was adjusted to pH 7 and shaken (180 rpm) at 28 C for 7 days. Extraction of lovastatin was done on the 7-day samples according to Samiee et al.(2003) where fifty ml production culture was adjusted to pH 3.0 using conc HCl, followed by the addition of ethyl acetate and shaking at 180 rpm, ambient temperature for 2 h. The samples were collected from the organic phase after centrifuging at 1500g for 15 minutes.
Thin layer chromatography (TLC) was used to detect the presence of lovastatin. One and a half ml of the organic phase concentrated to 50 uL using a block heater, 45 C and spotted onto a PET-backed TLC plate. The mobile phase was dichloromethane:ethyl acetate (70:30, v/v) and visualization of the spots was done by exposing the plates to iodine vapour. Lovastatin standard (98% pure, Sigma) was used for Rf comparison.
Quantificaiton of lovastatin was done using HPLC. One and a half mL of the organic phase of each positive sample in the TLC was completely evaporated and was dissolved in 1.5mL acetonitrile analyzed by HPLC-LC20AD (Shimadzu Prominence, Kyoto, Japan), UV-Vis detector (23 5nm), a 150×4.6mm Shim-pack CLC-ODS C18). The mobile phase was acetonitrile:water (60:40 v/v) and a flowrate of 1.5ml/min.
Angkak rice sample was obtained from the Department of Food Science and Chemistry from an on-going study on angkak production for use as food ingredient. The method used for the solid state fermentation using rice as substrate was from Dizon et al. (1984).Powdered angkak rice was extracted with 15ml of 75% ethanol at 30 C for one hour with shaking, 250 rpm (Chen and Hu 2005) and then the suspension was centrifuged for 10min, 10,000 rpm prior to TLC analysis.
Results for the screening of lovastatin from Monascus and quantification of lovastatin were subjected to one-way ANOVA at 0.05 level of significance and Duncan’s Multiple Range Test.
Results and Discussion
Table 1 shows the eight Monascus strains used in this study. Thin layer chromatography (TLC) was carried out for the initial detection of the presence of lovastatin. Samples extracted by ethyl acetate, and the standard were spotted onto a plastic-backed TLC plate (10cmx10cm) was developed inside a glass chamber equilibrated with dichloromethane and ethyl acetate (70:30, v/v).
|Monascus purpureus, Went||JCM 6934|
|Monascus purpureus, Went||JCM 22616|
|Monascus purpureus, Went||JCM22621|
|Monascus anka, Nakazawa and Saito||UPLB-MNH-MCC 2105|
|Monascus purpureus Went||UPLB-MNH-MCC2106|
|Monascus purpureus Went||UPLB-MNH-MCC 2108|
|Monascus sp.||UPLB-MNH-MCC 2196|
|Monascus sp.||UPLB-MNH-MCC 2197|
Table 1. The eight Monascus strains used. (JCM=Japan Culture of Microorganisms; UPLB-Museum of Natural History Microbial Culture Collection)
The spots were visualized using iodine vapor which turned lovastatin to golden yellow. The Rf values of the isolates were found to be 0.413± (0.0043). Using one-way ANOVA, results showed that there was no significant difference among the eight cultures and the standard. All strains were shown to be positive for lovastatin production. For confirmation and quantification of lovastatin, the extracts were analyzed by RP-HPLC. Figure 1 shows the chromatogram of the isolates together with the standard.
|Fig 1. Thin layer chromatograms of the extracts with the standard lovastatin to confirm the spot.|
Confirmation of the identity of lovastatin using HPLC
After screening for lovastatin using TLC, results showed that all eight strains were positive for lovastatin production. For confirmation and quantification of lovastatin, All of the eight TLC positive cultures were analyzed using HPLC. The analysis was done in triplicate. The peak of lovastatin was identified by comparing the retention time with the corresponding lovastatin standard. All the cultures showed a peak with retention times of 5.408 ± (0.038) minutes for culture A, 5.416 ± (0.0043) minutes for culture B, 5.412 ± (0.0014) minutes for culture C, 5.409 ± (0.0112) minutes for culture D, 5.422 ± (0.008) minutes for culture E, 5.423 ± (0.027) minutes for culture F, 5.421 ± (0.016) minutes for culture G, and 5.410 ± (0.047) minutes for culture H. The retention time of the lovastatin standard was 5.415 ± (0.008) minutes. Using one-way Anova, results showed that there was no significant difference among the retention time of the eight cultures and the standard. Thus, it can be stated that all the eight cultures produced lovastatin.
Quantification of lovastatin
Lovastatin was quantified using the equation generated from a standard curve. Figure 2 shows a graph comparing the lovastatin content of the eight strains. As shown, strain A yielding the highest lovastatin content (84.85 ppm).
Using one-way Anova, results showed that there was a significant difference among the eight cultures used in this study F(7,16) = 412.608, 0.00<0.05. Results were then subjected to Duncan’s Multiple Range Test (DMRT) to determine which cultures are significantly different among the rest.
|Fig 2. Lovastatin content of the eight cultures studied, analyzed by HJPLC, the highest Culture A showing a lovastatin content of 84.85 ppm. Among the local starins Culture F showed the highest, at 38.9 ppm.|
A comparison with high-yielding microbial sources reported in literature showed that lovastatin content produced by the fungi-producing Aspergillus terreus (55 ppm), Penicillium funiculosom (19.3 ppm), (Becker 2008), were very much less compared to our high-yielding strain A.
Identification of Lovastatin in Angkak Rice
Sample Produced Locally Using TLC
Powdered angkak rice was extracted with 75% ethanol and the extract was subjected to TLC using the method described earlier. The Rf values of the rice-ethanol extract was calculated to be 0.418 ± (0.01511) while the Rf value of the standard was 0.41533 ± (0.00379). Using one-way Anova, results showed that there was no significant difference on the Rf value of the angkak rice sample and the standard. Figure 3 shows the chromatogram of the angkak rice sample extract with the lovastatin standard and that the rice samples also contained lovastatin similar to the submerged culture extracts.
Fig. 3. Thin Layer Chromatogram of the angkak rice sample
extract together with the standard.
This detection method revealed that lovastatin could be a characteristic constituent of angkak rice or rice fermented with Monascus strains. However, lovastatin from solid-state fermentation could further be quantified more accurately using HPLC, with photodiode array detector, and this now being undertaken in the lab.
Utilization of angkak in popular biscuits
A preliminary study was done to apply powdered angkak in such popular snack item as biscuits, just like in Taiwan. Although angkak has been proven to contain compounds contributing to good health, such as Monacolin K, issues on its safety is currently being considered. Presence of citrinin should be monitored and controlled in angkak. Thin-layer chromatography was used to detect citrinin in angkak samples. Standard citrinin has an Rf value of 0.686. A compound in angkak which was suspected to be citrinin emitted a weak fluorescence and has an Rf value of 0.711 and 0.701 for samples extracted with 70% ethanol and 95% ethanol, respectively. Standard citrinin emitted yellow green fluorescence but a weak fluorescence was emitted by the angkak sample. By comparing the fluorescence emission of standard citrinin and angkak samples and its corresponding Rf values, it was found out that citrinin in our angkak sample was almost undetectable. Therefore, it was considered that angkak from Monascus purpureus UPLB-MNH-MCC 2106 could be safe for human consumption. For the general acceptability test, sign test was used to determine if the product was acceptable. Appearance/color, aroma, texture, taste and overall acceptability from the sensory tests resulted in all acceptable. Figure 4 shows the locally made angkak samples, the TLC for citrinin content and the biscuits made using the angkak as additive.
|Figure 4. The rice angkak samples (a) and biscuits made in the laboratory, and the test for presence of citirinin using TLC (b). Standard citrinin is expected to emit a yellow-green chromophore, the blue fluorescent emissions could be due to presence of monopurflouore, according to Hsu et al (2010.)|
A strain of Monascus which our lab acquired from the Japan Culture of Microorganisms showed a very high content (85ppm) of lovastatin in submerged culture, even higher than those reported in literatures. A strain of Monascus purpureus acquired from the UPLB-MNH Culture collection was the highest among the local Philippine strains at 39 ppm. The strain was cultured onto rice to make angkak and determine if lovastatin was still synthesized. A TLC method was used and lovastatin was detected. A preliminary utilization of the angkak rice in a popular biscuit formulation showed that the inclusion of 3g angkak did not affect the acceptability of the product. However, before sensory tests were done, a test for presence of citrinin content was done and which showed an almost undetectable presence. Further tests on lovastatin and citrinin, especially on solid substrates, are now being done using Photodiode Array as detector for HPLC as especially recommended in literatures .